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In Silico Drug Repurposing for SARS-CoV-2 Main Proteinase and Spike Proteins

  • Irene Maffucci*
    Irene Maffucci
    Université de technologie de Compiègne, UPJV, CNRS, Enzyme and Cell Engineering, Centre de recherche Royallieu - CS 60 319 - 60 203 Compiègne Cedex, France
    *Email: [email protected]
    More by Irene Maffucci
  •  and 
  • Alessandro Contini*
    Alessandro Contini
    Università degli Studi di Milano, Dipartimento di Scienze Farmaceutiche, Sezione di Chimica Generale e Organica “A. Marchesini”, Via Venezian, 21 20133 Milano, Italy
    *Email: [email protected]
    More by Alessandro Contini
    Orcidhttp://orcid.org/0000-0002-4394-8956
Cite this: J. Proteome Res. 2020, 19, 11, 4637–4648
Publication Date (Web):September 7, 2020
https://doi.org/10.1021/acs.jproteome.0c00383

Copyright © 2020 American Chemical Society. This publication is licensed under

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Abstract

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The pandemic caused by SARS-CoV-2 is currently representing a major health and economic threat to humanity. So far, no specific treatment to this viral infection has been developed and the emergency still requires an efficient intervention. In this work, we used virtual screening to facilitate drug repurposing against SARS-CoV-2, targeting viral main proteinase and spike protein with 3000 existing drugs. We used a protocol based on a docking step followed by a short molecular dynamic simulation and rescoring by the Nwat-MMGBSA approach. Our results provide suggestions for prioritizing in vitro and/or in vivo tests of already available compounds.

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SPECIAL ISSUE

This article is part of the Proteomics in Pandemic Disease special issue.

Introduction

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The outbreak of a novel β-coronavirus, SARS-CoV-2, is currently a pandemic threat, with already more than 23 million confirmed cases and more than 800 000 deaths all over the world, according to the World Health Organization (data of August 2020, https://covid19.who.int). Unfortunately, although many clinical and preclinical studies are ongoing, to date there is not a validated treatment to this infection.
As for other known coronaviruses, such as SARS-CoV and MERS-CoV, the SARS-CoV-2 entry into host cells is mediated by its transmembrane spike glycoprotein (S-protein). This is a trimeric protein belonging to the class I fusion proteins, whose structure for SARS-CoV-2 has been partially resolved by cryo-electron microscopy (code PDB 6VXX and 6VSB). (1,2) The S-protein is divided into two functional subunits: the S1 subunit, which contains the receptor binding domain (RBD) responsible for the interaction with host cell’s receptors, and the S2 subunit, which is implicated in the fusion of the viral and cellular membranes.
Recent works showed that SARS-CoV-2 S-protein is able to bind the human angiotensin-converting enzyme 2 (hACE2), (3−5) explaining the symptoms linked to the SARS-CoV-2 infection (COVID-19), since hACE2 is widely expressed in endothelial cells from small and large arteries, in lung alveolar epithelial cells, but also in the heart, kidney, testis, and gastrointestinal system. (6,7) Moreover, the crystallographic structure of the RBD in complex with the hACE2 has been recently resolved (PDB code 6M0J), (4) giving molecular details about this interaction (Figure 2). The binding to the host cell receptor triggers a series of conformational changes which allow the fusion with the host cell and the entry of the virus. (1)
In addition, a recognized target for coronaviruses treatments is the main proteinase Mpro, also known as 3CLpro. (8,9) This protein processes the polyprotein 1ab into mature nonstructural proteins that are essential for viral replication (10) and is rather conserved among coronaviruses. Moreover, human proteases with the same specificity have not been discovered so far, making Mpro an ideal target to treat coronavirus infections. The crystal structure of the SARS-CoV-2 Mpro in complex with a covalent peptidomimetic inhibitor (PDB code 6LU7 (11)) was made available. Additionally, the Zhang group, developer of the popular homology-modeling software I-TASSER, (12) made available 24 3D structural models (13) of proteins in the SARS-CoV-2 genome. (14) Among these, the model of the Mpro (code QHD43415) was made available before the release of the crystal and was characterized by a very high reliability score (TM-score = 0.96).
It is clear that both spike and Mpro proteins represent potential targets for anti-SARS-CoV-2 drugs: on one side, hampering the interaction between hACE2 and the viral RBD will block the entry of the virus into the human cells. On the other side, inhibiting the viral proteases, as done with many antiviral drugs currently used in the therapy of HIV infection, (15) will interfere with the viral replication.
However, the experimental procedure to conceive a new drug is long (up to decades) and expensive (up to several millions of dollars). Such a time and resources price is not affordable in the current emergency situation; therefore, a promising alternative consists in a drug repurposing investigation exploiting in silico techniques, such as Virtual Screening (VS), which already proved to be able to identify active molecules against a target. (16,17)
Within this context and aiming to give our contribution to the current sanitary crisis, we designed a VS campaign of currently worldwide approved drugs. Despite the fact that similar studies have been recently published, (18,19) in this work we independently screened more than 3000 molecules against the two SARS-CoV-2 proteins mentioned above to provide information useful for a multiple treatment approach. In addition, we applied a solid VS procedure we recently developed and which was shown to be successful in discriminating active from inactive compounds within the screening of classical small molecules and protein–protein interaction inhibitors. (20)

Methods

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Receptor Preparation

Receptor models for the SARS-CoV-2 Mpro were prepared starting from both the 6LU7 crystal structure and the QHD43415 I-Tasser model. This choice was made to take binding site flexibility into account through an ensemble docking approach (21) but without the need to perform time-consuming molecular dynamic (MD) simulations to generate reliable conformational ensembles. The two Mpro models were prepared using the MOE2019 software, (22) with the following protocol:
6LU7: all water molecules were deleted. The covalently bound peptidomimetic ligand was then unbound from Cys145, and the α,β double bond of the ligand, that behaves as a Michael acceptor, was restored. The Structure Preparation module of MOE was used to correct PDB inconsistencies and to assign the protonation state at pH = 7.0. The default Amber10EHT force field, coupled to the Born solvation model was assigned to the system. The ligand was then minimized, keeping the receptor constrained. Then, the receptor was minimized by applying backbone restraints and keeping the ligand constrained. Finally, the complex was minimized in two separate steps, first by keeping backbone restraints, second by removing all restraints. All minimizations were performed up to a gradient of 0.1 kcal mol–1 Å–2. The receptor and the ligand were then saved for future use.
4MDS: the crystal structure of SARS-CoV 3CLpro proteinase, (23) a close homologue of SARS-CoV-2 Mpro, in complex with a carboxamide inhibitor was also modeled to be used as an additional reference; this was done because no specific SARS-Cov2Mpro noncovalent inhibitors were published at the time of this screening. (24) The system was prepared for calculations as follow: the PDB was corrected and protonated at pH = 7.0 using MOE as stated above. The ligand was minimized, keeping the receptor constrained, using the MMFF94x force field coupled with the Born solvation model. The receptor was then minimized, keeping the ligand constrained, using Amber10EHT+Born. Finally, the complex was minimized in two steps, as described above.
QHD43415: the I-TASSER model QHD43415_5 (13) was superposed to 4MDS (prepared as previously described) in order to precisely define the binding site. Since we observed that the 4MDS ligand also fitted QHD43415_5, the ligand was transferred, and the complex was prepared as described for 4MDS. The resulting structure was used for docking.
The RBD of the S-protein was obtained by the recently resolved X-ray structure of the complex between the SARS-CoV-2 RBD and the human ACE2 (code PDB 6M0J). (4) After the deletion of this latter, the RBD has been protonated at physiological conditions using the H++ server. (25)

RBD binding site definition

In order to determine the RBD residues playing the most important role in the binding to ACE2 (hot spots), the complex between RBD and ACE2 has been initially protonated as the single RBD. Successively, it has been submitted to a molecular dynamics (MD) simulation using the AMBER18 (26) package and the ff14SB (27) force field. The system has been neutralized by adding the proper number of Na+ ions and solvated adding a cubic box of TIP3P water up to a distance of 10 Å from the solute. The system has been relaxed by optimizing the geometry of hydrogens, ions, and water molecules (1000 cycles of steepest descent and 4000 cycles of conjugated gradient). The solvent box has been equilibrated at 300 K by 100 ps of NVT (constant volume and temperature) and 100 ps of NPT (constant pressure and temperature) simulation. Then, a minimization of side chains, water, and ions (2500 cycles of steepest descent and 2500 cycles of conjugated gradient) and a global minimization (2500 cycles of steepest descent and 2500 cycles of conjugated gradient) were performed with a restraint of 10 kcal/mol applied on the backbone atoms. Successively the system has been heated up to 300 K in 6 steps of 20 ps each (ΔT = 50 K) during which the backbone restraints were reduced progressively from 10 to 5 kcal/mol. The systems were then equilibrated for 100 ps in the NVT ensemble and for 200 ps in the NPT ensemble keeping a 5 kcal/mol restraint on the backbone atoms. This was followed by a 4 steps NPT equilibration during which the restraints were progressively reduced to 1 kcal/mol. Finally, after a 500 ps unrestrained NPT equilibration, a production run of 20 ns was performed. During the whole simulation, an electrostatic cutoff of 8 Å, a time step of 2 fs, and the SHAKE algorithm were applied. (28) The root mean squared deviation (RMSD) of the backbone atoms using the X-ray structure as reference was used as a metric of simulation convergence (Figure S1). Hydrogen bonds (H-bond) analysis was performed on the last 10 ns of the simulation using the cpptraj module of AmberTools and using a donor–acceptor distance cutoff of 3.5 Å and a donor–donor hydrogen-acceptor angle cutoff of 150 deg (Table S2).
After having defined as interfacial those RBD residues whose difference in the solvent accessible area when going from the complex to the isolated state was greater than 0.75 Å, an in silico alanine scanning was performed on the last 10 ns of the production run. The mutated complexes have been built using PyMol, (29) and the alanine scanning was run with the Amber mmpbsa.py code on one frame every 100 ps and by choosing the GB-Neck2 (30) as implicit solvent model (igb = 8), the mbondi2 as radii set, and a salt concentration of 0.15 M. The ΔΔG was calculated as the difference between the ΔG of the mutated system and the one of the native system (Table S1). The residues giving ΔΔG greater than 2.5 kcal/mol were considered as hot spots and were used to define the RBD potential binding site for small molecules.

Database Preparation

Two separate databases were downloaded from the corresponding sources (31,32) and merged. The database was then checked and redundant molecules, identified by CAS number, were removed. The database was then processed by MOE in order to build the 3D structures and to minimize the geometry of each molecule. The wash function of the MOE database tool was used with the MMFF94x+Born force field, requesting the dominant protonation state at pH = 7.0 and preserving existing chirality. The final database, consisting of 3118 unique molecules, was saved in SDF format.

Virtual Screening

The Virtual Screening (VS) was done according to our recently developed protocol. (20) This is applied using a set of scripts (available for download as Supporting Information within ref (20)) that does the following steps automatically: (1) Preparation of the screening library, including the generation of tautomers, alternative protonation states, stereoisomers and ring conformers, if requested. (2) Docking of all molecules using PLANTS. (33) (3) Analysis of results. (4) Parameterization of docked ligands selected for rescoring. (5) Molecular dynamics of complexes selected for rescoring, using Amber. (26) Rescoring using the Nwat-MMGBSA method. (20,34,35) All dockings were performed by PLANTS, requesting a search speed = 1 (maximum accuracy) and the ChemPLP scoring function. (36) Only the principal tautomer and protonation state predicted at pH = 7 were considered for the docking. The following receptor-specific parameters were also set up: 6LU7: binding site center (b.s.c.; x,y,z) = −10.2858, 12.3088, 69.3271; binding site radius (b.s.r.; Å) = 16. QHD43415: b.s.c. = −15.124, 15.0521, −24.6152; b.s.r. = 14. RBD-BS1: b.s.c. = −38.621, 39.731, 1.564; b.s.r. = 17. RBD-BS2: b.s.c. = −36.355, 20.471, 2.322; b.s.r. = 17.
Nwat-MMGBSA rescoring was requested for the top 2% of compounds (about 60 molecules for each target). Rescoring consists in performing a short MD simulation (about 2.5 ns, including 1.5 ns of equilibration and 1 ns of production), followed by calculation of binding energy by MMGBSA. (37) In a previous work we demonstrated that longer MD simulations are not necessary for this purpose. (20) Nwat-MMGBSA binding energies were computed by including no explicit waters (Nwat = 0, corresponding to standard MMGBSA calculations) or by selecting a certain number of explicit waters to be included in the calculation (Nwat = 10, 20, 30, 60, and 100).
The same protocol was applied to 4MDS also, since the binding energy computed for the 4MDS crystallographic ligand was used as a reference. Analogously, the binding energy of the 6LU7 ligand (whose covalent bond was broken as described above), was computed as a reference.

Results and Discussion

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Virtual Screening on Mpro

The VS campaign on SARS-CoV-2 Mpro was conducted on two different models (e.g., 6LU7 and QHD43415) to take binding site flexibility into account through an ensemble docking approach, increasing the solidity of the procedure with respect to previous VSs on the same protein.
The results of the VS campaign are summarized in Table 1 and Table 2, respectively, while Tables S2 and S3, Supporting Information, report compounds selected by docking but that failed during the MD/Nwat-MMGBSA rescoring step. Results of Nwat-MMGBSA rescoring, using 30 explicit waters, are the only reported, since Nwat = 30 was considered a reasonable value in previous publications. (20,34)
Table 1. Results of the VS Campaign on the Crystal Structure of SARS-CoV-2 Mpro (6LU7)a
Drug Name Dock score Nwat-MMGBSAb
Angiotensin II –124.4 –120.3 ± 10.1
GHRP-2 –132.6 –106.0 ± 8.3
Indinavir –122.4 –86.5 ± 5.7
Polymyxin B –107.9 –84.2 ± 8.3
Fexofenadine –107.8 –77.0 ± 7.8
Atazanavir –109.6 –73.0 ± 7.6
Cobicistat –124.3 –72.8 ± 8.3
Aliskiren –109.9 –70.9 ± 6.5
Lercanidipine –106.6 –67.4 ± 8.4
Darunavir –108.1 –66.6 ± 6.8
Montelukast –112.8 –54.9 ± 6.8
Latanoprost –108.5 –52.5 ± 4.2
Octenidine –114.0 –50.8 ± 4.9
Velpatasvir –108.4 –46.5 ± 8.1
Tyloxapol –112.3 –42.5 ± 6.5
Salvianolic acid B –124.4 –41.1 ± 11.0
Nilotinib –106.6 –40.1 ± 8.6
Siponimod –105.9 –38.5 ± 6.0
Travoprost –114.9 –35.6 ± 6.1
Vitamin A Palmitate –107.6 –35.5 ± 6.1
Penfluridol –110.1 –30.2 ± 7.3
Clindamycin –106.2 –20.5 ± 15.4
Ledipasvir –109.6 –20.1 ± 7.8
Elbasvir –106.3 –19.8 ± 9.9
a

Top 2% of compounds selected from the docking of 3118 FDA approved drugs and rescored by Nwat-MMGBSA (Nwat = 30) are shown. Compounds that ranked better than the reference are highlighted in bold. The 6LU7 crystallographic ligand of the SARS-CoV-2 main protease (6LU7) was used as the reference. Docking and Nwat-MMGBSA scores are −132.7 and −70.6 ± 8.0 kcal/mol, respectively.

b

Nwat-MMGBSA rescoring was done considering 30 explicit water molecules around the ligand (Nwat = 30).

Table 2. Results of the VS Campaign on the Homology Model of SARS-CoV-2 Mpro (QHD43415)a,b
Drug Name Dock score Nwat-MMGBSAc,d
Caspofungin –108.3 –97.9 ± 12.4
Lopinavir –106.5 –89.9 ± 5.9
Atazanavir –109.9 –86.0 ± 7.0
GHRP-2 –116.7 –79.2 ± 11.1
Indinavir –105.4 –78.6 ± 6.5
Angiotensin II –125.7 –75.7 ± 9.2
Dehydroandrographolide Succinate –99.4 –61.1
Ritonavir –112.3 –58.3 ± 7.8
Azilsartan medoxomil –102.1 –54.4
Salvianolic acid B –116.0 –51.0 ± 7.7
Vilanterol –100.7 –50.9
Elbasvir –110.2 –48.0 ± 7.7
Clindamycin –99.6 –47.8
Montelukast –110.1 –47.5 ± 6.9
Latanoprost –101.0 –46.8
Cobicistat –119.3 –45.4 ± 11.6
Octenidine –104.8 –43.6
Mupirocin –98.1 –42.3
Tyloxapol –105.5 –41.1 ± 8.3
Echinacoside –103.1 –40.0
Salmeterol Xinafoate –105.3 –37.9 ± 7.3
Ledipasvir –101.5 –37.3
Thonzonium Bromide –99.3 –36.7
Lomitapide –98.1 –34.2
Travoprost –99.2 –34.0
Itraconazole –100.2 –32.6
Penfluridol –106.2 –31.8 ± 9.6
Cisatracurium besylate –100.3 –23.6
Retinol palmitate –100.1 –21.8
Terfenadine –98.1 –17.7
a

The homology model of SARS-CoV-2 Mpro was made available by the Zhang group at https://zhanglab.ccmb.med.umich.edu/C-I-TASSER/2019-nCov/

b

Top 2% of compounds selected from the docking of 3118 FDA approved drugs and rescored by Nwat-MMGBSA (Nwat = 30) are shown. Compounds that ranked better than the reference are highlighted in bold. The 4MDS crystallographic ligand in complex with SARS-CoV 3CLpro, a close homologue of SARS-Cov-2 Mpro, was used to compute reference scorings. Docking and Nwat-MMGBSA scores are −96.4 and −59.8 ± 5.3 kcal/mol, respectively.

c

Nwat-MMGBSA rescoring was done considering 30 explicit water molecules around the ligand (Nwat = 30).

As can be observed, the protease inhibitors indinavir and atazanavir, currently used to treat HIV infections, have been selected by both models. Conversely, the protease inhibitor lopinavir is top ranked for the QHD43415 model only, while it failed during MD for 6LU7 (Table S2, SI), probably due to steric clashes originating from a position of the side chains in the crystal structure not favorable for a stable binding of this compound. Figure 1 shows the predicted binding mode for lopinavir, that anchors to the Mpro binding site by multiple H-bonds. The first H-bond is observed between the catalytic His41 residue and the aryloxyacetylamido carbonyl (H-bond length = 2.1 Å). Additional H-bonds are observed with Glu166 and the hydroxyl group at C-4, that acts both as a donor and an acceptor (lop-O(H)···(H)N-Glu166 and lop-OH···O═C-Glu166 distances = 1.8 Å and 2.2, respectively). Finally, a dual H-bond is observed between the side chain of Gln189 and both the butanamido NH (lop-NH···O═C-Gln189 = 1.9 Å) and the 2-oxo-1,3-diazinanyl carbonyl (lop-C═O···H2N-Gln189 = 2.0 Å).

Figure 1

Figure 1. Predicted binding mode of lopinavir to Mpro. The model was obtained by performing a cluster analysis of the MD trajectory of the docking pose, followed by a backbone-restrained geometry minimization of the main cluster using MOE.

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In addition, within an in vitro study against SARS-CoV-2, it has been shown that lopinavir has an estimated 50% effective concentration (EC50) of 26.63 μM in Vero E6 cells. (38)
Other HIV protease inhibitors such as darunavir and ritonavir were selected by one of the models, but failed for the other. Indeed, darunavir scored rather well within 6LU7 screening, while ritonavir was high-ranked by QHD43415 although the Nwat-MMGBSA score was slightly lower than the chosen thresholds for both compounds. Interestingly, similar results were also obtained by another group using artificial intelligence. (39) However, ritonavir alone showed an EC50 greater than 100 μM in Vero E6 cells. (38)
Although some clinical studies on the use of HIV protease inhibitors in COVID-19 were already terminated when this screening was made, their results were not already available (https://clinicaltrials.gov/ct2/results?cond=COVID-19). Now, a publication showing that a combination of lopinavir and ritonavir succeeded in alleviating symptoms and shortening the hospitalization in patients with mild to moderate COVID-19, especially when used in association with ribavirin. (40) Nevertheless, it should also be noted that no benefits were observed for hospitalized patients with severe COVID-19 when treated with lopinavir–ritonavir, beyond standard care, (41) suggesting that the treatment is only effective when given at an early stage of the disease. Cobicistat, another drug that is approved for the treatment of HIV infection, has been selected by the VS on the 6LU7 receptor model as a potential Mpro inhibitor, even if its main mechanism is claimed to be the inhibition of CYP3A. (42) At the moment, a combination of darunavir and cobicistat is under clinical evaluation for COVID-19, but preliminary results on efficacy and safety are not encouraging. However, final results are expected for August 31st, 2020. (43)
Some drugs already approved for the treatment of hepatitis C were also identified. These includes elbasvir, (44) ledipasvir, (45) and velpatasvir, (46) that were also identified in other in silico screenings. (19,47) Notably, clinical trials to evaluate the efficacy of ledipasvir on COVID-19 are currently ongoing (https://clinicaltrials.gov/ct2/results?cond=COVID-19). (48)
Interestingly, angiotensin II and GHRP-2 are selected in both screenings. Since the Mpro catalytic activity is to cleave the polyprotein, a peptide of about 15 amino acids, it is plausible that peptides, such as angiotensin II and GHRP-2 of 8 and 6 amino acids, respectively, can fit into the Mpro active site. Although the use of these hormones might not be indicated for the treatment of SARS-CoV-2, their structures might be used as templates for further drug development. Similarly, the antibiotic polymyxin B was also picked as a high-rank hit, although by the 6LU7 model only, probably due to its peptide nature. Interestingly, polymyxin B was top-ranked within the S-protein screening also, as discussed later in this article. Another lipopeptide, caspofungin, has also been identified, but by the QHD43415 model only. Caspofungin is an antifungal drug specifically used in HIV-infected individuals. (49) It was also identified in another independent study as an inhibitor of SARS-CoV-2 replication, (50) even if the Nsp12 polymerase has been claimed as the target.
A hit that might be worthy of attention, although only identified by the QHD43415 model, is dehydroandrographolide succinate (DAS). DAS is a natural product extracted from Andrographis paniculate, well-known by traditional Chinese medicine. (51) Indeed, while the herb has long been used to treat cold and fever, purified andrographolides, including DAS and analogues, have been prepared and used to treat respiratory diseases. (52,53) Antibacterial, antiviral, anti-inflammatory, and immune-stimulatory activities were claimed for DAS, (54) including the inhibition of HIV and H5N1 viruses in vitro. (55,56)
Several other compounds were selected by initial docking but failed during the MD simulation phase (Tables S2 and S3). Such a failure might be due to several causes, among all poor parametrization (the BCC charge parametrization method, (57) instead of the more rigorous RESP method, (58) was chosen for time constraints). However, the reason for the failure might also be due to severe steric clashes or mispositioning during the docking stage. Thus, although some good hits might be found within the “F” series, these selections are to be considered as the least reliable.

Virtual Screening on Spike Protein

When this work was realized, no S-protein RBD-targeting small molecule was known either for SARS-CoV-2 or other coronaviruses. Conversely, few antibodies recognizing the RBD (59−62) and a recombinant ACE2 enzyme (63) are reported. In addition, a 23-mer peptide derived from the ACE2 showed an affinity of 47 nM toward the SARS-CoV-2 RBD, (64) and a few other peptides were developed to inhibit the interactions of the S2 subunit during the fusion process in both SARS-CoV and SARS-CoV-2. (65) Nonetheless, considering that several compounds that are currently being tested, some with positive results, were identified by our VS campaign on Mpro, the results reported hereafter can also represent a step torward the treatment against SARS-CoV-2.

RBD Binding Sites Definition

As for most of the protein–protein interaction (PPIs) interfaces, (66) the one between hACE2 and RBD is quite extended. However, it is possible to target only the residues making the major contribution to the binding free energy between the two proteins (hot spots). In light of this, an alanine scanning analysis was performed by individually mutating the RBD residues at the interface with hACE2 (see Methods, Figure 2 and Table S4), in order to determine which are the RBD hot spot residues. This allowed us to define two clusters of hot spots (Figures 2 and 3), in agreement with a recently published preprint article. (67) More in detail, the first cluster (cluster 1) involves Leu455, Phe456, Phe486, Asn487, Tyr489, and Gln493, while the second one (cluster 2) includes Tyr449, Gln498, Thr500, Asn501, and Tyr505 (Figures 2 and 3). It has to be noted that the found hot spots are not included in the observed mutations found so far, (68) suggesting that they can be safely targeted by potential inhibitors of the ACE2-RBD interaction.

Figure 2

Figure 2. Complex between hACE2 (yellow) and SARS-CoV-2 RBD (green) from the X-ray structure (PDB code 6M0J). The hot spot residues are represented in sticks and labeled in the inset. Hot spots of cluster 1 are represented in orange, while those of cluster 2 are represented in cyan.

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Figure 3

Figure 3. Difference in the binding free energy between the mutated system and the native one computed on the last 10 ns of the MD simulation of the hACE2-RBD complex. All the residues for which the ΔΔG is greater than the threshold (2.5 kcal/mol) have been considered as hot spots. The hot spots of cluster 1 are highlighted in orange and those of cluster 2 in cyan.

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In the second cluster we can find Gln498: when it is mutated to alanine, the complex ΔG has a loss of ∼9 kcal/mol, indicating that this residue is fundamental for the interaction with hACE2. Indeed, the Gln498 side chain is involved in a highly stable H-bond (occupancy >80%, Table S2) with the hACE2 Asp38 side chain, and for the remaining ∼20% it interacts with the hACE2 Lys353 side chain.
Since cluster 1 and 2 are well separated and localized at the two extremities of the RBD interface to hACE2, it is possible to determine two distinct binding sites to target by VS (see Methods), called BS1 and BS2, respectively, in the following discussion. Working with two different possible RBD binding sites represents an advantage as compared to other similar studies, where a single but larger binding site on the RBD was defined. (18,69) Indeed, as previously said, most of the currently available drugs are small molecules which are able to interact with a limited surface and only with a few residues. Therefore, selecting two binding sites corresponding to specific hot spot clusters makes the docking pose search more efficient, by limiting the search space. In addition, it allows us to discard molecules which are predicted to strongly interact with the protein target but on a protein region without hot spots, not assuring the PPI inhibition.

Virtual Screening on RBD-BS1

The results of the VS are reported in Table 3, after the deletion of those molecules which, although being part of the top 2% during the docking step, left the binding during the MD simulation (namely, GHRP-2, cobicistat, oxytocin, and vitamin B12). Although different Nwat values have been evaluated, we will limit our discussion to the results provided by Nwat-MMGBSA with Nwat = 60, since this value resulted to be appropriate when dealing with PPI inhibitors. (20,34)
Table 3. Results of the VS Campaign on the Crystal Structure of SARS-CoV-2 S-Protein RBD Binding Site 1a
Drug name Dock score Nwat-MMGBSAb
Polymyxin B –107.6 –152.1 ± 11.5
Colistin –101.7 –149.4 ± 12.5
Daptomycin –95.2 –137.8 ± 13.1
Oritavancin –93.6 –126.8 ± 13.3
Thymopentin –92.4 –121.9 ± 14.6
Terlipressin –103.7 –118.0 ± 9.6
Lypressin –103.2 –111.3 ± 12.6
Vancomycin –96.2 –104.6 ± 17.9
Leuprolide –110.7 –101.3 ± 10.8
Alarelin –104.6 –98.3 ± 8.9
Deferoxamine –90.8 –97.4 ± 9.0
Bacitracin –93.9 –97.0 ± 11.8
Sennoside B –91.3 –94.9 ± 8.9
Angiotensin II –104.2 –94.8 ± 11.6
Salvianolic acid B –104.5 –93.9 ± 10.2
Gonadorelin –104.2 –93.5 ± 8.7
Nafarelin –111.8 –90.3 ± 11.5
Amphotericin B –108.0 –89.2 ± 11.8
Madecassoside –96.0 –88.5 ± 10.2
Micafungin –95.6 –86.0 ± 12.4
Mupirocin –91.4 –82.1 ± 7.0
Goserelin –107.9 –81.0 ± 12.7
Nystatin –102.2 –78.8 ± 12.8
Echinacoside –93.2 –71.9 ± 9.5
Dalbavancin –90.7 –69.7 ± 12.5
Tyloxapol –106.6 –68.5 ± 7.7
Icatibant –115.8 –67.9 ± 10.4
Landiolol –91.6 –67.3 ± 12.4
Venetoclax –92.5 –66.9 ± 7.3
Vilanterol –95.1 –65.5 ± 8.7
Montelukast –97.6 –65.3 ± 11.9
Salmeterol –100.4 –64.6 ± 7.5
Ginsenoside Rb1 –96.2 –64.2 ± 10.9
Somatostatin –98.6 –61.2 ± 10.2
Ledipasvir –97.4 –60.8 ± 8.5
Zafirlukast –91.0 –57.7 ± 6.0
Latanoprost –98.0 –56.5 ± 7.4
Fexofenadine –91.0 –53.3 ± 17.9
Velpatasvir –91.3 –53.1 ± 8.7
Nebivolol –90.9 –52.2 ± 7.5
Azelnidipine –91.1 –51.6 ± 7.6
Astemizole –91.9 –51.1 ± 5.5
Pranlukast –91.5 –50.3 ± 5.6
Travoprost –89.9 –49.1 ± 8.5
Vilazodone –97.0 –48.6 ± 5.9
Aclidinium –90.0 –48.5 ± 6.4
Octenidine –102.4 –48.1 ± 9.5
Elbasvir –97.4 –47.8 ± 10.0
L-Ascorbyl 6-palmitate –90.8 –47.7 ± 8.0
Silodosin –90.3 –47.1 ± 9.2
Ponatinib –96.6 –44.6 ± 7.7
Ebastine –95.2 –44.3 ± 7.9
Vitamin K2 –95.7 –41.5 ± 6.1
Posaconazole –99.5 –32.6 ± 7.7
Penfluridol –90.3 –31.5 ± 9.1
Vitamin A –96.6 –31.1 ± 8.1
Lapatinib –100.8 –31.1 ± 7.2
Behenic alcohol –93.4 –28.8 ± 7.2
Gefarnate –89.8 –26.2 ± 10.4
Azilsartan –90.2 –24.5 ± 12.4
a

Top 2% of compounds selected from the docking of 3118 FDA approved drugs and rescored by Nwat-MMGBSA are shown ranked by Nwat-MMGBSA scores.

b

Energy obtained by using Nwat = 60, ± standard deviation.

It is not surprising that the best ranked ligands are peptide-like molecules, since these are usually larger than small molecules and allow a better interaction with the PPI binding partner. Most of the top ranked peptide-like molecules, such as the polymyxin B, colistin, and daptomycin, are currently used as antibiotics because of their ability in disrupting the bacterial membrane. Among these, polymyxin B has been tested within a compassionate use protocol for patients with an immediately life-threatening condition. (70)
Others compounds identified herein as potential binders of the S-protein are terlipressin and lypressin, analogs of vasopressin and used against hypotension. We can also find hormone-peptides, such as alarelin or leuprorelin, which belong to the gonadotropin-releasing hormone family, or somatostatin, an endocrine system regulator. Furthermore, we can observe the presence of peptidomimetics, such as icatibant, which acts as an antagonist of B2 bradykinine receptors. This last compound was also identified by an independent study as a potential disruptor of the S-protein-hACE2 PPI. (71)
The only non-peptide molecules found in the top 2% are large compounds (molecular weight >500 g/mol) rich in H-bond acceptor and donor atoms. This is not surprising, indeed it has been suggested that the SARS-CoV-2 binds to the host heparan sulfate chains of the heparan sulfate proteoglycan receptor also, initiating the internalization. (72) In addition, it has been recently shown that the RBD can bind to the heparin (73−75) and that an octosaccharide sequence strongly inhibits this interaction (IC50 = 38 nM). (73) In our VS campaign, we found the salvianolic acid B (also found in Mpro screening), used as antioxidant, antifungal drugs (amphotericin B, micafungin, nystatin, micafungin), the madecassoside and ginsenoside Rb1, molecules with anti-inflammatory properties, and the tensioactive tyloxapol in the top 2%. Interestingly, a combination of amphotericin B and deoxycholate was shown to have an effect in decreasing the infectivity of transmissible gastroenteritis coronavirus. (76) We also found at #11 deferoxamine, a chelating agent under clinical study against COVID-19 (ClinicalTrials.gov Identifier: NCT04333550). However, this molecule is claimed as responsible of chelating the iron whose dissociation from heme is increased by SARS-CoV-2, causing oxidative stress and damage to the lung. (77) The top 2% also contains antivirals such as ledipasvir and elbasvir, used against hepatitis C. However, their mechanism of action involves the inhibition of viral proteins. Additionally, both elbasvir and ledipasvir were identified on the same target in another independent study, using a different computational protocol. (47)
Unexpectedly, among the best 60 RBD ligands there is salmeterol and vilanterol, which are agonists of β2-adrenergic receptors, a class of molecules which showed a minor amplification of the viral phenotype in a recent preprinted study. (78) Conversely, another antiasthmatic drug, montelukast, has been shown to cause the disruption of the viral integrity of the Zika virus; (79) thus, its presence in the top 2% (#39) enhances the interest of this compound against SARS-CoV-2 also.
In addition, we also found a few beta-adrenergic blockers, namely landiolol and nebivolol; this class of molecules, although it is not known to bind to the S-protein, has been hypothesized to be able to decrease the SARS-CoV-2 entry into the cells by downregulating ACE2 receptors. (78,80)
Peptides are usually highly flexible and require an extensive conformational sampling before the VS procedure. However, most of the peptides herein considered are partially cyclic: this creates a structural constraint, making feasible and globally reliable their docking to RBD.
The best scored compounds interact with the RBD through hydrophobic interactions and stable direct and water-mediated H-bond with BS1 hot spots and neighboring residues, creating a stable network of interactions, as shown in Figure 4 for the four top-ranked ligands. For example, polymyxin B can create direct H-bonds with Glu484, Phe486, Asn487, Tyr489, and Gln493 and water-mediated H-bonds with Asn487, Glu484, and Phe490. In addition, we can observe hydrophobic interactions between polymyxin B and Val483 and Phe486.

Figure 4

Figure 4. Snapshot of the MD simulation between RBD (gray) and one of the top four ligands (green). RBD BS1 hot spots are highlighted in orange. Direct and water (spheres) mediated H-bonds are also displayed as dashed lines. Additional RBD residues interacting with the ligand are displayed as sticks.

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Virtual Screening on RBD-BS2

Similar results were obtained for the BS2, as shown in Table 4. In this case also, the ligands which left the binding site during the MD simulation, namely colistin, ritonavir, salmeterol, dalbavancin, and atazanavir, were removed from the list. It is interesting to notice that colistin was the second best ligand for BS1; conversely for BS2, even if the docking procedure ranked this ligand in the top 2%, it could not maintain the favorable interactions during the MD simulation. This highlights the importance of performing MD simulations on the complexes obtained by the docking procedure and provides a further confirmation of the quality of our protocol. (20) In addition, although the amino acid composition of the two binding sites is quite similar (Figure 2), their conformational organization is specific and exploitable for further studies on the development of new potential inhibitors of the RBD-hACE2 interaction.
Table 4. Results of the VS Campaign on the Crystal Structure of SARS-CoV-2 S-Protein RBD Binding Site 2a
Drug name Dock score Nwat-MMGBSAb
Polymyxin B –99.4 –164.3 ± 11.3
Thymopentin –97.7 –154.5 ± 12.9
Icatibant –107.6 –143.1 ± 12.0
Octreotide –94.6 –127.2 ± 10.9
Oritavancin –98.3 –123.6 ± 14.1
Nystatin –110.8 –123.2 ± 10.5
Terlipressin –98.2 –122.8 ± 10.7
Salvianolic acid B –112.0 –121.6 ± 10.6
Echinacoside –104.6 –113.3 ± 8.2
Bleomycin –103.4 –110.1 ± 15.3
Angiotensin II –100.3 –107.3 ± 12.1
Nafarelin –121.9 –106.4 ± 10.6
Leuprorelin –114.5 –106.2 ± 9.8
Sennoside B –91.9 –99.1 ± 10.6
Aliskiren –99.6 –96.3 ± 6.7
Caspofungin –99.1 –95.4 ± 14.3
Alarelin –103.7 –94.6 ± 10.3
GHRP-2 –104.9 –93.8 ± 9.9
Lentinan –96.5 –93.4 ± 12.5
Leuprolide –109.6 –93.4 ± 11.1
Hederacoside C –98.5 –89.1 ± 10.1
Gonadorelin –111.4 –88.8 ± 13.0
Pneumocandin –95.3 –86.4 ± 11.4
Daptomycin –94.4 –85.4 ± 18.5
NAD+ –96.9 –83.6 ± 33.4
Deferoxamine –97.2 –83.3 ± 8.5
Goserelin –99.2 –80.4 ± 10.9
Neohesperidin –94.2 –79.8 ± 8.0
Gramicidin –98.5 –79.3 ± 11.8
Somatostatin –110.7 –77.2 ± 10.7
Vilanterol –96.3 –75.5 ± 6.3
Desmopressin –95.1 –74.9 ± 11.7
Elbasvir –108.7 –73.4 ± 7.3
Manidipine –92.6 –72.3 ± 6.4
Ginsenoside Rb1 –93.8 –72.3 ± 10.8
Lercanidipine –95.5 –71.3 ± 6.5
Atazanavir –98.1 –70.8 ± 7.4
Cobicistat –100.3 –69.5 ± 8.7
Montelukast –100.8 –67.5 ± 7.6
Vitamin B12 –93.5 –65.9 ± 11.7
Tyloxapol –104.1 –64.5 ± 7.1
Micafungin –95.4 –63.2 ± 12.7
Salmeterol –99.8 –62.8 ± 8.6
Zafirlukast –94.6 –61.8 ± 5.8
Labetalol –91.8 –61.4 ± 5.9
Indinavir –105.0 –60.0 ± 8.7
Latanoprost –94.7 –57.2 ± 6.5
Amphotericin B –132.7 –57.0 ± 7.8
Ombitasvir –94.3 –53.2 ± 12.6
Tocofersolan –91.6 –52.5 ± 6.8
Haloperidol –91.9 –52.5 ± 9.2
Tafluprost –94.3 –51.6 ± 6.3
Itraconazole –96.0 –46.5 ± 7.3
Avanafil –96.7 –46.2 ± 5.8
Ledipasvir –92.6 –43.4 ± 8.2
Octenidine –99.1 –43.2 ± 9.1
Thonzonium –92.4 –41.0 ± 8.1
Fulvestrant –96.5 –40.9 ± 7.1
Gefarnate –91.7 –39.3 ± 6.6
Clindamycin –91.9 –33.4 ± 7.8
a

Top 2% of compounds selected from the docking of 3118 FDA approved drugs and rescored by Nwat-MMGBSA are shown ranked by Nwat-MMGBSA scores.

b

Energy obtained by using Nwat = 60, ± standard deviation.

Globally, the top 2% of ligands binding to BS2 is similar in composition to the one binding to BS1: most of the ligands are peptide-like molecules known to be antibiotics, antifungals, peptide hormones, and pressure regulators. As noticed for BS1, we can also find molecules containing both large hydrophobic groups and H-bond donors and acceptors, such as echinacoside and aliskiren (Table 4). The best ranked ligand poses show a tight network of direct and water-mediated H-bonds with both the hot spot residues and the neighboring ones, in addition to additional hydrophobic interactions (Figure 5). For example, the best ranked molecule, which is polymyxin B also in this case, creates direct H-bonds with Arg403, Tyr449, Gly496, the most relevant hot spot Gln498, and Asn501, together with water-mediated H-bonds with Glu406, Tyr449, Tyr453, Gln493, Gln498, and Thr500. Except for polymyxin B, the ranking is quite different from that for BS1, with molecules which were not present in the top 10 for BS1 being ranked in the top positions for BS2, such as icatibant (#3) and octeotride (#4).

Figure 5

Figure 5. Snapshot of the MD simulation between RBD (gray) and one of the top four ligands (green). RBD BS2 hot spots are highlighted in cyan. Direct and water (spheres) mediated H-bonds are also displayed as dashed lines. Additional RBD residues interacting with the ligand are displayed as sticks.

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Thymopentin is the only linear peptide found in the top 2% docked molecules for both BS. In order to verify if the docked conformation properly took into account for the peptide flexibility and its accessible conformations, we predicted the 3D structure of the peptide using the PEPFOLD3 (81) server, and the best model has a backbone RMSD of 1.3 Å (Figure S2) from the thymopentin docked to RBD and ranked second in the VS campaign targeting RBD BS2. It should be underscored that thymopentin is an immunostimulant peptide applied in numerous clinical studies during the AIDS pandemic between 1983 and 1985. (82,83) Therefore, together with the good binding to the SARS-CoV-2 S-protein RBD shown within this VS campaign, a potential immunostimulant effect of this peptide could be helpful in enhancing the immune response to the viral infection.

Conclusions

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SARS-CoV-2 currently represents a major threat to human health, having caused hundreds of thousands of deaths in a few months. At the moment a cure against this pandemic infection is still lacking, together with a vaccine against this virus. In order to rapidly face the emergency, testing the efficacy against SARS-CoV-2 of drugs already approved for the treatment of other diseases (drug repurposing) is a good option. Indeed, it has the advantage of exploiting molecules which have already been tested in terms of toxicity and which are usually easy to purchase for clinical tests and patient administration. Therefore, positive results from this kind of procedure can speed up the process of finding a treatment against SARS-CoV-2. However, the number of approved drugs by the major jurisdictions is huge and directly performing either in vitro or in vivo studies on all of them would be time-consuming; thus a fundamental contribution to accelerate the screening can come from in silico techniques.
Within this context, we proposed a multiple VS campaign aimed to prioritize the testing against SARS-CoV-2 of already approved molecules. More in detail, we performed 4 independent VS procedures of more than 3000 approved drugs using two different SARS-CoV-2 proteins: the main proteinase Mpro and the RBD of the S-protein. Inhibiting the former would block the viral replication, while targeting the S-protein domain (i.e., RBD) would hamper the viral entry into the human cells. We applied an advanced VS procedure, which already proved to better discriminate between active and inactive compounds on multiple systems, compared to standard docking procedures. (20)
The VS campaign against Mpro ranked in the top 2% of inhibitors of the HIV protease, such as indinavir, atazanavir, and lopinavir, which recently proved to be able to alleviate the symptoms of mild-to-moderate SARS-CoV-2 infection in combination with ritonavir. (40)
The VS campaign on Spike protein RBD indicated that peptides or peptidomimetics actually used as antibiotics (i.e., polymyxin B, colistin, and daptomycin), pressure regulators (i.e., terlipressin and lypressin), hormone-peptides (i.e., alarelin and leuprorelin), and immunostimulants, such as the thymopentin, could be evaluated against SARS-Cov-2 also. Currently, there are not clinical studies on molecules known to specifically disrupt the interaction between the human ACE2 and the RBD; however, a few peptides were designed with this aim and successfully tested in vitro, validating our hypothesis that peptide-based molecules can be adapted to inhibit the ACE2-RBD interaction.
In conclusion, together with providing a good starting point for future in vitro and in vivo investigations on the resulting top compounds, the results of this extensive VS can support the design of selective and specific molecules to treat SARS-CoV-2 infection by targeting different viral proteins.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jproteome.0c00383.

  • Figure S1. RMSD of the RBD-hACE2 complex backbone atoms from the X-ray structure during the MD simulation. Table S1. Difference in the binding free energies between the mutated and the native RBD-hACE2 complex obtained from the alanine scanning. Table S2. Compounds selected by docking on Mpro (6LU7 model) that failed during the MD/Nwat-MMGBSA rescoring step. Table S3. Compounds selected by docking on Mpro (QHD43415 homology model) that failed during the MD/Nwat-MMGBSA rescoring step. Table S4. Hydrogen bonds between RBD and hACE2 during the last half of the 20 ns MD simulation. Figure S2. Superposition of thymopentin docked to RBD BS2 and the PEPFOLD3 structure prediction. (PDF)

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Author Information

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  • Corresponding Authors
    • Irene Maffucci - Université de technologie de Compiègne, UPJV, CNRS, Enzyme and Cell Engineering, Centre de recherche Royallieu - CS 60 319 - 60 203 Compiègne Cedex, France;  Email: [email protected]
    • Alessandro Contini - Università degli Studi di Milano, Dipartimento di Scienze Farmaceutiche, Sezione di Chimica Generale e Organica “A. Marchesini”, Via Venezian, 21 20133 Milano, Italy;  Orcidhttp://orcid.org/0000-0002-4394-8956;  Email: [email protected]
    • Funding

      No specific funding was received for this work.

    • Notes
      The authors declare no competing financial interest.

    Acknowledgments

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    A.C. thanks Dr. Andrea Bazzoli for useful discussions that lead to the idea of performing a screening on Mpro.

    Abbreviations

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    VS

    virtual screening
    MD

    molecular dynamics
    RBD

    receptor binding domain
    ACE2

    angiotensin converting enzyme 2
    H-bond

    hydrogen bond

    References

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      Hamming, I.; Timens, W.; Bulthuis, M. L. C.; Lely, A. T.; Navis, G. J.; van Goor, H. Tissue Distribution of ACE2 Protein, the Functional Receptor for SARS Coronavirus. A First Step in Understanding SARS Pathogenesis. J. Pathol. 2004, 203, 631– 637,  DOI: 10.1002/path.1570
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      6
      Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis
      Hamming, I.; Timens, W.; Bulthuis, M. L. C.; Lely, A. T.; Navis, G. J.; van Goor, H.
      Journal of Pathology (2004), 203 (2), 631-637CODEN: JPTLAS; ISSN:0022-3417. (John Wiley & Sons Ltd.)
      Severe acute respiratory syndrome (SARS) is an acute infectious disease that spreads mainly via the respiratory route. A distinct coronavirus (SARS-CoV) has been identified as the etiol. agent of SARS. Recently, a metallopeptidase named angiotensin-converting enzyme 2 (ACE2) has been identified as the functional receptor for SARS-CoV. Although ACE2 mRNA is known to be present in virtually all organs, its protein expression is largely unknown. Since identifying the possible route of infection has major implications for understanding the pathogenesis and future treatment strategies for SARS, the present study investigated the localization of ACE2 protein in various human organs (oral and nasal mucosa, nasopharynx, lung, stomach, small intestine, colon, skin, lymph nodes, thymus, bone marrow, spleen, liver, kidney, and brain). The most remarkable finding was the surface expression of ACE2 protein on lung alveolar epithelial cells and enterocytes of the small intestine. Furthermore, ACE2 was present in arterial and venous endothelial cells and arterial smooth muscle cells in all organs studied. In conclusion, ACE2 is abundantly present in humans in the epithelia of the lung and small intestine, which might provide possible routes of entry for the SARS-CoV. This epithelial expression, together with the presence of ACE2 in vascular endothelium, also provides a first step in understanding the pathogenesis of the main SARS disease manifestations.
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    7. 7
      Douglas, G. C.; O’Bryan, M. K.; Hedger, M. P.; Lee, D. K. L.; Yarski, M. A.; Smith, A. I.; Lew, R. A. The Novel Angiotensin-Converting Enzyme (ACE) Homolog, ACE2, Is Selectively Expressed by Adult Leydig Cells of the Testis. Endocrinology 2004, 145, 4703– 4711,  DOI: 10.1210/en.2004-0443
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      7
      The novel angiotensin-converting enzyme (ACE) homolog, ACE2, is selectively expressed by adult Leydig cells of the testis
      Douglas, Gabrielle C.; O'Bryan, Moira K.; Hedger, Mark P.; Lee, David K. L.; Yarski, Michael A.; Smith, A. Ian; Lew, Rebecca A.
      Endocrinology (2004), 145 (10), 4703-4711CODEN: ENDOAO; ISSN:0013-7227. (Endocrine Society)
      The metallopeptidase angiotensin-converting enzyme (ACE) plays a pivotal role in the cardiovascular system by generating the vasoconstrictor peptide angiotensin II. A homolog of ACE with different substrate specificity, ACE2, has recently been cloned that shows an expression pattern restricted to endothelial cells of the heart and kidney, epithelial cells of the distal tubule of the kidney, and the testis. Although the importance of ACE2 to cardiac function is already evident, its role in the testis remains unknown. In this study, we report the cloning and expression of human testicular ACE2 and confirm that it is identical to the somatic form of the enzyme. ACE2 catalytic activity was present in membrane prepns. of whole testes and Leydig cells from adult rats; expression of the protein in Leydig cells was confirmed by Western immunoblot anal. Using immunohistochem., ACE2 expression was confined to the Leydig cells in the rat testis and to Leydig and Sertoli cells in the human testis. Ablation of the Leydig cells in the rat by the specific toxin, ethane dimethane sulfonate, eliminated ACE2-pos. cells from the interstitium. Expression of ACE2 in rat Leydig cells was up-regulated during the development of adult-type Leydig cells at puberty and after ethane dimethane sulfonate treatment. Expression of ACE2 activity in the testis was not significantly altered by manipulation of the pituitary-testicular hormonal axis with s.c. testosterone implants. These data suggest that ACE2 is a constitutive product of adult-type Leydig cells and may participate in the control of testicular function by as yet unknown mechanisms.
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    8. 8
      Anand, K.; Ziebuhr, J.; Wadhwani, P.; Mesters, J. R.; Hilgenfeld, R. Coronavirus Main Proteinase (3CLpro) Structure: Basis for Design of Anti-SARS Drugs. Science (Washington, DC, U. S.) 2003, 300, 1763– 1767,  DOI: 10.1126/science.1085658
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      8
      Coronavirus Main Proteinase (3CLpro) Structure: Basis for Design of Anti-SARS Drugs
      Anand, Kanchan; Ziebuhr, John; Wadhwani, Parvesh; Mesters, Jeroen R.; Hilgenfeld, Rolf
      Science (Washington, DC, United States) (2003), 300 (5626), 1763-1767CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      A novel coronavirus has been identified as the causative agent of severe acute respiratory syndrome (SARS). The viral main proteinase (Mpro, also called 3CLpro), which controls the activities of the coronavirus replication complex, is an attractive target for therapy. The authors detd. crystal structures for human coronavirus (strain 229E) Mpro and for an inhibitor complex of porcine coronavirus [transmissible gastroenteritis virus (TGEV)] Mpro, and the authors constructed a homol. model for SARS coronavirus (SARS-CoV) Mpro. The structures reveal a remarkable degree of conservation of the substrate-binding sites, which is further supported by recombinant SARS-CoV Mpro-mediated cleavage of a TGEV Mpro substrate. Mol. modeling suggests that available rhinovirus 3Cpro inhibitors may be modified to make them useful for treating SARS.
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    9. 9
      Zhang, L.; Lin, D.; Sun, X.; Curth, U.; Drosten, C.; Sauerhering, L.; Becker, S.; Rox, K.; Hilgenfeld, R. Crystal Structure of SARS-CoV-2 Main Protease Provides a Basis for Design of Improved a-Ketoamide Inhibitors. Science (Washington, DC, U. S.) 2020, 368, 409– 412,  DOI: 10.1126/science.abb3405
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      9
      Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors
      Zhang, Linlin; Lin, Daizong; Sun, Xinyuanyuan; Curth, Ute; Drosten, Christian; Sauerhering, Lucie; Becker, Stephan; Rox, Katharina; Hilgenfeld, Rolf
      Science (Washington, DC, United States) (2020), 368 (6489), 409-412CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
      The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is a global health emergency. An attractive drug target among coronaviruses is the main protease (Mpro, also called 3CLpro) because of its essential role in processing the polyproteins that are translated from the viral RNA. We report the x-ray structures of the unliganded SARS-CoV-2 Mpro and its complex with an α-ketoamide inhibitor. This was derived from a previously designed inhibitor but with the P3-P2 amide bond incorporated into a pyridone ring to enhance the half-life of the compd. in plasma. On the basis of the unliganded structure, we developed the lead compd. into a potent inhibitor of the SARS-CoV-2 Mpro. The pharmacokinetic characterization of the optimized inhibitor reveals a pronounced lung tropism and suitability for administration by the inhalative route.
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    10. 10
      Hilgenfeld, R. From SARS to MERS: Crystallographic Studies on Coronaviral Proteases Enable Antiviral Drug Design. FEBS J. 2014, 281, 4085– 4096,  DOI: 10.1111/febs.12936
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      10
      From SARS to MERS: crystallographic studies on coronaviral proteases enable antiviral drug design
      Hilgenfeld, Rolf
      FEBS Journal (2014), 281 (18), 4085-4096CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)
      A review. Here, the author focuses on the important contributions that macromol. crystallog. has made over the past 12 yr to elucidating structures and mechanisms of the essential proteases of coronaviruses, the main protease (Mpro) and the papain-like protease (PLpro). The role of x-ray crystallog. in structure-assisted drug discovery against these targets is discussed. Aspects dealt with in this review include the emergence of the SARS coronavirus in 2002-2003 and of the MERS coronavirus 10 yr later and the origins of these viruses. The crystal structure of the free SARS coronavirus Mpro and its dependence on pH is discussed, as are efforts to design inhibitors on the basis of these structures. The mechanism of maturation of the enzyme from the viral polyprotein is still a matter of debate. The crystal structure of the SARS coronavirus PLpro and its complex with ubiquitin is also discussed, as is its ortholog from MERS coronavirus. Efforts at predictive structure-based inhibitor development for bat coronavirus Mpros to increase the preparedness against zoonotic transmission to man are described as well. The paper closes with a brief discussion of structure-based discovery of antivirals in an academic setting.
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    11. 11
      Jin, Z.; Du, X.; Xu, Y.; Deng, Y.; Liu, M.; Zhao, Y.; Zhang, B.; Li, X.; Zhang, L.; Peng, C.; Duan, Y.; Yu, J.; Wang, L.; Yang, K.; Liu, F.; Jiang, R.; Yang, X.; You, T.; Liu, X.; Yang, X.; Bai, F.; Liu, H.; Liu, X.; Guddat, L. W.; Xu, W.; Xiao, G.; Qin, C.; Shi, Z.; Jiang, H.; Rao, Z.; Yang, H. Structure of Mpro from SARS-CoV-2 and Discovery of Its Inhibitors. Nature 2020, 582, 289– 293,  DOI: 10.1038/s41586-020-2223-y
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      11
      Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors
      Jin, Zhenming; Du, Xiaoyu; Xu, Yechun; Deng, Yongqiang; Liu, Meiqin; Zhao, Yao; Zhang, Bing; Li, Xiaofeng; Zhang, Leike; Peng, Chao; Duan, Yinkai; Yu, Jing; Wang, Lin; Yang, Kailin; Liu, Fengjiang; Jiang, Rendi; Yang, Xinglou; You, Tian; Liu, Xiaoce; Yang, Xiuna; Bai, Fang; Liu, Hong; Liu, Xiang; Guddat, Luke W.; Xu, Wenqing; Xiao, Gengfu; Qin, Chengfeng; Shi, Zhengli; Jiang, Hualiang; Rao, Zihe; Yang, Haitao
      Nature (London, United Kingdom) (2020), 582 (7811), 289-293CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
      Abstr.: A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the etiol. agent responsible for the 2019-2020 viral pneumonia outbreak of coronavirus disease 2019 (COVID-19). Currently, there are no targeted therapeutic agents for the treatment of this disease, and effective treatment options remain very limited. Here, we describe the results of a program that aimed to rapidly discover lead compds. for clin. use, by combining structure-assisted drug design, virtual drug screening and high-throughput screening. This program focused on identifying drug leads that target main protease (Mpro) of SARS-CoV-2: Mpro is a key enzyme of coronaviruses and has a pivotal role in mediating viral replication and transcription, making it an attractive drug target for SARS-CoV-2. We identified a mechanism-based inhibitor (N3) by computer-aided drug design, and then detd. the crystal structure of Mpro of SARS-CoV-2 in complex with this compd. Through a combination of structure-based virtual and high-throughput screening, we assayed more than 10,000 compds.-including approved drugs, drug candidates in clin. trials and other pharmacol. active compds.-as inhibitors of Mpro. Six of these compds. inhibited Mpro, showing half-maximal inhibitory concn. values that ranged from 0.67 to 21.4μM. One of these compds. (ebselen) also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of our screening strategy, which can lead to the rapid discovery of drug leads with clin. potential in response to new infectious diseases for which no specific drugs or vaccines are available.
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    12. 12
      Roy, A.; Kucukural, A.; Zhang, Y. I-TASSER: A Unified Platform for Automated Protein Structure and Function Prediction. Nat. Protoc. 2010, 5, 725– 738,  DOI: 10.1038/nprot.2010.5
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      12
      I-TASSER: a unified platform for automated protein structure and function prediction
      Roy, Ambrish; Kucukural, Alper; Zhang, Yang
      Nature Protocols (2010), 5 (4), 725-738CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)
      The iterative threading assembly refinement (I-TASSER) server is an integrated platform for automated protein structure and function prediction based on the sequence-to-structure-to-function paradigm. Starting from an amino acid sequence, I-TASSER first generates three-dimensional (3D) at. models from multiple threading alignments and iterative structural assembly simulations. The function of the protein is then inferred by structurally matching the 3D models with other known proteins. The output from a typical server run contains full-length secondary and tertiary structure predictions, and functional annotations on ligand-binding sites, Enzyme Commission nos. and Gene Ontol. terms. An est. of accuracy of the predictions is provided based on the confidence score of the modeling. This protocol provides new insights and guidelines for designing of online server systems for the state-of-the-art protein structure and function predictions. The server is available at http://zhanglab.ccmb.med.umich.edu/I-TASSER.
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    13. 13
      Zhang, C.; Zheng, W.; Huang, X.; Bell, E. W.; Zhou, X.; Zhang, Y. Protein Structure and Sequence Reanalysis of 2019-NCoV Genome Refutes Snakes as Its Intermediate Host and the Unique Similarity between Its Spike Protein Insertions and HIV-1. J. Proteome Res. 2020, 19, 1351– 1360,  DOI: 10.1021/acs.jproteome.0c00129
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      13
      Protein Structure and Sequence Reanalysis of 2019-nCoV Genome Refutes Snakes as Its Intermediate Host and the Unique Similarity between Its Spike Protein Insertions and HIV-1
      Zhang, Chengxin; Zheng, Wei; Huang, Xiaoqiang; Bell, Eric W.; Zhou, Xiaogen; Zhang, Yang
      Journal of Proteome Research (2020), 19 (4), 1351-1360CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
      As the infection of 2019-nCoV coronavirus is quickly developing into a global pneumonia epidemic, the careful anal. of its transmission and cellular mechanisms is sorely needed. In this Communication, we first analyzed two recent studies that concluded that snakes are the intermediate hosts of 2019-nCoV and that the 2019-nCoV spike protein insertions share a unique similarity to HIV-1. However, the reimplementation of the analyses, built on larger scale data sets using state-of-the-art bioinformatics methods and databases, presents clear evidence that rebuts these conclusions. Next, using metagenomic samples from Manis javanica, we assembled a draft genome of the 2019-nCoV-like coronavirus, which shows 73% coverage and 91% sequence identity to the 2019-nCoV genome. In particular, the alignments of the spike surface glycoprotein receptor-binding domain revealed four times more variations in the bat coronavirus RaTG13 than in the Manis coronavirus compared with 2019-nCoV, suggesting the pangolin as a missing link in the transmission of 2019-nCoV from bats to human.
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    14. 14
      Wu, F.; Zhao, S.; Yu, B.; Chen, Y. M.; Wang, W.; Song, Z. G.; Hu, Y.; Tao, Z. W.; Tian, J. H.; Pei, Y. Y.; Yuan, M. L.; Zhang, Y. L.; Dai, F. H.; Liu, Y.; Wang, Q. M.; Zheng, J. J.; Xu, L.; Holmes, E. C.; Zhang, Y. Z. A New Coronavirus Associated with Human Respiratory Disease in China. Nature 2020, 579, 265– 269,  DOI: 10.1038/s41586-020-2008-3
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      14
      A new coronavirus associated with human respiratory disease in China
      Wu, Fan; Zhao, Su; Yu, Bin; Chen, Yan-Mei; Wang, Wen; Song, Zhi-Gang; Hu, Yi; Tao, Zhao-Wu; Tian, Jun-Hua; Pei, Yuan-Yuan; Yuan, Ming-Li; Zhang, Yu-Ling; Dai, Fa-Hui; Liu, Yi; Wang, Qi-Min; Zheng, Jiao-Jiao; Xu, Lin; Holmes, Edward C.; Zhang, Yong-Zhen
      Nature (London, United Kingdom) (2020), 579 (7798), 265-269CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
      Emerging infectious diseases, such as severe acute respiratory syndrome (SARS) and Zika virus disease, present a major threat to public health. Despite intense research efforts, how, when and where new diseases appear are still a source of considerable uncertainty. A severe respiratory disease was recently reported in Wuhan, Hubei province, China. As of 25 Jan. 2020, at least 1,975 cases had been reported since the first patient was hospitalized on 12 Dec. 2019. Epidemiol. investigations have suggested that the outbreak was assocd. with a seafood market in Wuhan. Here we study a single patient who was a worker at the market and who was admitted to the Central Hospital of Wuhan on 26 Dec. 2019 while experiencing a severe respiratory syndrome that included fever, dizziness and a cough. Metagenomic RNA sequencing of a sample of bronchoalveolar lavage fluid from the patient identified a new RNA virus strain from the family Coronaviridae, which is designated here 'WH-Human 1' coronavirus (and has also been referred to as '2019-nCoV'). Phylogenetic anal. of the complete viral genome (29,903 nucleotides) revealed that the virus was most closely related (89.1% nucleotide similarity) to a group of SARS-like coronaviruses (genus Betacoronavirus, subgenus Sarbecovirus) that had previously been found in bats in China. This outbreak highlights the ongoing ability of viral spill-over from animals to cause severe disease in humans.
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    15. 15
      Patick, A. K.; Potts, K. E. Protease Inhibitors as Antiviral Agents. Clin. Microbiol. Rev. 1998, 11, 614,  DOI: 10.1128/CMR.11.4.614
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      15
      Protease inhibitors as antiviral agents
      Patick, A. K.; Potts, K. E.
      Clinical Microbiology Reviews (1998), 11 (4), 614-627CODEN: CMIREX; ISSN:0893-8512. (American Society for Microbiology)
      A review with 169 refs.
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    16. 16
      Cavasotto, C. N.; Orry, A. J. W. Ligand Docking and Structure-Based Virtual Screening in Drug Discovery. Curr. Top. Med. Chem. 2007, 7, 1006– 1014,  DOI: 10.2174/156802607780906753
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      Ligand docking and structure-based virtual screening in drug discovery
      Cavasotto, Claudio N.; Orry, Andrew J. W.
      Current Topics in Medicinal Chemistry (Sharjah, United Arab Emirates) (2007), 7 (10), 1006-1014CODEN: CTMCCL; ISSN:1568-0266. (Bentham Science Publishers Ltd.)
      A review. Ligand-docking-based methods are starting to play a crit. role in lead discovery and optimization, thus resulting in new 'drug-candidates'. They offer the possibility to go beyond the pool of existing active compds., and thus find novel chemotypes. A brief turorial on ligand docking and structure-based virtual screening is presented highlighting current problems and limitations, together with the most recent methodol. and algorithmic developments in the field. Recent successful applications of docking-based tools for hit doscovery, lead optimization and target-biased library design are also presented. Special consideration is devoted to ongoing efforts to account for protein flexibility in structure-based virtual screening.
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      Clark, D. E. What Has Virtual Screening Ever Done for Drug Discovery?. Expert Opin. Drug Discovery 2008, 3, 841– 851,  DOI: 10.1517/17460441.3.8.841
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      17
      What has virtual screening ever done for drug discovery?
      Clark, David E.
      Expert Opinion on Drug Discovery (2008), 3 (8), 841-851CODEN: EODDBX; ISSN:1746-0441. (Informa Healthcare)
      A review. Background: Although virtual screening is now widely applied as a hit-finding methodol. within drug discovery programs, there are relatively few reports of its contributing to compds. on the market or in the clinic. Objective: To assess the impact of virtual screening on drug discovery. Method: Such cases as can be found in the public domain at the current time are reviewed. Addnl., some of the current challenges in structure- and ligand-based virtual screening are discussed. Conclusion: It is concluded that virtual screening has contributed to the discovery of several compds. that have either reached the market or entered clin. trials. In terms of praxis, there is 'no free lunch' in virtual screening and as many methods as possible should be applied to maximize the likelihood of success.
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    18. 18
      Choudhary, S.; Malik, Y. S.; Tomar, S. Identification of SARS-CoV-2 Cell Entry Inhibitors by Drug Repurposing Using in Silico Structure-Based Virtual Screening Approach. Front. Immunol. 2020, 11, 1664,  DOI: 10.3389/fimmu.2020.01664
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      18
      Identification of SARS-CoV-2 cell entry inhibitors by drug repurposing using in silico structure-based virtual screening approach
      Choudhary, Shweta; Malik, Yashpal S.; Tomar, Shailly
      Frontiers in Immunology (2020), 11 (), 1664CODEN: FIRMCW; ISSN:1664-3224. (Frontiers Media S.A.)
      The rapidly spreading, highly contagious and pathogenic SARS-coronavirus 2 (SARS-CoV-2) assocd. Coronavirus Disease 2019 (COVID-19) has been declared as a pandemic by the World Health Organization (WHO). The novel 2019 SARS-CoV-2 enters the host cell by binding of the viral surface spike glycoprotein (S-protein) to cellular angiotensin converting enzyme 2 (ACE2) receptor. The virus specific mol. interaction with the host cell represents a promising therapeutic target for identifying SARS-CoV-2 antiviral drugs. The repurposing of drugs can provide a rapid and potential cure toward exponentially expanding COVID-19. Thereto, high throughput virtual screening approach was used to investigate FDA approved LOPAC library drugs against both the receptor binding domain of spike protein (S-RBD) and ACE2 host cell receptor. Primary screening identified a few promising mols. for both the targets, which were further analyzed in details by their binding energy, binding modes through mol. docking, dynamics and simulations. Evidently, GR 127935 hydrochloride hydrate, GNF-5, RS504393, TNP, and eptifibatide acetate were found binding to virus binding motifs of ACE2 receptor. Addnl., KT203, BMS195614, KT185, RS504393, and GSK1838705A were identified to bind at the receptor binding site on the viral S-protein. These identified mols. may effectively assist in controlling the rapid spread of SARS-CoV-2 by not only potentially inhibiting the virus at entry step but are also hypothesized to act as anti-inflammatory agents, which could impart relief in lung inflammation. Timely identification and detn. of an effective drug to combat and tranquilize the COVID-19 global crisis is the utmost need of hour. Further, prompt in vivo testing to validate the anti-SARS-CoV-2 inhibition efficiency by these mols. could save lives is justified.
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    19. 19
      Chen, Y. W.; Yiu, C. P. B.; Wong, K. Y. Prediction of the SARS-CoV-2 (2019-NCoV) 3C-like Protease (3CLpro) Structure: Virtual Screening Reveals Velpatasvir, Ledipasvir, and Other Drug Repurposing Candidates. F1000Research 2020, 9, 9,  DOI: 10.12688/f1000research.22457.2
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      There is no corresponding record for this reference.
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      Maffucci, I.; Hu, X.; Fumagalli, V.; Contini, A. An Efficient Implementation of the Nwat-MMGBSA Method to Rescore Docking Results in Medium-Throughput Virtual Screenings. Front. Chem. 2018, 6, 43,  DOI: 10.3389/fchem.2018.00043
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      20
      An efficient implementation of the nwat-mmgbsa method to rescore docking results in medium-throughput virtual screenings
      Maffucci, Irene; Hu, Xiao; Fumagalli, Valentina; Contini, Alessandro
      Frontiers in Chemistry (Lausanne, Switzerland) (2018), 6 (), 43/1-43/14CODEN: FCLSAA; ISSN:2296-2646. (Frontiers Media S.A.)
      Nwat-MMGBSA is a variant of MM-PB/GBSA based on the inclusion of a no. of explicit water mols. that are the closest to the ligand in each frame of a mol. dynamics trajectory. This method demonstrated improved correlations between calcd. and exptl. binding energies in both protein-protein interactions and ligand-receptor complexes, in comparison to the std. MM-GBSA. A protocol optimization, aimed to maximize efficacy and efficiency, is discussed here considering penicillopepsin, HIV1-protease, and BCL-XL as test cases. Calcns. were performed in triplicates on both classic HPC environments and on std. workstations equipped by a GPU card, evidencing no statistical differences in the results. No relevant differences in correlation to expts. were also obsd. when performing Nwat-MMGBSA calcns. on 4 or 1 ns long trajectories. A fully automatic workflow for structure-based virtual screening, performing from library set-up to docking and Nwat-MMGBSA rescoring, has then been developed. The protocol has been tested against no rescoring or std. MM-GBSA rescoring within a retrospective virtual screening of inhibitors of AmpC β-lactamase and of the Rac1-Tiam1 protein-protein interaction. In both cases, Nwat-MMGBSA rescoring provided a statistically significant increase in the ROC AUCs of between 20 and 30%, compared to docking scoring or to std. MM-GBSA rescoring.
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    21. 21
      Amaro, R. E.; Baudry, J.; Chodera, J.; Demir, Ö.; McCammon, J. A.; Miao, Y.; Smith, J. C. Ensemble Docking in Drug Discovery. Biophys. J. Biophysical Society, May 22, 2018; pp 2271– 2278.
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      There is no corresponding record for this reference.
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      Molecular Operating Environment (MOE), 2019.0102; Chemical Computing Group Inc.: Montreal, 2019.
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      There is no corresponding record for this reference.
    23. 23
      Turlington, M.; Chun, A.; Tomar, S.; Eggler, A.; Grum-Tokars, V.; Jacobs, J.; Daniels, J. S.; Dawson, E.; Saldanha, A.; Chase, P.; Baez-Santos, Y. M.; Lindsley, C. W.; Hodder, P.; Mesecar, A. D.; Stauffer, S. R. Discovery of N-(Benzo[1,2,3]Triazol-1-Yl)-N-(Benzyl)Acetamido)Phenyl) Carboxamides as Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) 3CLpro Inhibitors: Identification of ML300 and Noncovalent Nanomolar Inhibitors with an Induced-Fit Binding. Bioorg. Med. Chem. Lett. 2013, 23, 6172– 6177,  DOI: 10.1016/j.bmcl.2013.08.112
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      23
      Discovery of N-(benzo[1,2,3]triazol-1-yl)-N-((benzyl)acetamido)phenyl carboxamides as severe acute respiratory syndrome coronavirus (SARS-CoV) 3CLpro inhibitors: Identification of ML300 and noncovalent nanomolar inhibitors with an induced-fit binding
      Turlington, Mark; Chun, Aspen; Tomar, Sakshi; Eggler, Aimee; Grum-Tokars, Valerie; Jacobs, Jon; Daniels, J. Scott; Dawson, Eric; Saldanha, Adrian; Chase, Peter; Baez-Santos, Yahira M.; Lindsley, Craig W.; Hodder, Peter; Mesecar, Andrew D.; Stauffer, Shaun R.
      Bioorganic & Medicinal Chemistry Letters (2013), 23 (22), 6172-6177CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)
      Herein we report the discovery and SAR of a novel series of SARS-CoV 3CLpro inhibitors identified through the NIH Mol. Libraries Probe Prodn. Centers Network (MLPCN). In addn. to ML188, ML300 represents the second probe declared for 3CLpro from this collaborative effort. The x-ray structure of SARS-CoV 3CLpro bound with a ML300 analog highlights a unique induced-fit reorganization of the S2-S4 binding pockets leading to the first sub-micromolar noncovalent 3CLpro inhibitors retaining a single amide bond.
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      Contini, A. Virtual Screening of an FDA Approved Drugs Database on Two COVID-19 Coronavirus Proteins. ChemRxiv Preprint. 2020,  DOI: 10.26434/Chemrxiv.11847381.V1 .
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      Anandakrishnan, R.; Aguilar, B.; Onufriev, A. V. H++ 3.0: Automating PK Prediction and the Preparation of Biomolecular Structures for Atomistic Molecular Modeling and Simulations. Nucleic Acids Res. 2012, 40, W537– 541,  DOI: 10.1093/nar/gks375
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      H++ 3.0: automating pK prediction and the preparation of biomolecular structures for atomistic molecular modeling and simulations
      Anandakrishnan, Ramu; Aguilar, Boris; Onufriev, Alexey V.
      Nucleic Acids Research (2012), 40 (W1), W537-W541CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      The accuracy of atomistic biomol. modeling and simulation studies depend on the accuracy of the input structures. Prepg. these structures for an atomistic modeling task, such as mol. dynamics (MD) simulation, can involve the use of a variety of different tools for: correcting errors, adding missing atoms, filling valences with hydrogens, predicting pK values for titratable amino acids, assigning predefined partial charges and radii to all atoms, and generating force field parameter/topol. files for MD. Identifying, installing and effectively using the appropriate tools for each of these tasks can be difficult for novice and time-consuming for experienced users. H++ (http://biophysics.cs.vt.edu/) is a free open-source web server that automates the above key steps in the prepn. of biomol. structures for mol. modeling and simulations. H++ also performs extensive error and consistency checking, providing error/warning messages together with the suggested corrections. In addn. to numerous minor improvements, the latest version of H++ includes several new capabilities and options: fix erroneous (flipped) side chain conformations for HIS, GLN and ASN, include a ligand in the input structure, process nucleic acid structures and generate a solvent box with specified no. of common ions for explicit solvent MD.
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    26. 26
      Case, D. A.; Ben-Shalom, I. Y.; Brozell, S. R.; Cerutti, D. S.; Cheatham, T. E., III, Cruzeiro, V. W. D.; Duke, T. A. D. R. E.; Ghoreishi, D.; Gilson, M.K.; Gohlke, H.; Goetz, A. W.; Greene, D.; Harris, R.; Homeyer, N.; Izadi, Y. H. S.; Kovalenko, A.; Kurtzman, T.; Lee, T. S.; LeGrand, S.; Li, P.; Lin, C.; Liu, J.; Luchko, T.; Luo, R.; D. J., Mermelstein, Merz, K. M.; Miao, Y.; Monard, G.; Nguyen, C.; Nguyen, H.; Omelyan, I.; Onufriev, A.; Pan, F.; R., Qi, Roe, D. R.; Roitberg, A.; Sagui, C.; Schott-Verdugo, S.; Shen, J.; Simmerling, C. L.; Smith, J.; SalomonFerrer, R.; Swails, J.; Walker, R. C.; Wang, J.; Wei, H.; Wolf, R. M.; Wu, X.; Xiao, L.; D. M, Y.; P. A, K. AMBER 2018; University of California, San Francisco, 2018.
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      Maier, J. A.; Martinez, C.; Kasavajhala, K.; Wickstrom, L.; Hauser, K. E.; Simmerling, C. Ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from Ff99SB. J. Chem. Theory Comput. 2015, 11, 3696– 3713,  DOI: 10.1021/acs.jctc.5b00255
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      ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB
      Maier, James A.; Martinez, Carmenza; Kasavajhala, Koushik; Wickstrom, Lauren; Hauser, Kevin E.; Simmerling, Carlos
      Journal of Chemical Theory and Computation (2015), 11 (8), 3696-3713CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
      Mol. mechanics is powerful for its speed in atomistic simulations, but an accurate force field is required. The Amber ff99SB force field improved protein secondary structure balance and dynamics from earlier force fields like ff99, but weaknesses in side chain rotamer and backbone secondary structure preferences have been identified. Here, we performed a complete refit of all amino acid side chain dihedral parameters, which had been carried over from ff94. The training set of conformations included multidimensional dihedral scans designed to improve transferability of the parameters. Improvement in all amino acids was obtained as compared to ff99SB. Parameters were also generated for alternate protonation states of ionizable side chains. Av. errors in relative energies of pairs of conformations were under 1.0 kcal/mol as compared to QM, reduced 35% from ff99SB. We also took the opportunity to make empirical adjustments to the protein backbone dihedral parameters as compared to ff99SB. Multiple small adjustments of φ and ψ parameters were tested against NMR scalar coupling data and secondary structure content for short peptides. The best results were obtained from a phys. motivated adjustment to the φ rotational profile that compensates for lack of ff99SB QM training data in the β-ppII transition region. Together, these backbone and side chain modifications (hereafter called ff14SB) not only better reproduced their benchmarks, but also improved secondary structure content in small peptides and reprodn. of NMR χ1 scalar coupling measurements for proteins in soln. We also discuss the Amber ff12SB parameter set, a preliminary version of ff14SB that includes most of its improvements.
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      Ryckaert, J. P.; Ciccotti, G.; Berendsen, H. J. C. Numerical Integration of the Cartesian Equations of Motion of a System with Constraints: Molecular Dynamics of n-Alkanes. J. Comput. Phys. 1977, 23, 327– 341,  DOI: 10.1016/0021-9991(77)90098-5
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      Numerical integration of the Cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes
      Ryckaert, Jean Paul; Ciccotti, Giovanni; Berendsen, Herman J. C.
      Journal of Computational Physics (1977), 23 (3), 327-41CODEN: JCTPAH; ISSN:0021-9991.
      A numerical algorithm integrating the 3N Cartesian equation of motion of a system of N points subject to holonomic constraints is applied to mol. dynamics simulation of a liq. of 64 butane mols.
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      Schrödinger, L. PyMOL Molecular Graphics System , Version 1.8; 2015.
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      Nguyen, H.; Roe, D. R.; Simmerling, C. Improved Generalized Born Solvent Model Parameters for Protein Simulations. J. Chem. Theory Comput. 2013, 9, 2020– 2034,  DOI: 10.1021/ct3010485
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      Improved Generalized Born Solvent Model Parameters for Protein Simulations
      Nguyen, Hai; Roe, Daniel R.; Simmerling, Carlos
      Journal of Chemical Theory and Computation (2013), 9 (4), 2020-2034CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
      The generalized Born (GB) model is one of the fastest implicit solvent models, and it has become widely adopted for Mol. Dynamics (MD) simulations. This speed comes with trade-offs, and many reports in the literature have pointed out weaknesses with GB models. Because the quality of a GB model is heavily affected by empirical parameters used in calcg. solvation energy, in this work we have refit these parameters for GB-Neck, a recently developed GB model, in order to improve the accuracy of both the solvation energy and effective radii calcns. The data sets used for fitting are significantly larger than those used in the past. Comparing to other pairwise GB models like GB-OBC and the original GB-Neck, the new GB model (GB-Neck2) has better agreement with Poisson-Boltzmann (PB) in terms of reproducing solvation energies for a variety of systems ranging from peptides to proteins. Secondary structure preferences are also in much better agreement with those obtained from explicit solvent MD simulations. We also obtain near-quant. reprodn. of exptl. structure and thermal stability profiles for several model peptides with varying secondary structure motifs. Extension to nonprotein systems will be explored in the future.
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      Korb, O.; Möller, H. M.; Exner, T. E. NMR-Guided Molecular Docking of a Protein-Peptide Complex Based on Ant Colony Optimization. ChemMedChem 2010, 5, 1001– 1006,  DOI: 10.1002/cmdc.201000090
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      NMR-Guided Molecular Docking of a Protein-Peptide Complex Based on Ant Colony Optimization
      Korb, Oliver; Moeller, Heiko M.; Exner, Thomas E.
      ChemMedChem (2010), 5 (7), 1001-1006CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Std. docking approaches used for the prediction of protein-ligand complexes in the drug development process have problems identifying the correct binding mode of large flexible ligands. Herein the authors show how addnl. exptl. data from NMR expts. can be used to predict the binding mode of a mucin 1 (MUC-1) pentapeptide recognized by the breast-cancer-selective monoclonal antibody SM3. Distance constraints derived from trNOE and satn. transfer difference NMR expts. are combined with the docking approach PLANTS. The resulting complex structures show excellent agreement with the NMR data and with a published X-ray crystal structure. The method was then further tested on two complexes in order to demonstrate its more general applicability: T-antigen disaccharide bound to Maclura pomifera agglutinin, and the inhibitor SBi279 bound to S100B protein. The authors' new approach has the advantages of being fully automatic, rapid, and unbiased; moreover, it is based on relatively easily obtainable exptl. data and can greatly increase the reliability of the generated structures.
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      Maffucci, I.; Contini, A. Explicit Ligand Hydration Shells Improve the Correlation between MM-PB/GBSA Binding Energies and Experimental Activities. J. Chem. Theory Comput. 2013, 9, 2706– 2717,  DOI: 10.1021/ct400045d
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      Explicit Ligand Hydration Shells Improve the Correlation between MM-PB/GBSA Binding Energies and Experimental Activities
      Maffucci, Irene; Contini, Alessandro
      Journal of Chemical Theory and Computation (2013), 9 (6), 2706-2717CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
      Mol. Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) and Mol. Mechanics Generalized Born Surface Area (MM-GBSA) methods are widely used for drug design/discovery purposes. However, it is not clear if the correlation between predicted and exptl. binding affinities can be improved by explicitly considering selected water mols. in the calcn. of binding energies, since different and sometimes diverging opinions are found in the literature. The authors evaluated how variably populated hydration shells explicitly considered around the ligands may affect the correlation between MM-PB/GBSA computed binding energy and biol. activities (IC50 and ΔGbind, depending on the available exptl. data). Four different systems--namely, the DNA-topoisomerase complex, α-thrombin, penicillopepsin, and avidin--were considered and ligand hydration shells populated by 10-70 water mols. were systematically evaluated. The consideration of a hydration shell populated by a no. of water residues (Nwat) between 30 and 70 provided, in all of the considered examples, a pos. effect on correlation between MM-PB/GBSA calcd. binding affinities and exptl. activities, with a negligible increment of computational cost.
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      Maffucci, I.; Contini, A. Improved Computation of Protein-Protein Relative Binding Energies with the Nwat-MMGBSA Method. J. Chem. Inf. Model. 2016, 56, 1692– 1704,  DOI: 10.1021/acs.jcim.6b00196
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      Improved Computation of Protein-Protein Relative Binding Energies with the Nwat-MMGBSA Method
      Maffucci, Irene; Contini, Alessandro
      Journal of Chemical Information and Modeling (2016), 56 (9), 1692-1704CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)
      A MMGBSA variant (here referred to as Nwat-MMGBSA), based on the inclusion of a certain no. of explicit water mols. (Nwat) during the calcns., has been tested on a set of 20 protein-protein complexes, using the correlation between predicted and exptl. binding energy as the evaluation metric. Beside the Nwat parameter, the effect of the force field, the mol. dynamics simulation length, and the implicit solvent model used in the MMGBSA anal. have been also evaluated. Considering 30 interfacial water mols. improved the correlation between predicted and exptl. binding energies by up to 30%, compared to the std. approach. Moreover, the correlation resulted rather sensitively to the force field and, to a minor extent, to the implicit solvent model, and to the length of the MD simulation.
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      Korb, O.; Stützle, T.; Exner, T. E. Empirical Scoring Functions for Advanced Protein-Ligand Docking with PLANTS. J. Chem. Inf. Model. 2009, 49, 84– 96,  DOI: 10.1021/ci800298z
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      Empirical Scoring Functions for Advanced Protein-Ligand Docking with PLANTS
      Korb, Oliver; Stuetzle, Thomas; Exner, Thomas E.
      Journal of Chemical Information and Modeling (2009), 49 (1), 84-96CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)
      In this paper we present two empirical scoring functions, PLANTSCHEMPLP and PLANTSPLP, designed for our docking algorithm PLANTS (Protein-Ligand ANT System), which is based on ant colony optimization (ACO). They are related, regarding their functional form, to parts of already published scoring functions and force fields. The parametrization procedure described here was able to identify several parameter settings showing an excellent performance for the task of pose prediction on two test sets comprising 298 complexes in total. Up to 87% of the complexes of the Astex diverse set and 77% of the CCDC/Astex clean listnc (noncovalently bound complexes of the clean list) could be reproduced with root-mean-square deviations of less than 2 Å with respect to the exptl. detd. structures. A comparison with the state-of-the-art docking tool GOLD clearly shows that this is, esp. for the druglike Astex diverse set, an improvement in pose prediction performance. Addnl., optimized parameter settings for the search algorithm were identified, which can be used to balance pose prediction reliability and search speed.
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      Massova, I.; Kollman, P. A. Combined Molecular Mechanical and Continuum Solvent Approach (MM- PBSA/GBSA) to Predict Ligand Binding. Perspectives in Drug Discovery and Design; Springer, 2000; pp 113– 135.
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      Choy, K.-T.; Wong, A. Y.-L.; Kaewpreedee, P.; Sia, S. F.; Chen, D.; Hui, K. P. Y.; Chu, D. K. W.; Chan, M. C. W.; Cheung, P. P.-H.; Huang, X.; Peiris, M.; Yen, H.-L. Remdesivir, Lopinavir, Emetine, and Homoharringtonine Inhibit SARS-CoV-2 Replication in Vitro. Antiviral Res. 2020, 178, 104786,  DOI: 10.1016/j.antiviral.2020.104786
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      Remdesivir, lopinavir, emetine, and homoharringtonine inhibit SARS-CoV-2 replication in vitro
      Choy, Ka-Tim; Wong, Alvina Yin-Lam; Kaewpreedee, Prathanporn; Sia, Sin Fun; Chen, Dongdong; Hui, Kenrie Pui Yan; Chu, Daniel Ka Wing; Chan, Michael Chi Wai; Cheung, Peter Pak-Hang; Huang, Xuhui; Peiris, Malik; Yen, Hui-Ling
      Antiviral Research (2020), 178 (), 104786CODEN: ARSRDR; ISSN:0166-3542. (Elsevier B.V.)
      An escalating pandemic by the novel SARS-CoV-2 virus is impacting global health and effective therapeutic options are urgently needed. We evaluated the in vitro antiviral effect of compds. that were previously reported to inhibit coronavirus replication and compds. that are currently under evaluation in clin. trials for SARS-CoV-2 patients. We report the antiviral effect of remdesivir, lopinavir, homorringtonine, and emetine against SARS-CoV-2 virus in Vero E6 cells with the estd. 50% effective concn. at 23.15μM, 26.63μM, 2.55μM and 0.46μM, resp. Ribavirin or favipiravir that are currently evaluated under clin. trials showed no inhibition at 100μM. Synergy between remdesivir and emetine was obsd., and remdesivir at 6.25μM in combination with emetine at 0.195μM may achieve 64.9% inhibition in viral yield. Combinational therapy may help to reduce the effective concn. of compds. below the therapeutic plasma concns. and provide better clin. benefits.
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      Beck, B. R.; Shin, B.; Choi, Y.; Park, S.; Kang, K. Predicting Commercially Available Antiviral Drugs That May Act on the Novel Coronavirus (SARS-CoV-2) through a Drug-Target Interaction Deep Learning Model. Comput. Struct. Biotechnol. J. 2020, 18, 784– 790,  DOI: 10.1016/j.csbj.2020.03.025
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      Predicting commercially available antiviral drugs that may act on the novel coronavirus (SARS-CoV-2) through a drug-target interaction deep learning model
      Beck, Bo Ram; Shin, Bonggun; Choi, Yoonjung; Park, Sungsoo; Kang, Keunsoo
      Computational and Structural Biotechnology Journal (2020), 18 (), 784-790CODEN: CSBJAC; ISSN:2001-0370. (Elsevier B.V.)
      The infection of a novel coronavirus found in Wuhan of China (SARS-CoV-2) is rapidly spreading, and the incidence rate is increasing worldwide. Due to the lack of effective treatment options for SARS-CoV-2, various strategies are being tested in China, including drug repurposing. In this study, we used our pre-trained deep learning-based drug-target interaction model called Mol. Transformer-Drug Target Interaction (MT-DTI) to identify com. available drugs that could act on viral proteins of SARS-CoV-2. The result showed that atazanavir, an antiretroviral medication used to treat and prevent the human immunodeficiency virus (HIV), is the best chem. compd., showing an inhibitory potency with Kd of 94.94 nM against the SARS-CoV-2 3C-like proteinase, followed by remdesivir (113.13 nM), efavirenz (199.17 nM), ritonavir (204.05 nM), and dolutegravir (336.91 nM). Interestingly, lopinavir, ritonavir, and darunavir are all designed to target viral proteinases. However, in our prediction, they may also bind to the replication complex components of SARS-CoV-2 with an inhibitory potency with Kd < 1000 nM. In addn., we also found that several antiviral agents, such as Kaletra (lopinavir/ritonavir), could be used for the treatment of SARS-CoV-2. Overall, we suggest that the list of antiviral drugs identified by the MT-DTI model should be considered, when establishing effective treatment strategies for SARS-CoV-2.
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    40. 40
      Hung, I. F.-N.; Lung, K.-C.; Tso, E. Y.-K.; Liu, R.; Chung, T. W.-H.; Chu, M.-Y.; Ng, Y.-Y.; Lo, J.; Chan, J.; Tam, A. R.; Shum, H.-P.; Chan, V.; Wu, A. K.-L.; Sin, K.-M.; Leung, W.-S.; Law, W.-L.; Lung, D. C.; Sin, S.; Yeung, P.; Yip, C. C.-Y.; Zhang, R. R.; Fung, A. Y.-F.; Yan, E. Y.-W.; Leung, K.-H.; Ip, J. D.; Chu, A. W.-H.; Chan, W.-M.; Ng, A. C.-K.; Lee, R.; Fung, K.; Yeung, A.; Wu, T.-C.; Chan, J. W.-M.; Yan, W.-W.; Chan, W.-M.; Chan, J. F.-W.; Lie, A. K.-W.; Tsang, O. T.-Y.; Cheng, V. C.-C.; Que, T.-L.; Lau, C.-S.; Chan, K.-H.; To, K. K.-W.; Yuen, K.-Y. Triple Combination of Interferon Beta-1b, Lopinavir–Ritonavir, and Ribavirin in the Treatment of Patients Admitted to Hospital with COVID-19: An Open-Label, Randomised, Phase 2 Trial. Lancet 2020, 395, 1695– 1704,  DOI: 10.1016/S0140-6736(20)31042-4
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      Triple combination of interferon beta-1b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with COVID-19: an open-label, randomised, phase 2 trial
      Hung, Ivan Fan-Ngai; Lung, Kwok-Cheung; Tso, Eugene Yuk-Keung; Liu, Raymond; Chung, Tom Wai-Hin; Chu, Man-Yee; Ng, Yuk-Yung; Lo, Jenny; Chan, Jacky; Tam, Anthony Raymond; Shum, Hoi-Ping; Chan, Veronica; Wu, Alan Ka-Lun; Sin, Kit-Man; Leung, Wai-Shing; Law, Wai-Lam; Lung, David Christopher; Sin, Simon; Yeung, Pauline; Yip, Cyril Chik-Yan; Zhang, Ricky Ruiqi; Fung, Agnes Yim-Fong; Yan, Erica Yuen-Wing; Leung, Kit-Hang; Ip, Jonathan Daniel; Chu, Allen Wing-Ho; Chan, Wan-Mui; Ng, Anthony Chin-Ki; Lee, Rodney; Fung, Kitty; Yeung, Alwin; Wu, Tak-Chiu; Chan, Johnny Wai-Man; Yan, Wing-Wah; Chan, Wai-Ming; Chan, Jasper Fuk-Woo; Lie, Albert Kwok-Wai; Tsang, Owen Tak-Yin; Cheng, Vincent Chi-Chung; Que, Tak-Lun; Lau, Chak-Sing; Chan, Kwok-Hung; To, Kelvin Kai-Wang; Yuen, Kwok-Yung
      Lancet (2020), 395 (10238), 1695-1704CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)
      Effective antiviral therapy is important for tackling the coronavirus disease 2019 (COVID-19) pandemic. We assessed the efficacy and safety of combined interferon beta-1b, lopinavir-ritonavir, and ribavirin for treating patients with COVID-19. This was a multicenter, prospective, open-label, randomized, phase 2 trial in adults with COVID-19 who were admitted to six hospitals in Hong Kong. Patients were randomly assigned (2:1) to a 14-day combination of lopinavir 400 mg and ritonavir 100 mg every 12 h, ribavirin 400 mg every 12 h, and three doses of 8 million IU of interferon beta-1b on alternate days (combination group) or to 14 days of lopinavir 400 mg and ritonavir 100 mg every 12 h (control group). The primary endpoint was the time to providing a nasopharyngeal swab neg. for severe acute respiratory syndrome coronavirus 2 RT-PCR, and was done in the intention-to-treat population. The study is registered with ClinicalTrials.gov, NCT04276688. Between Feb 10 and March 20, 2020, 127 patients were recruited; 86 were randomly assigned to the combination group and 41 were assigned to the control group. The median no. of days from symptom onset to start of study treatment was 5 days (IQR 3-7). The combination group had a significantly shorter median time from start of study treatment to neg. nasopharyngeal swab (7 days [IQR 5-11]) than the control group (12 days [8-15]; hazard ratio 4·37 [95% CI 1·86-10·24], p=0·0010). Adverse events included self-limited nausea and diarrhea with no difference between the two groups. One patient in the control group discontinued lopinavir-ritonavir because of biochem. hepatitis. No patients died during the study. Early triple antiviral therapy was safe and superior to lopinavir-ritonavir alone in alleviating symptoms and shortening the duration of viral shedding and hospital stay in patients with mild to moderate COVID-19. Future clin. study of a double antiviral therapy with interferon beta-1b as a backbone is warranted. The Shaw-Foundation, Richard and Carol Yu, May Tam Mak Mei Yin, and Sanming Project of Medicine.
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    41. 41
      Cao, B.; Wang, Y.; Wen, D.; Liu, W.; Wang, J.; Fan, G.; Ruan, L.; Song, B.; Cai, Y.; Wei, M.; Li, X.; Xia, J.; Chen, N.; Xiang, J.; Yu, T.; Bai, T.; Xie, X.; Zhang, L.; Li, C.; Yuan, Y.; Chen, H.; Li, H.; Huang, H.; Tu, S.; Gong, F.; Liu, Y.; Wei, Y.; Dong, C.; Zhou, F.; Gu, X.; Xu, J.; Liu, Z.; Zhang, Y.; Li, H.; Shang, L.; Wang, K.; Li, K.; Zhou, X.; Dong, X.; Qu, Z.; Lu, S.; Hu, X.; Ruan, S.; Luo, S.; Wu, J.; Peng, L.; Cheng, F.; Pan, L.; Zou, J.; Jia, C.; Wang, J.; Liu, X.; Wang, S.; Wu, X.; Ge, Q.; He, J.; Zhan, H.; Qiu, F.; Guo, L.; Huang, C.; Jaki, T.; Hayden, F. G.; Horby, P. W.; Zhang, D.; Wang, C. A Trial of Lopinavir-Ritonavir in Adults Hospitalized with Severe Covid-19. N. Engl. J. Med. 2020, 382, 1787– 1799,  DOI: 10.1056/NEJMoa2001282
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      A Trial of Lopinavir-Ritonavir in Adults Hospitalized with Severe Covid-19
      Cao Bin; Wang Yeming; Wen Danning; Liu Wen; Wang Jingli; Fan Guohui; Ruan Lianguo; Song Bin; Cai Yanping; Wei Ming; Li Xingwang; Xia Jiaan; Chen Nanshan; Xiang Jie; Yu Ting; Bai Tao; Xie Xuelei; Zhang Li; Li Caihong; Yuan Ye; Chen Hua; Li Huadong; Huang Hanping; Tu Shengjing; Gong Fengyun; Liu Ying; Wei Yuan; Dong Chongya; Zhou Fei; Gu Xiaoying; Xu Jiuyang; Liu Zhibo; Zhang Yi; Li Hui; Shang Lianhan; Wang Ke; Li Kunxia; Zhou Xia; Dong Xuan; Qu Zhaohui; Lu Sixia; Hu Xujuan; Ruan Shunan; Luo Shanshan; Wu Jing; Peng Lu; Cheng Fang; Pan Lihong; Zou Jun; Jia Chunmin; Wang Juan; Liu Xia; Wang Shuzhen; Wu Xudong; Ge Qin; He Jing; Zhan Haiyan; Qiu Fang; Guo Li; Huang Chaolin; Jaki Thomas; Hayden Frederick G; Horby Peter W; Zhang Dingyu; Wang Chen
      The New England journal of medicine (2020), 382 (19), 1787-1799 ISSN:.
      BACKGROUND: No therapeutics have yet been proven effective for the treatment of severe illness caused by SARS-CoV-2. METHODS: We conducted a randomized, controlled, open-label trial involving hospitalized adult patients with confirmed SARS-CoV-2 infection, which causes the respiratory illness Covid-19, and an oxygen saturation (Sao2) of 94% or less while they were breathing ambient air or a ratio of the partial pressure of oxygen (Pao2) to the fraction of inspired oxygen (Fio2) of less than 300 mm Hg. Patients were randomly assigned in a 1:1 ratio to receive either lopinavir-ritonavir (400 mg and 100 mg, respectively) twice a day for 14 days, in addition to standard care, or standard care alone. The primary end point was the time to clinical improvement, defined as the time from randomization to either an improvement of two points on a seven-category ordinal scale or discharge from the hospital, whichever came first. RESULTS: A total of 199 patients with laboratory-confirmed SARS-CoV-2 infection underwent randomization; 99 were assigned to the lopinavir-ritonavir group, and 100 to the standard-care group. Treatment with lopinavir-ritonavir was not associated with a difference from standard care in the time to clinical improvement (hazard ratio for clinical improvement, 1.31; 95% confidence interval [CI], 0.95 to 1.80). Mortality at 28 days was similar in the lopinavir-ritonavir group and the standard-care group (19.2% vs. 25.0%; difference, -5.8 percentage points; 95% CI, -17.3 to 5.7). The percentages of patients with detectable viral RNA at various time points were similar. In a modified intention-to-treat analysis, lopinavir-ritonavir led to a median time to clinical improvement that was shorter by 1 day than that observed with standard care (hazard ratio, 1.39; 95% CI, 1.00 to 1.91). Gastrointestinal adverse events were more common in the lopinavir-ritonavir group, but serious adverse events were more common in the standard-care group. Lopinavir-ritonavir treatment was stopped early in 13 patients (13.8%) because of adverse events. CONCLUSIONS: In hospitalized adult patients with severe Covid-19, no benefit was observed with lopinavir-ritonavir treatment beyond standard care. Future trials in patients with severe illness may help to confirm or exclude the possibility of a treatment benefit. (Funded by Major Projects of National Science and Technology on New Drug Creation and Development and others; Chinese Clinical Trial Register number, ChiCTR2000029308.).
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    42. 42
      Xu, L.; Liu, H.; Murray, B. P.; Callebaut, C.; Lee, M. S.; Hong, A.; Strickley, R. G.; Tsai, L. K.; Stray, K. M.; Wang, Y.; Rhodes, G. R.; Desai, M. C. Cobicistat (GS-9350): A Potent and Selective Inhibitor of Human CYP3A as a Novel Pharmacoenhancer. ACS Med. Chem. Lett. 2010, 1, 209– 213,  DOI: 10.1021/ml1000257
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      42
      Cobicistat (GS-9350): A Potent and Selective Inhibitor of Human CYP3A as a Novel Pharmacoenhancer
      Xu, Lianhong; Liu, Hongtao; Murray, Bernard P.; Callebaut, Christian; Lee, Melody S.; Hong, Allen; Strickley, Robert G.; Tsai, Luong K.; Stray, Kirsten M.; Wang, Yujin; Rhodes, Gerry R.; Desai, Manoj C.
      ACS Medicinal Chemistry Letters (2010), 1 (5), 209-213CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)
      Cobicistat (3, GS-9350) is a newly discovered, potent, and selective inhibitor of human cytochrome P 450 3A (CYP3A) enzymes. In contrast to ritonavir, 3 is devoid of anti-HIV activity and is thus more suitable for use in boosting anti-HIV drugs without risking selection of potential drug-resistant HIV variants. Compd. 3 shows reduced liability for drug interactions and may have potential improvements in tolerability over ritonavir. In addn., 3 has high aq. soly. and can be readily co-formulated with other agents.
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    43. 43
      Covid-19 clinical trials showing “encouraging” results, says analyst https://www.clinicaltrialsarena.com/analysis/covid-19-clinical-trials-results-2/ (accessed Jun 1, 2020).
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    44. 44
      Lawitz, E.; Gane, E.; Pearlman, B.; Tam, E.; Ghesquiere, W.; Guyader, D.; Alric, L.; Bronowicki, J. P.; Lester, L.; Sievert, W.; Ghalib, R.; Balart, L.; Sund, F.; Lagging, M.; Dutko, F.; Shaughnessy, M.; Hwang, P.; Howe, A. Y. M.; Wahl, J.; Robertson, M.; Barr, E.; Haber, B. Efficacy and Safety of 12 Weeks versus 18 Weeks of Treatment with Grazoprevir (MK-5172) and Elbasvir (MK-8742) with or without Ribavirin for Hepatitis C Virus Genotype 1 Infection in Previously Untreated Patients with Cirrhosis and Patients with Previous Null Response with or without Cirrhosis (C-WORTHY): A Randomised, Open-Label Phase 2 Trial. Lancet 2015, 385, 1075– 1086,  DOI: 10.1016/S0140-6736(14)61795-5
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      44
      Efficacy and safety of 12 weeks versus 18 weeks of treatment with grazoprevir (MK-5172) and elbasvir (MK-8742) with or without ribavirin for hepatitis C virus genotype 1 infection in previously untreated patients with cirrhosis and patients with previous null response with or without cirrhosis (C-WORTHY): a randomised, open-label phase 2 trial
      Lawitz, Eric; Gane, Edward; Pearlman, Brian; Tam, Edward; Ghesquiere, Wayne; Guyader, Dominique; Alric, Laurent; Bronowicki, Jean-Pierre; Lester, Laura; Sievert, William; Ghalib, Reem; Balart, Luis; Sund, Fredrik; Lagging, Martin; Dutko, Frank; Shaughnessy, Melissa; Hwang, Peggy; Howe, Anita Y. M.; Wahl, Janice; Robertson, Michael; Barr, Eliav; Haber, Barbara
      Lancet (2015), 385 (9973), 1075-1086CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)
      There is a high medical need for an interferon-free, all-oral, short-duration therapy for hepatitis C virus (HCV) that is highly effective across diverse patient populations, including patients with cirrhosis or previous null response to pegylated interferon (peginterferon) plus ribavirin (PR-null responders). We aimed to assess the efficacy, safety, and effective treatment duration of grazoprevir (an HCV NS3/4A protease inhibitor) combined with elbasvir (an HCV NS5A inhibitor) with or without ribavirin in patients with HCV genotype 1 infection with baseline characteristics of poor response. The C-WORTHY trial is a randomised, open-label phase 2 trial of grazoprevir plus elbasvir with or without ribavirin; here we report findings for two cohorts of previously untreated patients with cirrhosis (cohort 1) and those with previous PR-null response with or without cirrhosis (cohort 2) enrolled in part B of the study. Eligible patients were adults aged 18 years or older with chronic HCV genotype 1 infection and HCV RNA concns. of 10 000 IU/mL or higher in peripheral blood. We randomly assigned patients to receive grazoprevir (100 mg daily) and elbasvir (50 mg daily) with or without ribavirin for 12 or 18 wk. Randomisation was done centrally with an interactive voice response system; patients and study investigators were masked to treatment duration up to week 12 but not to treatment allocation. The primary endpoint was the proportion of patients achieving HCV RNA less than 25 IU/mL at 12 wk after end of treatment (SVR12), assessed by COBAS TaqMan version 2.0. This study is registered with ClinicalTrials.gov, no. NCT01717326. We describe findings for 253 patients enrolled in cohort 1 (n=123) or cohort 2 (n=130). In cohort 1, we randomly assigned 60 patients to the 12-wk regimen (31 with ribavirin and 29 with no ribavirin) and 63 to the 18-wk regimen (32 with ribavirin and 31 with no ribavirin); in cohort 2, we randomly assigned 65 patients to the 12-wk regimen (32 with ribavirin and 33 with no ribavirin) and 65 to the 18-wk regimen 33 with ribavirin and 32 with no ribavirin. High SVR12 rates were achieved irresp. of the use of ribavirin or extension of the treatment duration from 12 to 18 wk; SVR12 rates ranged from 90% (95% CI 74-98; 28/31; cohort 1, 12 wk, ribavirin-contg.) to 100% (95% CI 89-100; 33/33; cohort 2, 18 wk, ribavirin-contg.). Among patients treated for 12 wk with grazoprevir plus elbasvir without ribavirin, 97% (95% CI 82-100, 28/29) of patients in cohort 1 and 91% (76-98, 30/33) of patients in cohort 2 achieved SVR12. Adverse events reported in more than 10% of patients were fatigue (66 patients, 26% [95% CI 21-32]), headache (58 patients, 23% [95% CI 18-29]), and asthenia (35 patients, 14% [95% CI 10-19]). Treatment with grazoprevir plus elbasvir, both with and without ribavirin and for both 12 and 18 wk' treatment duration, showed high rates of efficacy in previously untreated patients with cirrhosis and previous PR-null responders with and without cirrhosis. These results support the phase 3 development of grazoprevir plus elbasvir.Merck & Co, Inc.
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    45. 45
      Waheed, Y. Ledipasvir and Sofosbuvir: Interferon Free Therapy for HCV Genotype 1 Infection. World J. Virol. 2015, 4, 33,  DOI: 10.5501/wjv.v4.i1.33
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      45
      Ledipasvir and sofosbuvir: Interferon free therapy for hepatitis C virus genotype 1 infection
      Waheed Yasir
      World journal of virology (2015), 4 (1), 33-5 ISSN:2220-3249.
      Hepatitis C virus (HCV) has infected more than 200 million people around the globe. From 2001-2011, interferon plus ribavirin remained the standard of care for patients with HCV infection. The therapy had a limited response with a number of side effects. Recently, results for phase III trials of ledipasvir and sofosbuvir combination therapy have been announced. In treatment naive patients, 12 wk of therapy with ledipasvir and sofosbuvir showed a sustained virological response (SVR) rate of 99%. In treatment experienced patients, 12-24 wk of therapy with ledipasvir and sofosbuvir in the absence or presence of ribavirin showed an SVR rate of 94%-99%. In cirrhotic patients the rate of SVR was 86% and 99% for 12 and 24 wk of therapy, respectively. The ledipasvir and sofosbuvir therapy showed very good results in different subgroups of patients regardless of patient's race, alanine aminotransferase levels, sex and host genetic factors. The combination therapy was well tolerated with no emergence of resistant mutants. The most common adverse effects were nausea, headache and fatigue. With the availability of interferon free therapy with minimal adverse effects, it will be easy to decrease the future morbidity and mortality caused by HCV infection.
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    46. 46
      Zignego, A. L.; Monti, M.; Gragnani, L. Sofosbuvir/Velpatasvir for the Treatment of Hepatitis C Virus Infection. Acta Biomedica. Mattioli 1885 September 1, 2018; pp 321– 331.
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      Vishal, M.; Pravin, D.; Himani, G.; Nilam, V.; Urvisha, B.; Rajesh, P. Drug Repurposing of Approved Drugs Elbasvir, Ledipasvir, Paritaprevir, Velpatasvir, Antrafenine and Ergotamine for Combating COVID19. Preprint. ChemRxiv 2020,  DOI: 10.26434/Chemrxiv.12115251.V1 .
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      Search of: ledipasvir | Covid19 - List Results - ClinicalTrials.gov; https://clinicaltrials.gov/ct2/results?cond=Covid19&term=ledipasvir&cntry=&state=&city=&dist= (accessed Aug 27, 2020).
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      Waters, L.; Nelson, M. The Use of Caspofungin in HIV-Infected Individuals. Expert Opin. Invest. Drugs 2007, 16, 899– 908,  DOI: 10.1517/13543784.16.6.899
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      The use of caspofungin in HIV-infected individuals
      Waters, Laura; Nelson, Mark
      Expert Opinion on Investigational Drugs (2007), 16 (6), 899-908CODEN: EOIDER; ISSN:1354-3784. (Informa Healthcare)
      A review. Fungal infections are a significant cause of HIV-related morbidity and mortality, particularly in the developing world, but also in countries with access to highly active antiretroviral therapy. New agents are essential to improve present efficacy rates, particularly in cases of drug resistance. Caspofungin is a new antifungal from the echinocandin class and is licensed for the treatment of candidal infections and as a second-line therapy for invasive aspergillosis. In this paper, the pharmacol., interaction and susceptibility data for this agent are reviewed and studies supporting the use of this agent in HIV-infected individuals are examd. Finally, evidence for the use of caspofungin for the treatment of Pneumocystis jiroveci pneumonia, an unlicensed indication, including a case series from our own unit is explored.
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    50. 50
      Wang, M.; Ye, F.; Su, J.; Zhao, J.; Yuan, B.; Huang, B.; Peng, Q.; Peng, R.; Sun, Y.; Bai, S.; Wang, X.; Yang, W.; Fan, Z.; Wang, W.; Wu, G.; Gao, G. F.; Tan, W.; Sh, Y. Caspofungin and LTX-315 Inhibit SARS-CoV-2 Replication by Targeting the Nsp12 Polymerase. Research Square. Preprint. 2020,  DOI: 10.21203/RS.3.RS-19872/V1 .
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      Yang, Y.; Cao, T.; Guo, Q. Characterization of a Traditional Chinese Medicine Plant the Chloroplast Genome of Andrographis Paniculata. Mitochondrial DNA Part B 2020, 5, 1949– 1951,  DOI: 10.1080/23802359.2020.1756949
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      Wei-Ya, C.; Yuan-Song, W.; Chun-Yu, L.; Yu-Bin, J.; Fei-Fei, Y.; Yong-Hong, L. Comparison of Pulmonary Availability and Anti-Inflammatory Effect of Dehydroandrographolide Succinate via Intratracheal and Intravenous Administration. Eur. J. Pharm. Sci. 2020, 147, 105290,  DOI: 10.1016/j.ejps.2020.105290
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      52
      Comparison of pulmonary availability and anti-inflammatory effect of dehydroandrographolide succinate via intratracheal and intravenous administration
      Wei-Ya Chen; Chun-Yu Liu; Yuan-Song Wang; Yu-Bin Ji; Fei-Fei Yang; Yong-Hong Liao
      European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences (2020), 147 (), 105290 ISSN:.
      Dehydroandrographolide succinate (DAS) injection, which was approved in China for the treatment of viral pneumonia and upper respiratory tract infections, is often off-label used for nebulization therapy to avoid the adverse drug reactions associated with the injection. However, the aerodynamic properties and pulmonary fate of nebulized DAS was largely uninvestigated. In this study, the main objectives were to evaluate the in vitro aerodynamic deposition profiles of nebulizer generated aerosols and comparatively investigate the local drug availability and anti-inflammatory efficacy of DAS between intratracheal and intravenous dosing. The in vitro evaluation of aerodynamic characteristics and droplet size distribution showed more than 50% aerosol particles with size being <5 μm, allowing the aerosols to reach the lower respiratory tract. Following intratracheal administration, the drug underwent pulmonary absorption into the bloodstream, rendering an absolute bioavailability of 47.3%. Compared to the intravenous delivery, the intratracheal administration dramatically increased the drug availability in the lung tissue in rats by more than 80-fold, leading to an improved and prolonged local anti-inflammatory efficacy in a lipopolysaccharide induced lung injury model in mice. The present results demonstrated that inhalation delivery of DAS is a convenient and effective alternative to intravenous injections.
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    53. 53
      Chen, Q.; Liu, Y.; Liu, Y. M.; Liu, G. Y.; Zhang, M. Q.; Jia, J. Y.; Lu, C.; Yu, C. Pharmacokinetics and Tolerance of Dehydroandrographolide Succinate Injection after Intravenous Administration in Healthy Chinese Volunteers. Acta Pharmacol. Sin. 2012, 33, 1332– 1336,  DOI: 10.1038/aps.2012.79
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      Pharmacokinetics and tolerance of dehydroandrographolide succinate injection after intravenous administration in healthy Chinese volunteers
      Chen, Qian; Liu, Yun; Liu, Yan-mei; Liu, Gang-yi; Zhang, Meng-qi; Jia, Jing-ying; Lu, Chuan; Yu, Chen
      Acta Pharmacologica Sinica (2012), 33 (10), 1332-1336CODEN: APSCG5; ISSN:1671-4083. (Nature Publishing Group)
      Aim: Dehydroandrographolide succinate (DAS) is extd. from herbal medicine Andrographis paniculata (Burm f) Nees. DAS injection is used in China for the treatment of viral pneumonia and upper respiratory tract infections. The aim of this study is to investigate the pharmacokinetics and tolerance of DAS injection in healthy Chinese volunteers. Methods: This was a single-center, randomized, single-dose, three-way crossover design study. Nine eligible subjects were randomly divided into 3 groups, and each group sequentially received 80, 160, or 320 mg of DAS infusion according to a three-way Latin square design. Plasma and urine samples were collected and detd. using an LC-MS/MS method. Safety and tolerability were detd. via clin. evaluation and adverse event monitoring. Results: For the 80, 160, and 320 mg dose groups, the mean Cmax were 4.82, 12.85, and 26.90 mg/L, resp., and the mean AUC0-12 were 6.18, 16.95, and 40.65 mg·L-1·h, resp. DAS was rapidly cleared, with a mean Tmax of 0.94-1.0 h and a t1/2 of approx. 1.51-1.89 h. Approx. 10.1%-15.5% of the i.v. DAS dose was excreted unchanged in urine within 24 h in the 3 groups, and more than 90% of unchanged DAS was excreted between 0 and 4 h. The pharmacokinetic profile was similar between male and female subjects. No serious or unexpected adverse events were found during the study, but one mild adverse event (stomachache) was reported. Conclusion: This study shows that DAS has nonlinear pharmacokinetic characteristics. To guarantee the effective concn., multiple small doses are recommended in clin. regimens.
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      Jayakumar, T.; Hsieh, C. Y.; Lee, J. J.; Sheu, J. R. Experimental and Clinical Pharmacology of Andrographis Paniculata and Its Major Bioactive Phytoconstituent Andrographolide. J. Evidence-Based Complementary Altern. Med. 2013, 2013, 1 DOI: 10.1155/2013/846740 .
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      Chang, R. S.; Ding, L.; Gai-Qing, C.; Qi-Choa, P.; Ze-Lin, Z.; Smith, K. M. Dehydroandrographolide Succinic Acid Monoester as an Inhibitor against the Human Immunodeficiency Virus. Exp. Biol. Med. 1991, 197, 59– 66,  DOI: 10.3181/00379727-197-43225
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      Dehydroandrographolide succinic acid monoester as an inhibitor against the human immunodeficiency virus
      Chang, R. Shihman; Ding, Lu; Chen, Gaiqing; Pan, Qichoa; Zhao, Zelin; Smith, Kevin M.
      Proceedings of the Society for Experimental Biology and Medicine (1991), 197 (1), 59-66CODEN: PSEBAA; ISSN:0037-9727.
      Dehydroandrographolide succinic acid monoester (DASM) is made from the Chinese medicinal herb Andrographis paniculata. DASM is an inhibitor against the human immunodeficiency virus (HIV) in vitro. It was nontoxic to the H9 cell at 50-200 μg/mL and was inhibitory to the HIV-1 (IIIB) at the minimal concn. of 1.6-3.1 μg/mL. It was also inhibitory to two other strains of HIV-1 and a strain of HIV-2. This inhibitory effect could also be demonstrated in cultures of activated human blood mononuclear cells; the 50% toxic dose and the 50% HIV ID were about 200-≥400 and 0.8-2 μg/mL, resp. At the subtoxic concn., DASM partially interfered with HIV-induced cell fusion and with the binding of HIV to the H9 cell. Presumably, it also interfered with HIV replication at another unidentified step(s).
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    56. 56
      Cai, W.; Li, Y.; Chen, S.; Wang, M.; Zhang, A.; Zhou, H.; Chen, H.; Jin, M. 14-Deoxy-11,12-Dehydroandrographolide Exerts Anti-Influenza A Virus Activity and Inhibits Replication of H5N1 Virus by Restraining Nuclear Export of Viral Ribonucleoprotein Complexes. Antiviral Res. 2015, 118, 82– 92,  DOI: 10.1016/j.antiviral.2015.03.008
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      56
      14-Deoxy-11,12-dehydroandrographolide exerts anti-influenza A virus activity and inhibits replication of H5N1 virus by restraining nuclear export of viral ribonucleoprotein complexes
      Cai, Wentao; Li, Yongtao; Chen, Sunrui; Wang, Mengli; Zhang, Anding; Zhou, Hongbo; Chen, Huanchun; Jin, Meilin
      Antiviral Research (2015), 118 (), 82-92CODEN: ARSRDR; ISSN:0166-3542. (Elsevier B.V.)
      The highly pathogenic avian influenza H5N1 virus has become a worldwide public health threat, and current antiviral therapies have limited activity against the emerging, resistant influenza viruses. Therefore, effective drugs with novel targets against influenza A viruses, H5N1 strains in particular, should be developed. In the present study, 14-deoxy-11,12-dehydroandrographolide (DAP), a major component of the traditional Chinese medicine Andrographis paniculata, exerted potent anti-influenza A virus activity against A/chicken/Hubei/327/2004 (H5N1), A/duck/Hubei/XN/2007 (H5N1), A/PR/8/34 (H1N1), A/NanChang/08/2010 (H1N1) and A/HuNan/01/2014 (H3N2) in vitro. To elucidate the underlying mechanisms, a series of expts. was conducted using A/chicken/Hubei/327/2004 (H5N1) as an example. Our results demonstrated that DAP strongly inhibited H5N1 replication by reducing the prodn. of viral nucleoprotein (NP) mRNA, NP and NS1proteins, whereas DAP had no effect on the absorption and release of H5N1 towards/from A549 cells. DAP also effectively restrained the nuclear export of viral ribonucleoprotein (vRNP) complexes. This inhibitory effect ought to be an important anti-H5N1 mechanism of DAP. Meanwhile, DAP significantly reduced the upregulated expression of all the tested proinflammatory cytokines (TNF-α, IL-6, IL-8, IFN-α, IL-1β and IFN-β) and chemokines (CXCL-10 and CCL-2) stimulated by H5N1. Overall results suggest that DAP impairs H5N1 replication at least in part by restraining nuclear export of vRNP complexes, and the inhibition of viral replication leads to a subsequent decrease of the intense proinflammatory cytokine/chemokine expression. In turn, the effect of modification of the host excessive immune response may contribute to overcoming H5N1. To our knowledge, this study is the first to reveal the antiviral and anti-inflammatory activities of DAP in vitro against H5N1 influenza A virus infection.
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      Jakalian, A.; Jack, D. B.; Bayly, C. I. Fast, Efficient Generation of High-Quality Atomic Charges. AM1-BCC Model: II. Parameterization and Validation. J. Comput. Chem. 2002, 23, 1623– 1641,  DOI: 10.1002/jcc.10128
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      Fast, efficient generation of high-quality atomic charges. AM1-BCC model: II. parameterization and validation
      Jakalian, Araz; Jack, David B.; Bayly, Christopher I.
      Journal of Computational Chemistry (2002), 23 (16), 1623-1641CODEN: JCCHDD; ISSN:0192-8651. (John Wiley & Sons, Inc.)
      We present the first global parameterization and validation of a novel charge model, called AM1-BCC, which quickly and efficiently generates high-quality at. charges for computer simulations of org. mols. in polar media. The goal of the charge model is to produce at. charges that emulate the HF/6-31G* electrostatic potential (ESP) of a mol. Underlying electronic structure features, including formal charge and electron delocalization, are first captured by AM1 population charges; simple additive bond charge corrections (BCCs) are then applied to these AM1 at. charges to produce the AM1-BCC charges. The parameterization of BCCs was carried out by fitting to the HF/6-31G* ESP of a training set of >2700 mols. Most org. functional groups and their combinations were sampled, as well as an extensive variety of cyclic and fused bicyclic heteroaryl systems. The resulting BCC parameters allow the AM1-BCC charging scheme to handle virtually all types of org. compds. listed in The Merck Index and the NCI Database. Validation of the model was done through comparisons of hydrogen-bonded dimer energies and relative free energies of solvation using AM1-BCC charges in conjunction with the 1994 Cornell et al. forcefield for AMBER. Homo-dimer and hetero-dimer hydrogen-bond energies of a diverse set of org. mols. were reproduced to within 0.95 kcal/mol RMS deviation from the ab initio values, and for DNA dimers the energies were within 0.9 kcal/mol RMS deviation from ab initio values. The calcd. relative free energies of solvation for a diverse set of monofunctional isosteres were reproduced to within 0.69 kcal/mol of expt. In all these validation tests, AMBER with the AM1-BCC charge model maintained a correlation coeff. above 0.96. Thus, the parameters presented here for use with the AM1-BCC method present a fast, accurate, and robust alternative to HF/6-31G* ESP-fit charges for general use with the AMBER force field in computer simulations involving org. small mols.
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      Bayly, C. I.; Cieplak, P.; Cornell, W. D.; Kollman, P. A. A Well-Behaved Electrostatic Potential Based Method Using Charge Restraints for Deriving Atomic Charges: The RESP Model. J. Phys. Chem. 1993, 97, 10269– 10280,  DOI: 10.1021/j100142a004
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      A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: the RESP model
      Bayly, Christopher I.; Cieplak, Piotr; Cornell, Wendy; Kollman, Peter A.
      Journal of Physical Chemistry (1993), 97 (40), 10269-80CODEN: JPCHAX; ISSN:0022-3654.
      The authors present a new approach to generating electrostatic potential (ESP) derived charges for mols. The major strength of electrostatic potential derived charges is that they optimally reproduce the intermol. interaction properties of mols. with a simple two-body additive potential, provided, of course, that a suitably accurate level of quantum mech. calcn. is used to derive the ESP around the mol. Previously, the major weaknesses of these charges have been that they were not easily transferably between common functional groups in related mols., they have often been conformationally dependent, and the large charges that frequently occur can be problematic for simulating intramol. interactions. Introducing restraints in the form of a penalty function into the fitting process considerably reduces the above problems, with only a minor decrease in the quality of the fit to the quantum mech. ESP. Several other refinements in addn. to the restrained electrostatic potential (RESP) fit yield a general and algorithmic charge fitting procedure for generating atom-centered point charges. This approach can thus be recommended for general use in mol. mechanics, mol. dynamics, and free energy calcns. for any org. or bioorg. system.
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      Xia, S.; Liu, Q.; Wang, Q.; Sun, Z.; Su, S.; Du, L.; Ying, T.; Lu, L.; Jiang, S. Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Entry Inhibitors Targeting Spike Protein. Virus Res. 2014, 194, 200– 210,  DOI: 10.1016/j.virusres.2014.10.007
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      Middle East respiratory syndrome coronavirus (MERS-CoV) entry inhibitors targeting spike protein
      Xia, Shuai; Liu, Qi; Wang, Qian; Sun, Zhiwu; Su, Shan; Du, Lanying; Ying, Tianlei; Lu, Lu; Jiang, Shibo
      Virus Research (2014), 194 (), 200-210CODEN: VIREDF; ISSN:0168-1702. (Elsevier B.V.)
      A review. The recent outbreak of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) infection has led to more than 800 lab.-confirmed MERS cases with a high case fatality rate (∼35%), posing a serious threat to global public health and calling for the development of effective and safe therapeutic and prophylactic strategies to treat and prevent MERS-CoV infection. Here we discuss the most recent studies on the structure of the MERS-CoV spike protein and its role in virus binding and entry, and the development of MERS-CoV entry/fusion inhibitors targeting the S1 subunit, particularly the receptor-binding domain (RBD), and the S2 subunit, esp. the HR1 region, of the MERS-CoV spike protein. We then look ahead to future applications of these viral entry/fusion inhibitors, either alone or in combination with specific and nonspecific MERS-CoV replication inhibitors, for the treatment and prevention of MERS-CoV infection.
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      Du, L.; Zhao, G.; Yang, Y.; Qiu, H.; Wang, L.; Kou, Z.; Tao, X.; Yu, H.; Sun, S.; Tseng, C.-T. K.; Jiang, S.; Li, F.; Zhou, Y. A Conformation-Dependent Neutralizing Monoclonal Antibody Specifically Targeting Receptor-Binding Domain in Middle East Respiratory Syndrome Coronavirus Spike Protein. J. Virol. 2014, 88, 7045– 7053,  DOI: 10.1128/JVI.00433-14
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      A conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in Middle East respiratory syndrome coronavirus spike protein
      Du, Lanying; Zhao, Guangyu; Yang, Yang; Qiu, Hongjie; Wang, Lili; Kou, Zhihua; Tao, Xinrong; Yu, Hong; Sun, Shihui; Tseng, Chien-Te K.; Jiang, Shibo; Li, Fang; Zhou, Yusen
      Journal of Virology (2014), 88 (12), 7045-7053, 10 pp.CODEN: JOVIAM; ISSN:1098-5514. (American Society for Microbiology)
      Prophylactic and therapeutic strategies are urgently needed to combat infections caused by the newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV). Here, we have developed a neutralizing monoclonal antibody (MAb), designated Mersmab1, which potently blocks MERS-CoV entry into human cells. Biochem. assays reveal that Mersmab1 specifically binds to the receptor-binding domain (RBD) of the MERS-CoV spike protein and thereby competitively blocks the binding of the RBD to its cellular receptor, dipeptidyl peptidase 4 (DPP4). Furthermore, alanine scanning of the RBD has identified several residues at the DPP4-binding surface that serve as neutralizing epitopes for Mersmab1. These results suggest that if humanized, Mersmab1 could potentially function as a therapeutic antibody for treating and preventing MERS-CoV infections. Addnl., Mersmab1 may facilitate studies of the conformation and antigenicity of MERS-CoV RBD and thus will guide rational design of MERS-CoV subunit vaccines.
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      Li, Y.; Wan, Y.; Liu, P.; Zhao, J.; Lu, G.; Qi, J.; Wang, Q.; Lu, X.; Wu, Y.; Liu, W.; Zhang, B.; Yuen, K. Y.; Perlman, S.; Gao, G. F.; Yan, J. A Humanized Neutralizing Antibody against MERS-CoV Targeting the Receptor-Binding Domain of the Spike Protein. Cell Res. 2015, 25, 1237– 1249,  DOI: 10.1038/cr.2015.113
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      A humanized neutralizing antibody against MERS-CoV targeting the receptor-binding domain of the spike protein
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      Cell Research (2015), 25 (11), 1237-1249CODEN: CREEB6; ISSN:1001-0602. (NPG Nature Asia-Pacific)
      The newly-emerging Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe and fatal acute respiratory disease in humans. Despite global efforts, the potential for an assocd. pandemic in the future cannot be excluded. The development of effective counter-measures is urgent. MERS-CoV-specific anti-viral drugs or vaccines are not yet available. Using the spike receptor-binding domain of MERS-CoV (MERS-RBD) to immunize mice, we identified two neutralizing monoclonal antibodies (mAbs) 4C2 and 2E6. Both mAbs potently bind to MERS-RBD and block virus entry in vitro with high efficacy. We further investigated their mechanisms of neutralization by crystg. the complex between the Fab fragments and the RBD, and solved the structure of the 4C2 Fab/MERS-RBD complex. The structure showed that 4C2 recognizes an epitope that partially overlaps the receptor-binding footprint in MERS-RBD, thereby interfering with the virus/receptor interactions by both steric hindrance and interface-residue competition. 2E6 also blocks receptor binding, and competes with 4C2 for binding to MERS-RBD. Based on the structure, we further humanized 4C2 by preserving only the paratope residues and substituting the remaining amino acids with the counterparts from human Igs. The humanized 4C2 (4C2h) antibody sustained similar neutralizing activity and biochem. characteristics to the parental mouse antibody. Finally, we showed that 4C2h can significantly abate the virus titers in lungs of Ad5-hCD26-transduced mice infected with MERS-CoV, therefore representing a promising agent for prophylaxis and therapy in clin. settings.
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      Coughlin, M.; Lou, G.; Martinez, O.; Masterman, S. K.; Olsen, O. A.; Moksa, A. A.; Farzan, M.; Babcook, J. S.; Prabhakar, B. S. Generation and Characterization of Human Monoclonal Neutralizing Antibodies with Distinct Binding and Sequence Features against SARS Coronavirus Using XenoMouse®. Virology 2007, 361, 93– 102,  DOI: 10.1016/j.virol.2006.09.029
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      Generation and characterization of human monoclonal neutralizing antibodies with distinct binding and sequence features against SARS coronavirus using XenoMouse
      Coughlin, Melissa; Lou, Gin; Martinez, Osvaldo; Masterman, Stephanie K.; Olsen, Ole A.; Moksa, Angelica A.; Farzan, Michael; Babcook, John S.; Prabhakar, Bellur S.
      Virology (2007), 361 (1), 93-102CODEN: VIRLAX; ISSN:0042-6822. (Elsevier)
      Passive therapy with neutralizing human monoclonal antibodies (mAbs) could be an effective therapy against severe acute respiratory syndrome coronavirus (SARS-CoV). Utilizing the human Ig transgenic mouse, XenoMouse, the authors produced fully human SARS-CoV spike (S) protein specific antibodies. Antibodies were examd. for reactivity against a recombinant S1 protein, to which 200 antibodies reacted. Twenty-seven antibodies neutralized 200TCID50 SARS-CoV (Urbani). Addnl., 57 neutralizing antibodies were found that are likely specific to S2. Mapping of the binding region was achieved with several S1 recombinant proteins. Most S1 reactive neutralizing mAbs bound to the RBD, aa 318-510. However, two S1 specific mAbs reacted with a domain upstream of the RBD between aa 12 and 261. Ig gene sequence analyses suggested at least 8 different binding specificities. Unique human mAbs could be used as a cocktail that would simultaneously target several neutralizing epitopes and prevent emergence of escape mutants.
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      Xia, S.; Liu, M.; Wang, C.; Xu, W.; Lan, Q.; Feng, S.; Qi, F.; Bao, L.; Du, L.; Liu, S.; Qin, C.; Sun, F.; Shi, Z.; Zhu, Y.; Jiang, S.; Lu, L. Inhibition of SARS-CoV-2 (Previously 2019-NCoV) Infection by a Highly Potent Pan-Coronavirus Fusion Inhibitor Targeting Its Spike Protein That Harbors a High Capacity to Mediate Membrane Fusion. Cell Res. 2020, 30, 343– 355,  DOI: 10.1038/s41422-020-0305-x
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      Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion
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      The recent outbreak of coronavirus disease (COVID-19) caused by SARS-CoV-2 infection in Wuhan, China has posed a serious threat to global public health. To develop specific anti-coronavirus therapeutics and prophylactics, the mol. mechanism that underlies viral infection must first be defined. Therefore, we herein established a SARS-CoV-2 spike (S) protein-mediated cell-cell fusion assay and found that SARS-CoV-2 showed a superior plasma membrane fusion capacity compared to that of SARS-CoV. We solved the X-ray crystal structure of six-helical bundle (6-HB) core of the HR1 and HR2 domains in the SARS-CoV-2 S protein S2 subunit, revealing that several mutated amino acid residues in the HR1 domain may be assocd. with enhanced interactions with the HR2 domain. We previously developed a pan-coronavirus fusion inhibitor, EK1, which targeted the HR1 domain and could inhibit infection by divergent human coronaviruses tested, including SARS-CoV and MERS-CoV. Here we generated a series of lipopeptides derived from EK1 and found that EK1C4 was the most potent fusion inhibitor against SARS-CoV-2 S protein-mediated membrane fusion and pseudovirus infection with IC50s of 1.3 and 15.8 nM, about 241- and 149-fold more potent than the original EK1 peptide, resp. EK1C4 was also highly effective against membrane fusion and infection of other human coronavirus pseudoviruses tested, including SARS-CoV and MERS-CoV, as well as SARSr-CoVs, and potently inhibited the replication of 5 live human coronaviruses examd., including SARS-CoV-2. Intranasal application of EK1C4 before or after challenge with HCoV-OC43 protected mice from infection, suggesting that EK1C4 could be used for prevention and treatment of infection by the currently circulating SARS-CoV-2 and other emerging SARSr-CoVs.
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      Viruses (2012), 4 (), 1011-1033CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)
      A review. Coronaviruses are enveloped pos.-stranded RNA viruses that replicate in the cytoplasm. To deliver their nucleocapsid into the host cell, they rely on the fusion of their envelope with the host cell membrane. The spike glycoprotein (S) mediates virus entry and is a primary determinant of cell tropism and pathogenesis. It is classified as a class I fusion protein, and is responsible for binding to the receptor on the host cell as well as mediating the fusion of host and viral membranes - A process driven by major conformational changes of the S protein. This review discusses coronavirus entry mechanisms focusing on the different triggers used by coronaviruses to initiate the conformational change of the S protein: receptor binding, low pH exposure and proteolytic activation. We also highlight commonalities between coronavirus S proteins and other class I viral fusion proteins, as well as distinctive features that confer distinct tropism, pathogenicity and host interspecies transmission characteristics to coronaviruses.
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      Nucleic Acids Research (2016), 44 (W1), W449-W454CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      Structure detn. of linear peptides of 5-50 amino acids in aq. soln. and interacting with proteins is a key aspect in structural biol. PEPFOLD3 is a novel computational framework, that allows both (i) de novo free or biased prediction for linear peptides between 5 and 50 amino acids, and (ii) the generation of native-like conformations of peptides interacting with a protein when the interaction site is known in advance. PEP-FOLD3 is fast, and usually returns solns. in a few minutes. Testing PEP-FOLD3 on 56 peptides in aq. soln. led to exptl.-like conformations for 80% of the targets. Using a benchmark of 61 peptide - protein targets starting from the unbound form of the protein receptor, PEP-FOLD3 was able to generate peptide poses deviating on av. by 3.3% from the exptl. conformation and return a native-like pose in the first 10 clusters for 52% of the targets.
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      Mascart-Lemone, F.; Huygen, K.; Clumeck, N.; Brenez, D.; Bolla, K.; Duchateau, J. Stimulation of Cellualr Function by Thymopentin (TP-5) in Three AIDS Patients. The Lancet; Elsevier, September 24, 1983; pp 735– 736.
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      Thymopentin treatment in AIDS and pre-AIDS patients
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      Three pilot studies testing thymopentin in AIDS patients are presented. One study included 5 patients with the full-blown syndrome, all treated with 50 mg thymopentin 3 times a week by intravenous slow infusion; no immunologically nor clinically positive results were observed, indicating that the T cell pool in such patients is severely depleted. Six other patients with the prodromal stage of AIDS were treated 1 month with 50 mg thymopentin administered as an intravenous bolus injection 3 times weekly and thereafter for another month with same dose regimen as intravenous slow infusions. The patients on infusion therapy experienced statistically significant immunological improvements; these positive findings were paralleled with an improvement of the patients' clinical condition. These positive responses persisted for an average of 8 months. In another group of 5 pre-AIDS patients thymopentin was administered via the subcutaneous route using 15 mg 3 times weekly; only 1 patient revealed immunological and clinical improvement. In summary, only patients with the pre-AIDS syndrome are likely to benefit from immunomodulation therapy with thymopentin, and the mode of administration seems to be crucial.
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    • Abstract

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      Figure 1

      Figure 1. Predicted binding mode of lopinavir to Mpro. The model was obtained by performing a cluster analysis of the MD trajectory of the docking pose, followed by a backbone-restrained geometry minimization of the main cluster using MOE.

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      Figure 2

      Figure 2. Complex between hACE2 (yellow) and SARS-CoV-2 RBD (green) from the X-ray structure (PDB code 6M0J). The hot spot residues are represented in sticks and labeled in the inset. Hot spots of cluster 1 are represented in orange, while those of cluster 2 are represented in cyan.

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      Figure 3

      Figure 3. Difference in the binding free energy between the mutated system and the native one computed on the last 10 ns of the MD simulation of the hACE2-RBD complex. All the residues for which the ΔΔG is greater than the threshold (2.5 kcal/mol) have been considered as hot spots. The hot spots of cluster 1 are highlighted in orange and those of cluster 2 in cyan.

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      Figure 4

      Figure 4. Snapshot of the MD simulation between RBD (gray) and one of the top four ligands (green). RBD BS1 hot spots are highlighted in orange. Direct and water (spheres) mediated H-bonds are also displayed as dashed lines. Additional RBD residues interacting with the ligand are displayed as sticks.

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      Figure 5

      Figure 5. Snapshot of the MD simulation between RBD (gray) and one of the top four ligands (green). RBD BS2 hot spots are highlighted in cyan. Direct and water (spheres) mediated H-bonds are also displayed as dashed lines. Additional RBD residues interacting with the ligand are displayed as sticks.

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    • References

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      This article references 83 other publications.

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        Wrapp, Daniel; Wang, Nianshuang; Corbett, Kizzmekia S.; Goldsmith, Jory A.; Hsieh, Ching-Lin; Abiona, Olubukola; Graham, Barney S.; McLellan, Jason S.
        Science (Washington, DC, United States) (2020), 367 (6483), 1260-1263CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
        The outbreak of a novel coronavirus (2019-nCoV) represents a pandemic threat that has been declared a public health emergency of international concern. The CoV spike (S) glycoprotein is a key target for vaccines, therapeutic antibodies, and diagnostics. To facilitate medical countermeasure development, we detd. a 3.5-angstrom-resoln. cryo-electron microscopy structure of the 2019-nCoV S trimer in the prefusion conformation. The predominant state of the trimer has one of the three receptor-binding domains (RBDs) rotated up in a receptor-accessible conformation. We also provide biophys. and structural evidence that the 2019-nCoV S protein binds angiotensin-converting enzyme 2 (ACE2) with higher affinity than does severe acute respiratory syndrome (SARS)-CoV S. Addnl., we tested several published SARS-CoV RBD-specific monoclonal antibodies and found that they do not have appreciable binding to 2019-nCoV S, suggesting that antibody cross-reactivity may be limited between the two RBDs. The structure of 2019-nCoV S should enable the rapid development and evaluation of medical countermeasures to address the ongoing public health crisis.
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        Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV
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        Since 2002, beta coronaviruses (CoV) have caused three zoonotic outbreaks, SARS-CoV in 2002-2003, MERS-CoV in 2012, and the newly emerged SARS-CoV-2 in late 2019. However, little is currently known about the biol. of SARS-CoV-2. Here, using SARS-CoV-2 S protein pseudovirus system, we confirm that human angiotensin converting enzyme 2 (hACE2) is the receptor for SARS-CoV-2, find that SARS-CoV-2 enters 293/hACE2 cells mainly through endocytosis, that PIKfyve, TPC2, and cathepsin L are crit. for entry, and that SARS-CoV-2 S protein is less stable than SARS-CoV S. Polyclonal anti-SARS S1 antibodies T62 inhibit entry of SARS-CoV S but not SARS-CoV-2 S pseudovirions. Further studies using recovered SARS and COVID-19 patients' sera show limited cross-neutralization, suggesting that recovery from one infection might not protect against the other. Our results present potential targets for development of drugs and vaccines for SARS-CoV-2.
        >> More from SciFinder ®
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        4
        Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor
        Lan, Jun; Ge, Jiwan; Yu, Jinfang; Shan, Sisi; Zhou, Huan; Fan, Shilong; Zhang, Qi; Shi, Xuanling; Wang, Qisheng; Zhang, Linqi; Wang, Xinquan
        Nature (London, United Kingdom) (2020), 581 (7807), 215-220CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
        Abstr.: A new and highly pathogenic coronavirus (severe acute respiratory syndrome coronavirus-2, SARS-CoV-2) caused an outbreak in Wuhan city, Hubei province, China, starting from Dec. 2019 that quickly spread nationwide and to other countries around the world1-3. Here, to better understand the initial step of infection at an at. level, we detd. the crystal structure of the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 bound to the cell receptor ACE2. The overall ACE2-binding mode of the SARS-CoV-2 RBD is nearly identical to that of the SARS-CoV RBD, which also uses ACE2 as the cell receptor4. Structural anal. identified residues in the SARS-CoV-2 RBD that are essential for ACE2 binding, the majority of which either are highly conserved or share similar side chain properties with those in the SARS-CoV RBD. Such similarity in structure and sequence strongly indicate convergent evolution between the SARS-CoV-2 and SARS-CoV RBDs for improved binding to ACE2, although SARS-CoV-2 does not cluster within SARS and SARS-related coronaviruses1-3,5. The epitopes of two SARS-CoV antibodies that target the RBD are also analyzed for binding to the SARS-CoV-2 RBD, providing insights into the future identification of cross-reactive antibodies.
        >> More from SciFinder ®
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        Shang, J.; Ye, G.; Shi, K.; Wan, Y.; Luo, C.; Aihara, H.; Geng, Q.; Auerbach, A.; Li, F. Structural Basis of Receptor Recognition by SARS-CoV-2. Nature 2020, 581, 221– 224,  DOI: 10.1038/s41586-020-2179-y
        5
        Structural basis of receptor recognition by SARS-CoV-2
        Shang, Jian; Ye, Gang; Shi, Ke; Wan, Yushun; Luo, Chuming; Aihara, Hideki; Geng, Qibin; Auerbach, Ashley; Li, Fang
        Nature (London, United Kingdom) (2020), 581 (7807), 221-224CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
        Abstr.: A novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) recently emerged and is rapidly spreading in humans, causing COVID-191,2. A key to tackling this pandemic is to understand the receptor recognition mechanism of the virus, which regulates its infectivity, pathogenesis and host range. SARS-CoV-2 and SARS-CoV recognize the same receptor-angiotensin-converting enzyme 2 (ACE2)-in humans3,4. Here we detd. the crystal structure of the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 (engineered to facilitate crystn.) in complex with ACE2. In comparison with the SARS-CoV RBD, an ACE2-binding ridge in SARS-CoV-2 RBD has a more compact conformation; moreover, several residue changes in the SARS-CoV-2 RBD stabilize two virus-binding hotspots at the RBD-ACE2 interface. These structural features of SARS-CoV-2 RBD increase its ACE2-binding affinity. Addnl., we show that RaTG13, a bat coronavirus that is closely related to SARS-CoV-2, also uses human ACE2 as its receptor. The differences among SARS-CoV-2, SARS-CoV and RaTG13 in ACE2 recognition shed light on the potential animal-to-human transmission of SARS-CoV-2. This study provides guidance for intervention strategies that target receptor recognition by SARS-CoV-2.
        >> More from SciFinder ®
        https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXoslOqtbs%253D&md5=33bc9151641b2adcfb0dbf446621a1dc
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        Hamming, I.; Timens, W.; Bulthuis, M. L. C.; Lely, A. T.; Navis, G. J.; van Goor, H.
        Journal of Pathology (2004), 203 (2), 631-637CODEN: JPTLAS; ISSN:0022-3417. (John Wiley & Sons Ltd.)
        Severe acute respiratory syndrome (SARS) is an acute infectious disease that spreads mainly via the respiratory route. A distinct coronavirus (SARS-CoV) has been identified as the etiol. agent of SARS. Recently, a metallopeptidase named angiotensin-converting enzyme 2 (ACE2) has been identified as the functional receptor for SARS-CoV. Although ACE2 mRNA is known to be present in virtually all organs, its protein expression is largely unknown. Since identifying the possible route of infection has major implications for understanding the pathogenesis and future treatment strategies for SARS, the present study investigated the localization of ACE2 protein in various human organs (oral and nasal mucosa, nasopharynx, lung, stomach, small intestine, colon, skin, lymph nodes, thymus, bone marrow, spleen, liver, kidney, and brain). The most remarkable finding was the surface expression of ACE2 protein on lung alveolar epithelial cells and enterocytes of the small intestine. Furthermore, ACE2 was present in arterial and venous endothelial cells and arterial smooth muscle cells in all organs studied. In conclusion, ACE2 is abundantly present in humans in the epithelia of the lung and small intestine, which might provide possible routes of entry for the SARS-CoV. This epithelial expression, together with the presence of ACE2 in vascular endothelium, also provides a first step in understanding the pathogenesis of the main SARS disease manifestations.
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      7. 7
        Douglas, G. C.; O’Bryan, M. K.; Hedger, M. P.; Lee, D. K. L.; Yarski, M. A.; Smith, A. I.; Lew, R. A. The Novel Angiotensin-Converting Enzyme (ACE) Homolog, ACE2, Is Selectively Expressed by Adult Leydig Cells of the Testis. Endocrinology 2004, 145, 4703– 4711,  DOI: 10.1210/en.2004-0443
        7
        The novel angiotensin-converting enzyme (ACE) homolog, ACE2, is selectively expressed by adult Leydig cells of the testis
        Douglas, Gabrielle C.; O'Bryan, Moira K.; Hedger, Mark P.; Lee, David K. L.; Yarski, Michael A.; Smith, A. Ian; Lew, Rebecca A.
        Endocrinology (2004), 145 (10), 4703-4711CODEN: ENDOAO; ISSN:0013-7227. (Endocrine Society)
        The metallopeptidase angiotensin-converting enzyme (ACE) plays a pivotal role in the cardiovascular system by generating the vasoconstrictor peptide angiotensin II. A homolog of ACE with different substrate specificity, ACE2, has recently been cloned that shows an expression pattern restricted to endothelial cells of the heart and kidney, epithelial cells of the distal tubule of the kidney, and the testis. Although the importance of ACE2 to cardiac function is already evident, its role in the testis remains unknown. In this study, we report the cloning and expression of human testicular ACE2 and confirm that it is identical to the somatic form of the enzyme. ACE2 catalytic activity was present in membrane prepns. of whole testes and Leydig cells from adult rats; expression of the protein in Leydig cells was confirmed by Western immunoblot anal. Using immunohistochem., ACE2 expression was confined to the Leydig cells in the rat testis and to Leydig and Sertoli cells in the human testis. Ablation of the Leydig cells in the rat by the specific toxin, ethane dimethane sulfonate, eliminated ACE2-pos. cells from the interstitium. Expression of ACE2 in rat Leydig cells was up-regulated during the development of adult-type Leydig cells at puberty and after ethane dimethane sulfonate treatment. Expression of ACE2 activity in the testis was not significantly altered by manipulation of the pituitary-testicular hormonal axis with s.c. testosterone implants. These data suggest that ACE2 is a constitutive product of adult-type Leydig cells and may participate in the control of testicular function by as yet unknown mechanisms.
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      8. 8
        Anand, K.; Ziebuhr, J.; Wadhwani, P.; Mesters, J. R.; Hilgenfeld, R. Coronavirus Main Proteinase (3CLpro) Structure: Basis for Design of Anti-SARS Drugs. Science (Washington, DC, U. S.) 2003, 300, 1763– 1767,  DOI: 10.1126/science.1085658
        8
        Coronavirus Main Proteinase (3CLpro) Structure: Basis for Design of Anti-SARS Drugs
        Anand, Kanchan; Ziebuhr, John; Wadhwani, Parvesh; Mesters, Jeroen R.; Hilgenfeld, Rolf
        Science (Washington, DC, United States) (2003), 300 (5626), 1763-1767CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
        A novel coronavirus has been identified as the causative agent of severe acute respiratory syndrome (SARS). The viral main proteinase (Mpro, also called 3CLpro), which controls the activities of the coronavirus replication complex, is an attractive target for therapy. The authors detd. crystal structures for human coronavirus (strain 229E) Mpro and for an inhibitor complex of porcine coronavirus [transmissible gastroenteritis virus (TGEV)] Mpro, and the authors constructed a homol. model for SARS coronavirus (SARS-CoV) Mpro. The structures reveal a remarkable degree of conservation of the substrate-binding sites, which is further supported by recombinant SARS-CoV Mpro-mediated cleavage of a TGEV Mpro substrate. Mol. modeling suggests that available rhinovirus 3Cpro inhibitors may be modified to make them useful for treating SARS.
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      9. 9
        Zhang, L.; Lin, D.; Sun, X.; Curth, U.; Drosten, C.; Sauerhering, L.; Becker, S.; Rox, K.; Hilgenfeld, R. Crystal Structure of SARS-CoV-2 Main Protease Provides a Basis for Design of Improved a-Ketoamide Inhibitors. Science (Washington, DC, U. S.) 2020, 368, 409– 412,  DOI: 10.1126/science.abb3405
        9
        Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors
        Zhang, Linlin; Lin, Daizong; Sun, Xinyuanyuan; Curth, Ute; Drosten, Christian; Sauerhering, Lucie; Becker, Stephan; Rox, Katharina; Hilgenfeld, Rolf
        Science (Washington, DC, United States) (2020), 368 (6489), 409-412CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
        The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is a global health emergency. An attractive drug target among coronaviruses is the main protease (Mpro, also called 3CLpro) because of its essential role in processing the polyproteins that are translated from the viral RNA. We report the x-ray structures of the unliganded SARS-CoV-2 Mpro and its complex with an α-ketoamide inhibitor. This was derived from a previously designed inhibitor but with the P3-P2 amide bond incorporated into a pyridone ring to enhance the half-life of the compd. in plasma. On the basis of the unliganded structure, we developed the lead compd. into a potent inhibitor of the SARS-CoV-2 Mpro. The pharmacokinetic characterization of the optimized inhibitor reveals a pronounced lung tropism and suitability for administration by the inhalative route.
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      10. 10
        Hilgenfeld, R. From SARS to MERS: Crystallographic Studies on Coronaviral Proteases Enable Antiviral Drug Design. FEBS J. 2014, 281, 4085– 4096,  DOI: 10.1111/febs.12936
        10
        From SARS to MERS: crystallographic studies on coronaviral proteases enable antiviral drug design
        Hilgenfeld, Rolf
        FEBS Journal (2014), 281 (18), 4085-4096CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)
        A review. Here, the author focuses on the important contributions that macromol. crystallog. has made over the past 12 yr to elucidating structures and mechanisms of the essential proteases of coronaviruses, the main protease (Mpro) and the papain-like protease (PLpro). The role of x-ray crystallog. in structure-assisted drug discovery against these targets is discussed. Aspects dealt with in this review include the emergence of the SARS coronavirus in 2002-2003 and of the MERS coronavirus 10 yr later and the origins of these viruses. The crystal structure of the free SARS coronavirus Mpro and its dependence on pH is discussed, as are efforts to design inhibitors on the basis of these structures. The mechanism of maturation of the enzyme from the viral polyprotein is still a matter of debate. The crystal structure of the SARS coronavirus PLpro and its complex with ubiquitin is also discussed, as is its ortholog from MERS coronavirus. Efforts at predictive structure-based inhibitor development for bat coronavirus Mpros to increase the preparedness against zoonotic transmission to man are described as well. The paper closes with a brief discussion of structure-based discovery of antivirals in an academic setting.
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      11. 11
        Jin, Z.; Du, X.; Xu, Y.; Deng, Y.; Liu, M.; Zhao, Y.; Zhang, B.; Li, X.; Zhang, L.; Peng, C.; Duan, Y.; Yu, J.; Wang, L.; Yang, K.; Liu, F.; Jiang, R.; Yang, X.; You, T.; Liu, X.; Yang, X.; Bai, F.; Liu, H.; Liu, X.; Guddat, L. W.; Xu, W.; Xiao, G.; Qin, C.; Shi, Z.; Jiang, H.; Rao, Z.; Yang, H. Structure of Mpro from SARS-CoV-2 and Discovery of Its Inhibitors. Nature 2020, 582, 289– 293,  DOI: 10.1038/s41586-020-2223-y
        11
        Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors
        Jin, Zhenming; Du, Xiaoyu; Xu, Yechun; Deng, Yongqiang; Liu, Meiqin; Zhao, Yao; Zhang, Bing; Li, Xiaofeng; Zhang, Leike; Peng, Chao; Duan, Yinkai; Yu, Jing; Wang, Lin; Yang, Kailin; Liu, Fengjiang; Jiang, Rendi; Yang, Xinglou; You, Tian; Liu, Xiaoce; Yang, Xiuna; Bai, Fang; Liu, Hong; Liu, Xiang; Guddat, Luke W.; Xu, Wenqing; Xiao, Gengfu; Qin, Chengfeng; Shi, Zhengli; Jiang, Hualiang; Rao, Zihe; Yang, Haitao
        Nature (London, United Kingdom) (2020), 582 (7811), 289-293CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
        Abstr.: A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the etiol. agent responsible for the 2019-2020 viral pneumonia outbreak of coronavirus disease 2019 (COVID-19). Currently, there are no targeted therapeutic agents for the treatment of this disease, and effective treatment options remain very limited. Here, we describe the results of a program that aimed to rapidly discover lead compds. for clin. use, by combining structure-assisted drug design, virtual drug screening and high-throughput screening. This program focused on identifying drug leads that target main protease (Mpro) of SARS-CoV-2: Mpro is a key enzyme of coronaviruses and has a pivotal role in mediating viral replication and transcription, making it an attractive drug target for SARS-CoV-2. We identified a mechanism-based inhibitor (N3) by computer-aided drug design, and then detd. the crystal structure of Mpro of SARS-CoV-2 in complex with this compd. Through a combination of structure-based virtual and high-throughput screening, we assayed more than 10,000 compds.-including approved drugs, drug candidates in clin. trials and other pharmacol. active compds.-as inhibitors of Mpro. Six of these compds. inhibited Mpro, showing half-maximal inhibitory concn. values that ranged from 0.67 to 21.4μM. One of these compds. (ebselen) also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of our screening strategy, which can lead to the rapid discovery of drug leads with clin. potential in response to new infectious diseases for which no specific drugs or vaccines are available.
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      12. 12
        Roy, A.; Kucukural, A.; Zhang, Y. I-TASSER: A Unified Platform for Automated Protein Structure and Function Prediction. Nat. Protoc. 2010, 5, 725– 738,  DOI: 10.1038/nprot.2010.5
        12
        I-TASSER: a unified platform for automated protein structure and function prediction
        Roy, Ambrish; Kucukural, Alper; Zhang, Yang
        Nature Protocols (2010), 5 (4), 725-738CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)
        The iterative threading assembly refinement (I-TASSER) server is an integrated platform for automated protein structure and function prediction based on the sequence-to-structure-to-function paradigm. Starting from an amino acid sequence, I-TASSER first generates three-dimensional (3D) at. models from multiple threading alignments and iterative structural assembly simulations. The function of the protein is then inferred by structurally matching the 3D models with other known proteins. The output from a typical server run contains full-length secondary and tertiary structure predictions, and functional annotations on ligand-binding sites, Enzyme Commission nos. and Gene Ontol. terms. An est. of accuracy of the predictions is provided based on the confidence score of the modeling. This protocol provides new insights and guidelines for designing of online server systems for the state-of-the-art protein structure and function predictions. The server is available at http://zhanglab.ccmb.med.umich.edu/I-TASSER.
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      13. 13
        Zhang, C.; Zheng, W.; Huang, X.; Bell, E. W.; Zhou, X.; Zhang, Y. Protein Structure and Sequence Reanalysis of 2019-NCoV Genome Refutes Snakes as Its Intermediate Host and the Unique Similarity between Its Spike Protein Insertions and HIV-1. J. Proteome Res. 2020, 19, 1351– 1360,  DOI: 10.1021/acs.jproteome.0c00129
        13
        Protein Structure and Sequence Reanalysis of 2019-nCoV Genome Refutes Snakes as Its Intermediate Host and the Unique Similarity between Its Spike Protein Insertions and HIV-1
        Zhang, Chengxin; Zheng, Wei; Huang, Xiaoqiang; Bell, Eric W.; Zhou, Xiaogen; Zhang, Yang
        Journal of Proteome Research (2020), 19 (4), 1351-1360CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
        As the infection of 2019-nCoV coronavirus is quickly developing into a global pneumonia epidemic, the careful anal. of its transmission and cellular mechanisms is sorely needed. In this Communication, we first analyzed two recent studies that concluded that snakes are the intermediate hosts of 2019-nCoV and that the 2019-nCoV spike protein insertions share a unique similarity to HIV-1. However, the reimplementation of the analyses, built on larger scale data sets using state-of-the-art bioinformatics methods and databases, presents clear evidence that rebuts these conclusions. Next, using metagenomic samples from Manis javanica, we assembled a draft genome of the 2019-nCoV-like coronavirus, which shows 73% coverage and 91% sequence identity to the 2019-nCoV genome. In particular, the alignments of the spike surface glycoprotein receptor-binding domain revealed four times more variations in the bat coronavirus RaTG13 than in the Manis coronavirus compared with 2019-nCoV, suggesting the pangolin as a missing link in the transmission of 2019-nCoV from bats to human.
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      14. 14
        Wu, F.; Zhao, S.; Yu, B.; Chen, Y. M.; Wang, W.; Song, Z. G.; Hu, Y.; Tao, Z. W.; Tian, J. H.; Pei, Y. Y.; Yuan, M. L.; Zhang, Y. L.; Dai, F. H.; Liu, Y.; Wang, Q. M.; Zheng, J. J.; Xu, L.; Holmes, E. C.; Zhang, Y. Z. A New Coronavirus Associated with Human Respiratory Disease in China. Nature 2020, 579, 265– 269,  DOI: 10.1038/s41586-020-2008-3
        14
        A new coronavirus associated with human respiratory disease in China
        Wu, Fan; Zhao, Su; Yu, Bin; Chen, Yan-Mei; Wang, Wen; Song, Zhi-Gang; Hu, Yi; Tao, Zhao-Wu; Tian, Jun-Hua; Pei, Yuan-Yuan; Yuan, Ming-Li; Zhang, Yu-Ling; Dai, Fa-Hui; Liu, Yi; Wang, Qi-Min; Zheng, Jiao-Jiao; Xu, Lin; Holmes, Edward C.; Zhang, Yong-Zhen
        Nature (London, United Kingdom) (2020), 579 (7798), 265-269CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
        Emerging infectious diseases, such as severe acute respiratory syndrome (SARS) and Zika virus disease, present a major threat to public health. Despite intense research efforts, how, when and where new diseases appear are still a source of considerable uncertainty. A severe respiratory disease was recently reported in Wuhan, Hubei province, China. As of 25 Jan. 2020, at least 1,975 cases had been reported since the first patient was hospitalized on 12 Dec. 2019. Epidemiol. investigations have suggested that the outbreak was assocd. with a seafood market in Wuhan. Here we study a single patient who was a worker at the market and who was admitted to the Central Hospital of Wuhan on 26 Dec. 2019 while experiencing a severe respiratory syndrome that included fever, dizziness and a cough. Metagenomic RNA sequencing of a sample of bronchoalveolar lavage fluid from the patient identified a new RNA virus strain from the family Coronaviridae, which is designated here 'WH-Human 1' coronavirus (and has also been referred to as '2019-nCoV'). Phylogenetic anal. of the complete viral genome (29,903 nucleotides) revealed that the virus was most closely related (89.1% nucleotide similarity) to a group of SARS-like coronaviruses (genus Betacoronavirus, subgenus Sarbecovirus) that had previously been found in bats in China. This outbreak highlights the ongoing ability of viral spill-over from animals to cause severe disease in humans.
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      15. 15
        Patick, A. K.; Potts, K. E. Protease Inhibitors as Antiviral Agents. Clin. Microbiol. Rev. 1998, 11, 614,  DOI: 10.1128/CMR.11.4.614
        15
        Protease inhibitors as antiviral agents
        Patick, A. K.; Potts, K. E.
        Clinical Microbiology Reviews (1998), 11 (4), 614-627CODEN: CMIREX; ISSN:0893-8512. (American Society for Microbiology)
        A review with 169 refs.
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      16. 16
        Cavasotto, C. N.; Orry, A. J. W. Ligand Docking and Structure-Based Virtual Screening in Drug Discovery. Curr. Top. Med. Chem. 2007, 7, 1006– 1014,  DOI: 10.2174/156802607780906753
        16
        Ligand docking and structure-based virtual screening in drug discovery
        Cavasotto, Claudio N.; Orry, Andrew J. W.
        Current Topics in Medicinal Chemistry (Sharjah, United Arab Emirates) (2007), 7 (10), 1006-1014CODEN: CTMCCL; ISSN:1568-0266. (Bentham Science Publishers Ltd.)
        A review. Ligand-docking-based methods are starting to play a crit. role in lead discovery and optimization, thus resulting in new 'drug-candidates'. They offer the possibility to go beyond the pool of existing active compds., and thus find novel chemotypes. A brief turorial on ligand docking and structure-based virtual screening is presented highlighting current problems and limitations, together with the most recent methodol. and algorithmic developments in the field. Recent successful applications of docking-based tools for hit doscovery, lead optimization and target-biased library design are also presented. Special consideration is devoted to ongoing efforts to account for protein flexibility in structure-based virtual screening.
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      17. 17
        Clark, D. E. What Has Virtual Screening Ever Done for Drug Discovery?. Expert Opin. Drug Discovery 2008, 3, 841– 851,  DOI: 10.1517/17460441.3.8.841
        17
        What has virtual screening ever done for drug discovery?
        Clark, David E.
        Expert Opinion on Drug Discovery (2008), 3 (8), 841-851CODEN: EODDBX; ISSN:1746-0441. (Informa Healthcare)
        A review. Background: Although virtual screening is now widely applied as a hit-finding methodol. within drug discovery programs, there are relatively few reports of its contributing to compds. on the market or in the clinic. Objective: To assess the impact of virtual screening on drug discovery. Method: Such cases as can be found in the public domain at the current time are reviewed. Addnl., some of the current challenges in structure- and ligand-based virtual screening are discussed. Conclusion: It is concluded that virtual screening has contributed to the discovery of several compds. that have either reached the market or entered clin. trials. In terms of praxis, there is 'no free lunch' in virtual screening and as many methods as possible should be applied to maximize the likelihood of success.
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      18. 18
        Choudhary, S.; Malik, Y. S.; Tomar, S. Identification of SARS-CoV-2 Cell Entry Inhibitors by Drug Repurposing Using in Silico Structure-Based Virtual Screening Approach. Front. Immunol. 2020, 11, 1664,  DOI: 10.3389/fimmu.2020.01664
        18
        Identification of SARS-CoV-2 cell entry inhibitors by drug repurposing using in silico structure-based virtual screening approach
        Choudhary, Shweta; Malik, Yashpal S.; Tomar, Shailly
        Frontiers in Immunology (2020), 11 (), 1664CODEN: FIRMCW; ISSN:1664-3224. (Frontiers Media S.A.)
        The rapidly spreading, highly contagious and pathogenic SARS-coronavirus 2 (SARS-CoV-2) assocd. Coronavirus Disease 2019 (COVID-19) has been declared as a pandemic by the World Health Organization (WHO). The novel 2019 SARS-CoV-2 enters the host cell by binding of the viral surface spike glycoprotein (S-protein) to cellular angiotensin converting enzyme 2 (ACE2) receptor. The virus specific mol. interaction with the host cell represents a promising therapeutic target for identifying SARS-CoV-2 antiviral drugs. The repurposing of drugs can provide a rapid and potential cure toward exponentially expanding COVID-19. Thereto, high throughput virtual screening approach was used to investigate FDA approved LOPAC library drugs against both the receptor binding domain of spike protein (S-RBD) and ACE2 host cell receptor. Primary screening identified a few promising mols. for both the targets, which were further analyzed in details by their binding energy, binding modes through mol. docking, dynamics and simulations. Evidently, GR 127935 hydrochloride hydrate, GNF-5, RS504393, TNP, and eptifibatide acetate were found binding to virus binding motifs of ACE2 receptor. Addnl., KT203, BMS195614, KT185, RS504393, and GSK1838705A were identified to bind at the receptor binding site on the viral S-protein. These identified mols. may effectively assist in controlling the rapid spread of SARS-CoV-2 by not only potentially inhibiting the virus at entry step but are also hypothesized to act as anti-inflammatory agents, which could impart relief in lung inflammation. Timely identification and detn. of an effective drug to combat and tranquilize the COVID-19 global crisis is the utmost need of hour. Further, prompt in vivo testing to validate the anti-SARS-CoV-2 inhibition efficiency by these mols. could save lives is justified.
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      19. 19
        Chen, Y. W.; Yiu, C. P. B.; Wong, K. Y. Prediction of the SARS-CoV-2 (2019-NCoV) 3C-like Protease (3CLpro) Structure: Virtual Screening Reveals Velpatasvir, Ledipasvir, and Other Drug Repurposing Candidates. F1000Research 2020, 9, 9,  DOI: 10.12688/f1000research.22457.2
        There is no corresponding record for this reference.
      20. 20
        Maffucci, I.; Hu, X.; Fumagalli, V.; Contini, A. An Efficient Implementation of the Nwat-MMGBSA Method to Rescore Docking Results in Medium-Throughput Virtual Screenings. Front. Chem. 2018, 6, 43,  DOI: 10.3389/fchem.2018.00043
        20
        An efficient implementation of the nwat-mmgbsa method to rescore docking results in medium-throughput virtual screenings
        Maffucci, Irene; Hu, Xiao; Fumagalli, Valentina; Contini, Alessandro
        Frontiers in Chemistry (Lausanne, Switzerland) (2018), 6 (), 43/1-43/14CODEN: FCLSAA; ISSN:2296-2646. (Frontiers Media S.A.)
        Nwat-MMGBSA is a variant of MM-PB/GBSA based on the inclusion of a no. of explicit water mols. that are the closest to the ligand in each frame of a mol. dynamics trajectory. This method demonstrated improved correlations between calcd. and exptl. binding energies in both protein-protein interactions and ligand-receptor complexes, in comparison to the std. MM-GBSA. A protocol optimization, aimed to maximize efficacy and efficiency, is discussed here considering penicillopepsin, HIV1-protease, and BCL-XL as test cases. Calcns. were performed in triplicates on both classic HPC environments and on std. workstations equipped by a GPU card, evidencing no statistical differences in the results. No relevant differences in correlation to expts. were also obsd. when performing Nwat-MMGBSA calcns. on 4 or 1 ns long trajectories. A fully automatic workflow for structure-based virtual screening, performing from library set-up to docking and Nwat-MMGBSA rescoring, has then been developed. The protocol has been tested against no rescoring or std. MM-GBSA rescoring within a retrospective virtual screening of inhibitors of AmpC β-lactamase and of the Rac1-Tiam1 protein-protein interaction. In both cases, Nwat-MMGBSA rescoring provided a statistically significant increase in the ROC AUCs of between 20 and 30%, compared to docking scoring or to std. MM-GBSA rescoring.
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      21. 21
        Amaro, R. E.; Baudry, J.; Chodera, J.; Demir, Ö.; McCammon, J. A.; Miao, Y.; Smith, J. C. Ensemble Docking in Drug Discovery. Biophys. J. Biophysical Society, May 22, 2018; pp 2271– 2278.
        There is no corresponding record for this reference.
      22. 22
        Molecular Operating Environment (MOE), 2019.0102; Chemical Computing Group Inc.: Montreal, 2019.
        There is no corresponding record for this reference.
      23. 23
        Turlington, M.; Chun, A.; Tomar, S.; Eggler, A.; Grum-Tokars, V.; Jacobs, J.; Daniels, J. S.; Dawson, E.; Saldanha, A.; Chase, P.; Baez-Santos, Y. M.; Lindsley, C. W.; Hodder, P.; Mesecar, A. D.; Stauffer, S. R. Discovery of N-(Benzo[1,2,3]Triazol-1-Yl)-N-(Benzyl)Acetamido)Phenyl) Carboxamides as Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) 3CLpro Inhibitors: Identification of ML300 and Noncovalent Nanomolar Inhibitors with an Induced-Fit Binding. Bioorg. Med. Chem. Lett. 2013, 23, 6172– 6177,  DOI: 10.1016/j.bmcl.2013.08.112
        23
        Discovery of N-(benzo[1,2,3]triazol-1-yl)-N-((benzyl)acetamido)phenyl carboxamides as severe acute respiratory syndrome coronavirus (SARS-CoV) 3CLpro inhibitors: Identification of ML300 and noncovalent nanomolar inhibitors with an induced-fit binding
        Turlington, Mark; Chun, Aspen; Tomar, Sakshi; Eggler, Aimee; Grum-Tokars, Valerie; Jacobs, Jon; Daniels, J. Scott; Dawson, Eric; Saldanha, Adrian; Chase, Peter; Baez-Santos, Yahira M.; Lindsley, Craig W.; Hodder, Peter; Mesecar, Andrew D.; Stauffer, Shaun R.
        Bioorganic & Medicinal Chemistry Letters (2013), 23 (22), 6172-6177CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)
        Herein we report the discovery and SAR of a novel series of SARS-CoV 3CLpro inhibitors identified through the NIH Mol. Libraries Probe Prodn. Centers Network (MLPCN). In addn. to ML188, ML300 represents the second probe declared for 3CLpro from this collaborative effort. The x-ray structure of SARS-CoV 3CLpro bound with a ML300 analog highlights a unique induced-fit reorganization of the S2-S4 binding pockets leading to the first sub-micromolar noncovalent 3CLpro inhibitors retaining a single amide bond.
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      24. 24
        Contini, A. Virtual Screening of an FDA Approved Drugs Database on Two COVID-19 Coronavirus Proteins. ChemRxiv Preprint. 2020,  DOI: 10.26434/Chemrxiv.11847381.V1 .
        There is no corresponding record for this reference.
      25. 25
        Anandakrishnan, R.; Aguilar, B.; Onufriev, A. V. H++ 3.0: Automating PK Prediction and the Preparation of Biomolecular Structures for Atomistic Molecular Modeling and Simulations. Nucleic Acids Res. 2012, 40, W537– 541,  DOI: 10.1093/nar/gks375
        25
        H++ 3.0: automating pK prediction and the preparation of biomolecular structures for atomistic molecular modeling and simulations
        Anandakrishnan, Ramu; Aguilar, Boris; Onufriev, Alexey V.
        Nucleic Acids Research (2012), 40 (W1), W537-W541CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
        The accuracy of atomistic biomol. modeling and simulation studies depend on the accuracy of the input structures. Prepg. these structures for an atomistic modeling task, such as mol. dynamics (MD) simulation, can involve the use of a variety of different tools for: correcting errors, adding missing atoms, filling valences with hydrogens, predicting pK values for titratable amino acids, assigning predefined partial charges and radii to all atoms, and generating force field parameter/topol. files for MD. Identifying, installing and effectively using the appropriate tools for each of these tasks can be difficult for novice and time-consuming for experienced users. H++ (http://biophysics.cs.vt.edu/) is a free open-source web server that automates the above key steps in the prepn. of biomol. structures for mol. modeling and simulations. H++ also performs extensive error and consistency checking, providing error/warning messages together with the suggested corrections. In addn. to numerous minor improvements, the latest version of H++ includes several new capabilities and options: fix erroneous (flipped) side chain conformations for HIS, GLN and ASN, include a ligand in the input structure, process nucleic acid structures and generate a solvent box with specified no. of common ions for explicit solvent MD.
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      26. 26
        Case, D. A.; Ben-Shalom, I. Y.; Brozell, S. R.; Cerutti, D. S.; Cheatham, T. E., III, Cruzeiro, V. W. D.; Duke, T. A. D. R. E.; Ghoreishi, D.; Gilson, M.K.; Gohlke, H.; Goetz, A. W.; Greene, D.; Harris, R.; Homeyer, N.; Izadi, Y. H. S.; Kovalenko, A.; Kurtzman, T.; Lee, T. S.; LeGrand, S.; Li, P.; Lin, C.; Liu, J.; Luchko, T.; Luo, R.; D. J., Mermelstein, Merz, K. M.; Miao, Y.; Monard, G.; Nguyen, C.; Nguyen, H.; Omelyan, I.; Onufriev, A.; Pan, F.; R., Qi, Roe, D. R.; Roitberg, A.; Sagui, C.; Schott-Verdugo, S.; Shen, J.; Simmerling, C. L.; Smith, J.; SalomonFerrer, R.; Swails, J.; Walker, R. C.; Wang, J.; Wei, H.; Wolf, R. M.; Wu, X.; Xiao, L.; D. M, Y.; P. A, K. AMBER 2018; University of California, San Francisco, 2018.
        There is no corresponding record for this reference.
      27. 27
        Maier, J. A.; Martinez, C.; Kasavajhala, K.; Wickstrom, L.; Hauser, K. E.; Simmerling, C. Ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from Ff99SB. J. Chem. Theory Comput. 2015, 11, 3696– 3713,  DOI: 10.1021/acs.jctc.5b00255
        27
        ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB
        Maier, James A.; Martinez, Carmenza; Kasavajhala, Koushik; Wickstrom, Lauren; Hauser, Kevin E.; Simmerling, Carlos
        Journal of Chemical Theory and Computation (2015), 11 (8), 3696-3713CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
        Mol. mechanics is powerful for its speed in atomistic simulations, but an accurate force field is required. The Amber ff99SB force field improved protein secondary structure balance and dynamics from earlier force fields like ff99, but weaknesses in side chain rotamer and backbone secondary structure preferences have been identified. Here, we performed a complete refit of all amino acid side chain dihedral parameters, which had been carried over from ff94. The training set of conformations included multidimensional dihedral scans designed to improve transferability of the parameters. Improvement in all amino acids was obtained as compared to ff99SB. Parameters were also generated for alternate protonation states of ionizable side chains. Av. errors in relative energies of pairs of conformations were under 1.0 kcal/mol as compared to QM, reduced 35% from ff99SB. We also took the opportunity to make empirical adjustments to the protein backbone dihedral parameters as compared to ff99SB. Multiple small adjustments of φ and ψ parameters were tested against NMR scalar coupling data and secondary structure content for short peptides. The best results were obtained from a phys. motivated adjustment to the φ rotational profile that compensates for lack of ff99SB QM training data in the β-ppII transition region. Together, these backbone and side chain modifications (hereafter called ff14SB) not only better reproduced their benchmarks, but also improved secondary structure content in small peptides and reprodn. of NMR χ1 scalar coupling measurements for proteins in soln. We also discuss the Amber ff12SB parameter set, a preliminary version of ff14SB that includes most of its improvements.
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      28. 28
        Ryckaert, J. P.; Ciccotti, G.; Berendsen, H. J. C. Numerical Integration of the Cartesian Equations of Motion of a System with Constraints: Molecular Dynamics of n-Alkanes. J. Comput. Phys. 1977, 23, 327– 341,  DOI: 10.1016/0021-9991(77)90098-5
        28
        Numerical integration of the Cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes
        Ryckaert, Jean Paul; Ciccotti, Giovanni; Berendsen, Herman J. C.
        Journal of Computational Physics (1977), 23 (3), 327-41CODEN: JCTPAH; ISSN:0021-9991.
        A numerical algorithm integrating the 3N Cartesian equation of motion of a system of N points subject to holonomic constraints is applied to mol. dynamics simulation of a liq. of 64 butane mols.
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      29. 29
        Schrödinger, L. PyMOL Molecular Graphics System , Version 1.8; 2015.
        There is no corresponding record for this reference.
      30. 30
        Nguyen, H.; Roe, D. R.; Simmerling, C. Improved Generalized Born Solvent Model Parameters for Protein Simulations. J. Chem. Theory Comput. 2013, 9, 2020– 2034,  DOI: 10.1021/ct3010485
        30
        Improved Generalized Born Solvent Model Parameters for Protein Simulations
        Nguyen, Hai; Roe, Daniel R.; Simmerling, Carlos
        Journal of Chemical Theory and Computation (2013), 9 (4), 2020-2034CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
        The generalized Born (GB) model is one of the fastest implicit solvent models, and it has become widely adopted for Mol. Dynamics (MD) simulations. This speed comes with trade-offs, and many reports in the literature have pointed out weaknesses with GB models. Because the quality of a GB model is heavily affected by empirical parameters used in calcg. solvation energy, in this work we have refit these parameters for GB-Neck, a recently developed GB model, in order to improve the accuracy of both the solvation energy and effective radii calcns. The data sets used for fitting are significantly larger than those used in the past. Comparing to other pairwise GB models like GB-OBC and the original GB-Neck, the new GB model (GB-Neck2) has better agreement with Poisson-Boltzmann (PB) in terms of reproducing solvation energies for a variety of systems ranging from peptides to proteins. Secondary structure preferences are also in much better agreement with those obtained from explicit solvent MD simulations. We also obtain near-quant. reprodn. of exptl. structure and thermal stability profiles for several model peptides with varying secondary structure motifs. Extension to nonprotein systems will be explored in the future.
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      31. 31
        Compound Libraries for High Throughput/Content Screening | 96-Well. https://www.selleckchem.com/screening/fda-approved-drug-library.html (accessed May 27, 2020).
        There is no corresponding record for this reference.
      32. 32
        FDA-approved Drug Library|Targetmol|96-well. https://www.targetmol.com/compound-library/FDA-approved-Drug-Library (accessed May 27, 2020).
        There is no corresponding record for this reference.
      33. 33
        Korb, O.; Möller, H. M.; Exner, T. E. NMR-Guided Molecular Docking of a Protein-Peptide Complex Based on Ant Colony Optimization. ChemMedChem 2010, 5, 1001– 1006,  DOI: 10.1002/cmdc.201000090
        33
        NMR-Guided Molecular Docking of a Protein-Peptide Complex Based on Ant Colony Optimization
        Korb, Oliver; Moeller, Heiko M.; Exner, Thomas E.
        ChemMedChem (2010), 5 (7), 1001-1006CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)
        Std. docking approaches used for the prediction of protein-ligand complexes in the drug development process have problems identifying the correct binding mode of large flexible ligands. Herein the authors show how addnl. exptl. data from NMR expts. can be used to predict the binding mode of a mucin 1 (MUC-1) pentapeptide recognized by the breast-cancer-selective monoclonal antibody SM3. Distance constraints derived from trNOE and satn. transfer difference NMR expts. are combined with the docking approach PLANTS. The resulting complex structures show excellent agreement with the NMR data and with a published X-ray crystal structure. The method was then further tested on two complexes in order to demonstrate its more general applicability: T-antigen disaccharide bound to Maclura pomifera agglutinin, and the inhibitor SBi279 bound to S100B protein. The authors' new approach has the advantages of being fully automatic, rapid, and unbiased; moreover, it is based on relatively easily obtainable exptl. data and can greatly increase the reliability of the generated structures.
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      34. 34
        Maffucci, I.; Contini, A. Explicit Ligand Hydration Shells Improve the Correlation between MM-PB/GBSA Binding Energies and Experimental Activities. J. Chem. Theory Comput. 2013, 9, 2706– 2717,  DOI: 10.1021/ct400045d
        34
        Explicit Ligand Hydration Shells Improve the Correlation between MM-PB/GBSA Binding Energies and Experimental Activities
        Maffucci, Irene; Contini, Alessandro
        Journal of Chemical Theory and Computation (2013), 9 (6), 2706-2717CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
        Mol. Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) and Mol. Mechanics Generalized Born Surface Area (MM-GBSA) methods are widely used for drug design/discovery purposes. However, it is not clear if the correlation between predicted and exptl. binding affinities can be improved by explicitly considering selected water mols. in the calcn. of binding energies, since different and sometimes diverging opinions are found in the literature. The authors evaluated how variably populated hydration shells explicitly considered around the ligands may affect the correlation between MM-PB/GBSA computed binding energy and biol. activities (IC50 and ΔGbind, depending on the available exptl. data). Four different systems--namely, the DNA-topoisomerase complex, α-thrombin, penicillopepsin, and avidin--were considered and ligand hydration shells populated by 10-70 water mols. were systematically evaluated. The consideration of a hydration shell populated by a no. of water residues (Nwat) between 30 and 70 provided, in all of the considered examples, a pos. effect on correlation between MM-PB/GBSA calcd. binding affinities and exptl. activities, with a negligible increment of computational cost.
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      35. 35
        Maffucci, I.; Contini, A. Improved Computation of Protein-Protein Relative Binding Energies with the Nwat-MMGBSA Method. J. Chem. Inf. Model. 2016, 56, 1692– 1704,  DOI: 10.1021/acs.jcim.6b00196
        35
        Improved Computation of Protein-Protein Relative Binding Energies with the Nwat-MMGBSA Method
        Maffucci, Irene; Contini, Alessandro
        Journal of Chemical Information and Modeling (2016), 56 (9), 1692-1704CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)
        A MMGBSA variant (here referred to as Nwat-MMGBSA), based on the inclusion of a certain no. of explicit water mols. (Nwat) during the calcns., has been tested on a set of 20 protein-protein complexes, using the correlation between predicted and exptl. binding energy as the evaluation metric. Beside the Nwat parameter, the effect of the force field, the mol. dynamics simulation length, and the implicit solvent model used in the MMGBSA anal. have been also evaluated. Considering 30 interfacial water mols. improved the correlation between predicted and exptl. binding energies by up to 30%, compared to the std. approach. Moreover, the correlation resulted rather sensitively to the force field and, to a minor extent, to the implicit solvent model, and to the length of the MD simulation.
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      36. 36
        Korb, O.; Stützle, T.; Exner, T. E. Empirical Scoring Functions for Advanced Protein-Ligand Docking with PLANTS. J. Chem. Inf. Model. 2009, 49, 84– 96,  DOI: 10.1021/ci800298z
        36
        Empirical Scoring Functions for Advanced Protein-Ligand Docking with PLANTS
        Korb, Oliver; Stuetzle, Thomas; Exner, Thomas E.
        Journal of Chemical Information and Modeling (2009), 49 (1), 84-96CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)
        In this paper we present two empirical scoring functions, PLANTSCHEMPLP and PLANTSPLP, designed for our docking algorithm PLANTS (Protein-Ligand ANT System), which is based on ant colony optimization (ACO). They are related, regarding their functional form, to parts of already published scoring functions and force fields. The parametrization procedure described here was able to identify several parameter settings showing an excellent performance for the task of pose prediction on two test sets comprising 298 complexes in total. Up to 87% of the complexes of the Astex diverse set and 77% of the CCDC/Astex clean listnc (noncovalently bound complexes of the clean list) could be reproduced with root-mean-square deviations of less than 2 Å with respect to the exptl. detd. structures. A comparison with the state-of-the-art docking tool GOLD clearly shows that this is, esp. for the druglike Astex diverse set, an improvement in pose prediction performance. Addnl., optimized parameter settings for the search algorithm were identified, which can be used to balance pose prediction reliability and search speed.
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      37. 37
        Massova, I.; Kollman, P. A. Combined Molecular Mechanical and Continuum Solvent Approach (MM- PBSA/GBSA) to Predict Ligand Binding. Perspectives in Drug Discovery and Design; Springer, 2000; pp 113– 135.
        There is no corresponding record for this reference.
      38. 38
        Choy, K.-T.; Wong, A. Y.-L.; Kaewpreedee, P.; Sia, S. F.; Chen, D.; Hui, K. P. Y.; Chu, D. K. W.; Chan, M. C. W.; Cheung, P. P.-H.; Huang, X.; Peiris, M.; Yen, H.-L. Remdesivir, Lopinavir, Emetine, and Homoharringtonine Inhibit SARS-CoV-2 Replication in Vitro. Antiviral Res. 2020, 178, 104786,  DOI: 10.1016/j.antiviral.2020.104786
        38
        Remdesivir, lopinavir, emetine, and homoharringtonine inhibit SARS-CoV-2 replication in vitro
        Choy, Ka-Tim; Wong, Alvina Yin-Lam; Kaewpreedee, Prathanporn; Sia, Sin Fun; Chen, Dongdong; Hui, Kenrie Pui Yan; Chu, Daniel Ka Wing; Chan, Michael Chi Wai; Cheung, Peter Pak-Hang; Huang, Xuhui; Peiris, Malik; Yen, Hui-Ling
        Antiviral Research (2020), 178 (), 104786CODEN: ARSRDR; ISSN:0166-3542. (Elsevier B.V.)
        An escalating pandemic by the novel SARS-CoV-2 virus is impacting global health and effective therapeutic options are urgently needed. We evaluated the in vitro antiviral effect of compds. that were previously reported to inhibit coronavirus replication and compds. that are currently under evaluation in clin. trials for SARS-CoV-2 patients. We report the antiviral effect of remdesivir, lopinavir, homorringtonine, and emetine against SARS-CoV-2 virus in Vero E6 cells with the estd. 50% effective concn. at 23.15μM, 26.63μM, 2.55μM and 0.46μM, resp. Ribavirin or favipiravir that are currently evaluated under clin. trials showed no inhibition at 100μM. Synergy between remdesivir and emetine was obsd., and remdesivir at 6.25μM in combination with emetine at 0.195μM may achieve 64.9% inhibition in viral yield. Combinational therapy may help to reduce the effective concn. of compds. below the therapeutic plasma concns. and provide better clin. benefits.
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      39. 39
        Beck, B. R.; Shin, B.; Choi, Y.; Park, S.; Kang, K. Predicting Commercially Available Antiviral Drugs That May Act on the Novel Coronavirus (SARS-CoV-2) through a Drug-Target Interaction Deep Learning Model. Comput. Struct. Biotechnol. J. 2020, 18, 784– 790,  DOI: 10.1016/j.csbj.2020.03.025
        39
        Predicting commercially available antiviral drugs that may act on the novel coronavirus (SARS-CoV-2) through a drug-target interaction deep learning model
        Beck, Bo Ram; Shin, Bonggun; Choi, Yoonjung; Park, Sungsoo; Kang, Keunsoo
        Computational and Structural Biotechnology Journal (2020), 18 (), 784-790CODEN: CSBJAC; ISSN:2001-0370. (Elsevier B.V.)
        The infection of a novel coronavirus found in Wuhan of China (SARS-CoV-2) is rapidly spreading, and the incidence rate is increasing worldwide. Due to the lack of effective treatment options for SARS-CoV-2, various strategies are being tested in China, including drug repurposing. In this study, we used our pre-trained deep learning-based drug-target interaction model called Mol. Transformer-Drug Target Interaction (MT-DTI) to identify com. available drugs that could act on viral proteins of SARS-CoV-2. The result showed that atazanavir, an antiretroviral medication used to treat and prevent the human immunodeficiency virus (HIV), is the best chem. compd., showing an inhibitory potency with Kd of 94.94 nM against the SARS-CoV-2 3C-like proteinase, followed by remdesivir (113.13 nM), efavirenz (199.17 nM), ritonavir (204.05 nM), and dolutegravir (336.91 nM). Interestingly, lopinavir, ritonavir, and darunavir are all designed to target viral proteinases. However, in our prediction, they may also bind to the replication complex components of SARS-CoV-2 with an inhibitory potency with Kd < 1000 nM. In addn., we also found that several antiviral agents, such as Kaletra (lopinavir/ritonavir), could be used for the treatment of SARS-CoV-2. Overall, we suggest that the list of antiviral drugs identified by the MT-DTI model should be considered, when establishing effective treatment strategies for SARS-CoV-2.
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      40. 40
        Hung, I. F.-N.; Lung, K.-C.; Tso, E. Y.-K.; Liu, R.; Chung, T. W.-H.; Chu, M.-Y.; Ng, Y.-Y.; Lo, J.; Chan, J.; Tam, A. R.; Shum, H.-P.; Chan, V.; Wu, A. K.-L.; Sin, K.-M.; Leung, W.-S.; Law, W.-L.; Lung, D. C.; Sin, S.; Yeung, P.; Yip, C. C.-Y.; Zhang, R. R.; Fung, A. Y.-F.; Yan, E. Y.-W.; Leung, K.-H.; Ip, J. D.; Chu, A. W.-H.; Chan, W.-M.; Ng, A. C.-K.; Lee, R.; Fung, K.; Yeung, A.; Wu, T.-C.; Chan, J. W.-M.; Yan, W.-W.; Chan, W.-M.; Chan, J. F.-W.; Lie, A. K.-W.; Tsang, O. T.-Y.; Cheng, V. C.-C.; Que, T.-L.; Lau, C.-S.; Chan, K.-H.; To, K. K.-W.; Yuen, K.-Y. Triple Combination of Interferon Beta-1b, Lopinavir–Ritonavir, and Ribavirin in the Treatment of Patients Admitted to Hospital with COVID-19: An Open-Label, Randomised, Phase 2 Trial. Lancet 2020, 395, 1695– 1704,  DOI: 10.1016/S0140-6736(20)31042-4
        40
        Triple combination of interferon beta-1b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with COVID-19: an open-label, randomised, phase 2 trial
        Hung, Ivan Fan-Ngai; Lung, Kwok-Cheung; Tso, Eugene Yuk-Keung; Liu, Raymond; Chung, Tom Wai-Hin; Chu, Man-Yee; Ng, Yuk-Yung; Lo, Jenny; Chan, Jacky; Tam, Anthony Raymond; Shum, Hoi-Ping; Chan, Veronica; Wu, Alan Ka-Lun; Sin, Kit-Man; Leung, Wai-Shing; Law, Wai-Lam; Lung, David Christopher; Sin, Simon; Yeung, Pauline; Yip, Cyril Chik-Yan; Zhang, Ricky Ruiqi; Fung, Agnes Yim-Fong; Yan, Erica Yuen-Wing; Leung, Kit-Hang; Ip, Jonathan Daniel; Chu, Allen Wing-Ho; Chan, Wan-Mui; Ng, Anthony Chin-Ki; Lee, Rodney; Fung, Kitty; Yeung, Alwin; Wu, Tak-Chiu; Chan, Johnny Wai-Man; Yan, Wing-Wah; Chan, Wai-Ming; Chan, Jasper Fuk-Woo; Lie, Albert Kwok-Wai; Tsang, Owen Tak-Yin; Cheng, Vincent Chi-Chung; Que, Tak-Lun; Lau, Chak-Sing; Chan, Kwok-Hung; To, Kelvin Kai-Wang; Yuen, Kwok-Yung
        Lancet (2020), 395 (10238), 1695-1704CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)
        Effective antiviral therapy is important for tackling the coronavirus disease 2019 (COVID-19) pandemic. We assessed the efficacy and safety of combined interferon beta-1b, lopinavir-ritonavir, and ribavirin for treating patients with COVID-19. This was a multicenter, prospective, open-label, randomized, phase 2 trial in adults with COVID-19 who were admitted to six hospitals in Hong Kong. Patients were randomly assigned (2:1) to a 14-day combination of lopinavir 400 mg and ritonavir 100 mg every 12 h, ribavirin 400 mg every 12 h, and three doses of 8 million IU of interferon beta-1b on alternate days (combination group) or to 14 days of lopinavir 400 mg and ritonavir 100 mg every 12 h (control group). The primary endpoint was the time to providing a nasopharyngeal swab neg. for severe acute respiratory syndrome coronavirus 2 RT-PCR, and was done in the intention-to-treat population. The study is registered with ClinicalTrials.gov, NCT04276688. Between Feb 10 and March 20, 2020, 127 patients were recruited; 86 were randomly assigned to the combination group and 41 were assigned to the control group. The median no. of days from symptom onset to start of study treatment was 5 days (IQR 3-7). The combination group had a significantly shorter median time from start of study treatment to neg. nasopharyngeal swab (7 days [IQR 5-11]) than the control group (12 days [8-15]; hazard ratio 4·37 [95% CI 1·86-10·24], p=0·0010). Adverse events included self-limited nausea and diarrhea with no difference between the two groups. One patient in the control group discontinued lopinavir-ritonavir because of biochem. hepatitis. No patients died during the study. Early triple antiviral therapy was safe and superior to lopinavir-ritonavir alone in alleviating symptoms and shortening the duration of viral shedding and hospital stay in patients with mild to moderate COVID-19. Future clin. study of a double antiviral therapy with interferon beta-1b as a backbone is warranted. The Shaw-Foundation, Richard and Carol Yu, May Tam Mak Mei Yin, and Sanming Project of Medicine.
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      41. 41
        Cao, B.; Wang, Y.; Wen, D.; Liu, W.; Wang, J.; Fan, G.; Ruan, L.; Song, B.; Cai, Y.; Wei, M.; Li, X.; Xia, J.; Chen, N.; Xiang, J.; Yu, T.; Bai, T.; Xie, X.; Zhang, L.; Li, C.; Yuan, Y.; Chen, H.; Li, H.; Huang, H.; Tu, S.; Gong, F.; Liu, Y.; Wei, Y.; Dong, C.; Zhou, F.; Gu, X.; Xu, J.; Liu, Z.; Zhang, Y.; Li, H.; Shang, L.; Wang, K.; Li, K.; Zhou, X.; Dong, X.; Qu, Z.; Lu, S.; Hu, X.; Ruan, S.; Luo, S.; Wu, J.; Peng, L.; Cheng, F.; Pan, L.; Zou, J.; Jia, C.; Wang, J.; Liu, X.; Wang, S.; Wu, X.; Ge, Q.; He, J.; Zhan, H.; Qiu, F.; Guo, L.; Huang, C.; Jaki, T.; Hayden, F. G.; Horby, P. W.; Zhang, D.; Wang, C. A Trial of Lopinavir-Ritonavir in Adults Hospitalized with Severe Covid-19. N. Engl. J. Med. 2020, 382, 1787– 1799,  DOI: 10.1056/NEJMoa2001282
        41
        A Trial of Lopinavir-Ritonavir in Adults Hospitalized with Severe Covid-19
        Cao Bin; Wang Yeming; Wen Danning; Liu Wen; Wang Jingli; Fan Guohui; Ruan Lianguo; Song Bin; Cai Yanping; Wei Ming; Li Xingwang; Xia Jiaan; Chen Nanshan; Xiang Jie; Yu Ting; Bai Tao; Xie Xuelei; Zhang Li; Li Caihong; Yuan Ye; Chen Hua; Li Huadong; Huang Hanping; Tu Shengjing; Gong Fengyun; Liu Ying; Wei Yuan; Dong Chongya; Zhou Fei; Gu Xiaoying; Xu Jiuyang; Liu Zhibo; Zhang Yi; Li Hui; Shang Lianhan; Wang Ke; Li Kunxia; Zhou Xia; Dong Xuan; Qu Zhaohui; Lu Sixia; Hu Xujuan; Ruan Shunan; Luo Shanshan; Wu Jing; Peng Lu; Cheng Fang; Pan Lihong; Zou Jun; Jia Chunmin; Wang Juan; Liu Xia; Wang Shuzhen; Wu Xudong; Ge Qin; He Jing; Zhan Haiyan; Qiu Fang; Guo Li; Huang Chaolin; Jaki Thomas; Hayden Frederick G; Horby Peter W; Zhang Dingyu; Wang Chen
        The New England journal of medicine (2020), 382 (19), 1787-1799 ISSN:.
        BACKGROUND: No therapeutics have yet been proven effective for the treatment of severe illness caused by SARS-CoV-2. METHODS: We conducted a randomized, controlled, open-label trial involving hospitalized adult patients with confirmed SARS-CoV-2 infection, which causes the respiratory illness Covid-19, and an oxygen saturation (Sao2) of 94% or less while they were breathing ambient air or a ratio of the partial pressure of oxygen (Pao2) to the fraction of inspired oxygen (Fio2) of less than 300 mm Hg. Patients were randomly assigned in a 1:1 ratio to receive either lopinavir-ritonavir (400 mg and 100 mg, respectively) twice a day for 14 days, in addition to standard care, or standard care alone. The primary end point was the time to clinical improvement, defined as the time from randomization to either an improvement of two points on a seven-category ordinal scale or discharge from the hospital, whichever came first. RESULTS: A total of 199 patients with laboratory-confirmed SARS-CoV-2 infection underwent randomization; 99 were assigned to the lopinavir-ritonavir group, and 100 to the standard-care group. Treatment with lopinavir-ritonavir was not associated with a difference from standard care in the time to clinical improvement (hazard ratio for clinical improvement, 1.31; 95% confidence interval [CI], 0.95 to 1.80). Mortality at 28 days was similar in the lopinavir-ritonavir group and the standard-care group (19.2% vs. 25.0%; difference, -5.8 percentage points; 95% CI, -17.3 to 5.7). The percentages of patients with detectable viral RNA at various time points were similar. In a modified intention-to-treat analysis, lopinavir-ritonavir led to a median time to clinical improvement that was shorter by 1 day than that observed with standard care (hazard ratio, 1.39; 95% CI, 1.00 to 1.91). Gastrointestinal adverse events were more common in the lopinavir-ritonavir group, but serious adverse events were more common in the standard-care group. Lopinavir-ritonavir treatment was stopped early in 13 patients (13.8%) because of adverse events. CONCLUSIONS: In hospitalized adult patients with severe Covid-19, no benefit was observed with lopinavir-ritonavir treatment beyond standard care. Future trials in patients with severe illness may help to confirm or exclude the possibility of a treatment benefit. (Funded by Major Projects of National Science and Technology on New Drug Creation and Development and others; Chinese Clinical Trial Register number, ChiCTR2000029308.).
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      42. 42
        Xu, L.; Liu, H.; Murray, B. P.; Callebaut, C.; Lee, M. S.; Hong, A.; Strickley, R. G.; Tsai, L. K.; Stray, K. M.; Wang, Y.; Rhodes, G. R.; Desai, M. C. Cobicistat (GS-9350): A Potent and Selective Inhibitor of Human CYP3A as a Novel Pharmacoenhancer. ACS Med. Chem. Lett. 2010, 1, 209– 213,  DOI: 10.1021/ml1000257
        42
        Cobicistat (GS-9350): A Potent and Selective Inhibitor of Human CYP3A as a Novel Pharmacoenhancer
        Xu, Lianhong; Liu, Hongtao; Murray, Bernard P.; Callebaut, Christian; Lee, Melody S.; Hong, Allen; Strickley, Robert G.; Tsai, Luong K.; Stray, Kirsten M.; Wang, Yujin; Rhodes, Gerry R.; Desai, Manoj C.
        ACS Medicinal Chemistry Letters (2010), 1 (5), 209-213CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)
        Cobicistat (3, GS-9350) is a newly discovered, potent, and selective inhibitor of human cytochrome P 450 3A (CYP3A) enzymes. In contrast to ritonavir, 3 is devoid of anti-HIV activity and is thus more suitable for use in boosting anti-HIV drugs without risking selection of potential drug-resistant HIV variants. Compd. 3 shows reduced liability for drug interactions and may have potential improvements in tolerability over ritonavir. In addn., 3 has high aq. soly. and can be readily co-formulated with other agents.
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      43. 43
        Covid-19 clinical trials showing “encouraging” results, says analyst https://www.clinicaltrialsarena.com/analysis/covid-19-clinical-trials-results-2/ (accessed Jun 1, 2020).
        There is no corresponding record for this reference.
      44. 44
        Lawitz, E.; Gane, E.; Pearlman, B.; Tam, E.; Ghesquiere, W.; Guyader, D.; Alric, L.; Bronowicki, J. P.; Lester, L.; Sievert, W.; Ghalib, R.; Balart, L.; Sund, F.; Lagging, M.; Dutko, F.; Shaughnessy, M.; Hwang, P.; Howe, A. Y. M.; Wahl, J.; Robertson, M.; Barr, E.; Haber, B. Efficacy and Safety of 12 Weeks versus 18 Weeks of Treatment with Grazoprevir (MK-5172) and Elbasvir (MK-8742) with or without Ribavirin for Hepatitis C Virus Genotype 1 Infection in Previously Untreated Patients with Cirrhosis and Patients with Previous Null Response with or without Cirrhosis (C-WORTHY): A Randomised, Open-Label Phase 2 Trial. Lancet 2015, 385, 1075– 1086,  DOI: 10.1016/S0140-6736(14)61795-5
        44
        Efficacy and safety of 12 weeks versus 18 weeks of treatment with grazoprevir (MK-5172) and elbasvir (MK-8742) with or without ribavirin for hepatitis C virus genotype 1 infection in previously untreated patients with cirrhosis and patients with previous null response with or without cirrhosis (C-WORTHY): a randomised, open-label phase 2 trial
        Lawitz, Eric; Gane, Edward; Pearlman, Brian; Tam, Edward; Ghesquiere, Wayne; Guyader, Dominique; Alric, Laurent; Bronowicki, Jean-Pierre; Lester, Laura; Sievert, William; Ghalib, Reem; Balart, Luis; Sund, Fredrik; Lagging, Martin; Dutko, Frank; Shaughnessy, Melissa; Hwang, Peggy; Howe, Anita Y. M.; Wahl, Janice; Robertson, Michael; Barr, Eliav; Haber, Barbara
        Lancet (2015), 385 (9973), 1075-1086CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)
        There is a high medical need for an interferon-free, all-oral, short-duration therapy for hepatitis C virus (HCV) that is highly effective across diverse patient populations, including patients with cirrhosis or previous null response to pegylated interferon (peginterferon) plus ribavirin (PR-null responders). We aimed to assess the efficacy, safety, and effective treatment duration of grazoprevir (an HCV NS3/4A protease inhibitor) combined with elbasvir (an HCV NS5A inhibitor) with or without ribavirin in patients with HCV genotype 1 infection with baseline characteristics of poor response. The C-WORTHY trial is a randomised, open-label phase 2 trial of grazoprevir plus elbasvir with or without ribavirin; here we report findings for two cohorts of previously untreated patients with cirrhosis (cohort 1) and those with previous PR-null response with or without cirrhosis (cohort 2) enrolled in part B of the study. Eligible patients were adults aged 18 years or older with chronic HCV genotype 1 infection and HCV RNA concns. of 10 000 IU/mL or higher in peripheral blood. We randomly assigned patients to receive grazoprevir (100 mg daily) and elbasvir (50 mg daily) with or without ribavirin for 12 or 18 wk. Randomisation was done centrally with an interactive voice response system; patients and study investigators were masked to treatment duration up to week 12 but not to treatment allocation. The primary endpoint was the proportion of patients achieving HCV RNA less than 25 IU/mL at 12 wk after end of treatment (SVR12), assessed by COBAS TaqMan version 2.0. This study is registered with ClinicalTrials.gov, no. NCT01717326. We describe findings for 253 patients enrolled in cohort 1 (n=123) or cohort 2 (n=130). In cohort 1, we randomly assigned 60 patients to the 12-wk regimen (31 with ribavirin and 29 with no ribavirin) and 63 to the 18-wk regimen (32 with ribavirin and 31 with no ribavirin); in cohort 2, we randomly assigned 65 patients to the 12-wk regimen (32 with ribavirin and 33 with no ribavirin) and 65 to the 18-wk regimen 33 with ribavirin and 32 with no ribavirin. High SVR12 rates were achieved irresp. of the use of ribavirin or extension of the treatment duration from 12 to 18 wk; SVR12 rates ranged from 90% (95% CI 74-98; 28/31; cohort 1, 12 wk, ribavirin-contg.) to 100% (95% CI 89-100; 33/33; cohort 2, 18 wk, ribavirin-contg.). Among patients treated for 12 wk with grazoprevir plus elbasvir without ribavirin, 97% (95% CI 82-100, 28/29) of patients in cohort 1 and 91% (76-98, 30/33) of patients in cohort 2 achieved SVR12. Adverse events reported in more than 10% of patients were fatigue (66 patients, 26% [95% CI 21-32]), headache (58 patients, 23% [95% CI 18-29]), and asthenia (35 patients, 14% [95% CI 10-19]). Treatment with grazoprevir plus elbasvir, both with and without ribavirin and for both 12 and 18 wk' treatment duration, showed high rates of efficacy in previously untreated patients with cirrhosis and previous PR-null responders with and without cirrhosis. These results support the phase 3 development of grazoprevir plus elbasvir.Merck & Co, Inc.
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      45. 45
        Waheed, Y. Ledipasvir and Sofosbuvir: Interferon Free Therapy for HCV Genotype 1 Infection. World J. Virol. 2015, 4, 33,  DOI: 10.5501/wjv.v4.i1.33
        45
        Ledipasvir and sofosbuvir: Interferon free therapy for hepatitis C virus genotype 1 infection
        Waheed Yasir
        World journal of virology (2015), 4 (1), 33-5 ISSN:2220-3249.
        Hepatitis C virus (HCV) has infected more than 200 million people around the globe. From 2001-2011, interferon plus ribavirin remained the standard of care for patients with HCV infection. The therapy had a limited response with a number of side effects. Recently, results for phase III trials of ledipasvir and sofosbuvir combination therapy have been announced. In treatment naive patients, 12 wk of therapy with ledipasvir and sofosbuvir showed a sustained virological response (SVR) rate of 99%. In treatment experienced patients, 12-24 wk of therapy with ledipasvir and sofosbuvir in the absence or presence of ribavirin showed an SVR rate of 94%-99%. In cirrhotic patients the rate of SVR was 86% and 99% for 12 and 24 wk of therapy, respectively. The ledipasvir and sofosbuvir therapy showed very good results in different subgroups of patients regardless of patient's race, alanine aminotransferase levels, sex and host genetic factors. The combination therapy was well tolerated with no emergence of resistant mutants. The most common adverse effects were nausea, headache and fatigue. With the availability of interferon free therapy with minimal adverse effects, it will be easy to decrease the future morbidity and mortality caused by HCV infection.
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      46. 46
        Zignego, A. L.; Monti, M.; Gragnani, L. Sofosbuvir/Velpatasvir for the Treatment of Hepatitis C Virus Infection. Acta Biomedica. Mattioli 1885 September 1, 2018; pp 321– 331.
        There is no corresponding record for this reference.
      47. 47
        Vishal, M.; Pravin, D.; Himani, G.; Nilam, V.; Urvisha, B.; Rajesh, P. Drug Repurposing of Approved Drugs Elbasvir, Ledipasvir, Paritaprevir, Velpatasvir, Antrafenine and Ergotamine for Combating COVID19. Preprint. ChemRxiv 2020,  DOI: 10.26434/Chemrxiv.12115251.V1 .
        There is no corresponding record for this reference.
      48. 48
        Search of: ledipasvir | Covid19 - List Results - ClinicalTrials.gov; https://clinicaltrials.gov/ct2/results?cond=Covid19&term=ledipasvir&cntry=&state=&city=&dist= (accessed Aug 27, 2020).
        There is no corresponding record for this reference.
      49. 49
        Waters, L.; Nelson, M. The Use of Caspofungin in HIV-Infected Individuals. Expert Opin. Invest. Drugs 2007, 16, 899– 908,  DOI: 10.1517/13543784.16.6.899
        49
        The use of caspofungin in HIV-infected individuals
        Waters, Laura; Nelson, Mark
        Expert Opinion on Investigational Drugs (2007), 16 (6), 899-908CODEN: EOIDER; ISSN:1354-3784. (Informa Healthcare)
        A review. Fungal infections are a significant cause of HIV-related morbidity and mortality, particularly in the developing world, but also in countries with access to highly active antiretroviral therapy. New agents are essential to improve present efficacy rates, particularly in cases of drug resistance. Caspofungin is a new antifungal from the echinocandin class and is licensed for the treatment of candidal infections and as a second-line therapy for invasive aspergillosis. In this paper, the pharmacol., interaction and susceptibility data for this agent are reviewed and studies supporting the use of this agent in HIV-infected individuals are examd. Finally, evidence for the use of caspofungin for the treatment of Pneumocystis jiroveci pneumonia, an unlicensed indication, including a case series from our own unit is explored.
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      50. 50
        Wang, M.; Ye, F.; Su, J.; Zhao, J.; Yuan, B.; Huang, B.; Peng, Q.; Peng, R.; Sun, Y.; Bai, S.; Wang, X.; Yang, W.; Fan, Z.; Wang, W.; Wu, G.; Gao, G. F.; Tan, W.; Sh, Y. Caspofungin and LTX-315 Inhibit SARS-CoV-2 Replication by Targeting the Nsp12 Polymerase. Research Square. Preprint. 2020,  DOI: 10.21203/RS.3.RS-19872/V1 .
        There is no corresponding record for this reference.
      51. 51
        Yang, Y.; Cao, T.; Guo, Q. Characterization of a Traditional Chinese Medicine Plant the Chloroplast Genome of Andrographis Paniculata. Mitochondrial DNA Part B 2020, 5, 1949– 1951,  DOI: 10.1080/23802359.2020.1756949
        There is no corresponding record for this reference.
      52. 52
        Wei-Ya, C.; Yuan-Song, W.; Chun-Yu, L.; Yu-Bin, J.; Fei-Fei, Y.; Yong-Hong, L. Comparison of Pulmonary Availability and Anti-Inflammatory Effect of Dehydroandrographolide Succinate via Intratracheal and Intravenous Administration. Eur. J. Pharm. Sci. 2020, 147, 105290,  DOI: 10.1016/j.ejps.2020.105290
        52
        Comparison of pulmonary availability and anti-inflammatory effect of dehydroandrographolide succinate via intratracheal and intravenous administration
        Wei-Ya Chen; Chun-Yu Liu; Yuan-Song Wang; Yu-Bin Ji; Fei-Fei Yang; Yong-Hong Liao
        European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences (2020), 147 (), 105290 ISSN:.
        Dehydroandrographolide succinate (DAS) injection, which was approved in China for the treatment of viral pneumonia and upper respiratory tract infections, is often off-label used for nebulization therapy to avoid the adverse drug reactions associated with the injection. However, the aerodynamic properties and pulmonary fate of nebulized DAS was largely uninvestigated. In this study, the main objectives were to evaluate the in vitro aerodynamic deposition profiles of nebulizer generated aerosols and comparatively investigate the local drug availability and anti-inflammatory efficacy of DAS between intratracheal and intravenous dosing. The in vitro evaluation of aerodynamic characteristics and droplet size distribution showed more than 50% aerosol particles with size being <5 μm, allowing the aerosols to reach the lower respiratory tract. Following intratracheal administration, the drug underwent pulmonary absorption into the bloodstream, rendering an absolute bioavailability of 47.3%. Compared to the intravenous delivery, the intratracheal administration dramatically increased the drug availability in the lung tissue in rats by more than 80-fold, leading to an improved and prolonged local anti-inflammatory efficacy in a lipopolysaccharide induced lung injury model in mice. The present results demonstrated that inhalation delivery of DAS is a convenient and effective alternative to intravenous injections.
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      53. 53
        Chen, Q.; Liu, Y.; Liu, Y. M.; Liu, G. Y.; Zhang, M. Q.; Jia, J. Y.; Lu, C.; Yu, C. Pharmacokinetics and Tolerance of Dehydroandrographolide Succinate Injection after Intravenous Administration in Healthy Chinese Volunteers. Acta Pharmacol. Sin. 2012, 33, 1332– 1336,  DOI: 10.1038/aps.2012.79
        53
        Pharmacokinetics and tolerance of dehydroandrographolide succinate injection after intravenous administration in healthy Chinese volunteers
        Chen, Qian; Liu, Yun; Liu, Yan-mei; Liu, Gang-yi; Zhang, Meng-qi; Jia, Jing-ying; Lu, Chuan; Yu, Chen
        Acta Pharmacologica Sinica (2012), 33 (10), 1332-1336CODEN: APSCG5; ISSN:1671-4083. (Nature Publishing Group)
        Aim: Dehydroandrographolide succinate (DAS) is extd. from herbal medicine Andrographis paniculata (Burm f) Nees. DAS injection is used in China for the treatment of viral pneumonia and upper respiratory tract infections. The aim of this study is to investigate the pharmacokinetics and tolerance of DAS injection in healthy Chinese volunteers. Methods: This was a single-center, randomized, single-dose, three-way crossover design study. Nine eligible subjects were randomly divided into 3 groups, and each group sequentially received 80, 160, or 320 mg of DAS infusion according to a three-way Latin square design. Plasma and urine samples were collected and detd. using an LC-MS/MS method. Safety and tolerability were detd. via clin. evaluation and adverse event monitoring. Results: For the 80, 160, and 320 mg dose groups, the mean Cmax were 4.82, 12.85, and 26.90 mg/L, resp., and the mean AUC0-12 were 6.18, 16.95, and 40.65 mg·L-1·h, resp. DAS was rapidly cleared, with a mean Tmax of 0.94-1.0 h and a t1/2 of approx. 1.51-1.89 h. Approx. 10.1%-15.5% of the i.v. DAS dose was excreted unchanged in urine within 24 h in the 3 groups, and more than 90% of unchanged DAS was excreted between 0 and 4 h. The pharmacokinetic profile was similar between male and female subjects. No serious or unexpected adverse events were found during the study, but one mild adverse event (stomachache) was reported. Conclusion: This study shows that DAS has nonlinear pharmacokinetic characteristics. To guarantee the effective concn., multiple small doses are recommended in clin. regimens.
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      54. 54
        Jayakumar, T.; Hsieh, C. Y.; Lee, J. J.; Sheu, J. R. Experimental and Clinical Pharmacology of Andrographis Paniculata and Its Major Bioactive Phytoconstituent Andrographolide. J. Evidence-Based Complementary Altern. Med. 2013, 2013, 1 DOI: 10.1155/2013/846740 .
        There is no corresponding record for this reference.
      55. 55
        Chang, R. S.; Ding, L.; Gai-Qing, C.; Qi-Choa, P.; Ze-Lin, Z.; Smith, K. M. Dehydroandrographolide Succinic Acid Monoester as an Inhibitor against the Human Immunodeficiency Virus. Exp. Biol. Med. 1991, 197, 59– 66,  DOI: 10.3181/00379727-197-43225
        55
        Dehydroandrographolide succinic acid monoester as an inhibitor against the human immunodeficiency virus
        Chang, R. Shihman; Ding, Lu; Chen, Gaiqing; Pan, Qichoa; Zhao, Zelin; Smith, Kevin M.
        Proceedings of the Society for Experimental Biology and Medicine (1991), 197 (1), 59-66CODEN: PSEBAA; ISSN:0037-9727.
        Dehydroandrographolide succinic acid monoester (DASM) is made from the Chinese medicinal herb Andrographis paniculata. DASM is an inhibitor against the human immunodeficiency virus (HIV) in vitro. It was nontoxic to the H9 cell at 50-200 μg/mL and was inhibitory to the HIV-1 (IIIB) at the minimal concn. of 1.6-3.1 μg/mL. It was also inhibitory to two other strains of HIV-1 and a strain of HIV-2. This inhibitory effect could also be demonstrated in cultures of activated human blood mononuclear cells; the 50% toxic dose and the 50% HIV ID were about 200-≥400 and 0.8-2 μg/mL, resp. At the subtoxic concn., DASM partially interfered with HIV-induced cell fusion and with the binding of HIV to the H9 cell. Presumably, it also interfered with HIV replication at another unidentified step(s).
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      56. 56
        Cai, W.; Li, Y.; Chen, S.; Wang, M.; Zhang, A.; Zhou, H.; Chen, H.; Jin, M. 14-Deoxy-11,12-Dehydroandrographolide Exerts Anti-Influenza A Virus Activity and Inhibits Replication of H5N1 Virus by Restraining Nuclear Export of Viral Ribonucleoprotein Complexes. Antiviral Res. 2015, 118, 82– 92,  DOI: 10.1016/j.antiviral.2015.03.008
        56
        14-Deoxy-11,12-dehydroandrographolide exerts anti-influenza A virus activity and inhibits replication of H5N1 virus by restraining nuclear export of viral ribonucleoprotein complexes
        Cai, Wentao; Li, Yongtao; Chen, Sunrui; Wang, Mengli; Zhang, Anding; Zhou, Hongbo; Chen, Huanchun; Jin, Meilin
        Antiviral Research (2015), 118 (), 82-92CODEN: ARSRDR; ISSN:0166-3542. (Elsevier B.V.)
        The highly pathogenic avian influenza H5N1 virus has become a worldwide public health threat, and current antiviral therapies have limited activity against the emerging, resistant influenza viruses. Therefore, effective drugs with novel targets against influenza A viruses, H5N1 strains in particular, should be developed. In the present study, 14-deoxy-11,12-dehydroandrographolide (DAP), a major component of the traditional Chinese medicine Andrographis paniculata, exerted potent anti-influenza A virus activity against A/chicken/Hubei/327/2004 (H5N1), A/duck/Hubei/XN/2007 (H5N1), A/PR/8/34 (H1N1), A/NanChang/08/2010 (H1N1) and A/HuNan/01/2014 (H3N2) in vitro. To elucidate the underlying mechanisms, a series of expts. was conducted using A/chicken/Hubei/327/2004 (H5N1) as an example. Our results demonstrated that DAP strongly inhibited H5N1 replication by reducing the prodn. of viral nucleoprotein (NP) mRNA, NP and NS1proteins, whereas DAP had no effect on the absorption and release of H5N1 towards/from A549 cells. DAP also effectively restrained the nuclear export of viral ribonucleoprotein (vRNP) complexes. This inhibitory effect ought to be an important anti-H5N1 mechanism of DAP. Meanwhile, DAP significantly reduced the upregulated expression of all the tested proinflammatory cytokines (TNF-α, IL-6, IL-8, IFN-α, IL-1β and IFN-β) and chemokines (CXCL-10 and CCL-2) stimulated by H5N1. Overall results suggest that DAP impairs H5N1 replication at least in part by restraining nuclear export of vRNP complexes, and the inhibition of viral replication leads to a subsequent decrease of the intense proinflammatory cytokine/chemokine expression. In turn, the effect of modification of the host excessive immune response may contribute to overcoming H5N1. To our knowledge, this study is the first to reveal the antiviral and anti-inflammatory activities of DAP in vitro against H5N1 influenza A virus infection.
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      57. 57
        Jakalian, A.; Jack, D. B.; Bayly, C. I. Fast, Efficient Generation of High-Quality Atomic Charges. AM1-BCC Model: II. Parameterization and Validation. J. Comput. Chem. 2002, 23, 1623– 1641,  DOI: 10.1002/jcc.10128
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        Fast, efficient generation of high-quality atomic charges. AM1-BCC model: II. parameterization and validation
        Jakalian, Araz; Jack, David B.; Bayly, Christopher I.
        Journal of Computational Chemistry (2002), 23 (16), 1623-1641CODEN: JCCHDD; ISSN:0192-8651. (John Wiley & Sons, Inc.)
        We present the first global parameterization and validation of a novel charge model, called AM1-BCC, which quickly and efficiently generates high-quality at. charges for computer simulations of org. mols. in polar media. The goal of the charge model is to produce at. charges that emulate the HF/6-31G* electrostatic potential (ESP) of a mol. Underlying electronic structure features, including formal charge and electron delocalization, are first captured by AM1 population charges; simple additive bond charge corrections (BCCs) are then applied to these AM1 at. charges to produce the AM1-BCC charges. The parameterization of BCCs was carried out by fitting to the HF/6-31G* ESP of a training set of >2700 mols. Most org. functional groups and their combinations were sampled, as well as an extensive variety of cyclic and fused bicyclic heteroaryl systems. The resulting BCC parameters allow the AM1-BCC charging scheme to handle virtually all types of org. compds. listed in The Merck Index and the NCI Database. Validation of the model was done through comparisons of hydrogen-bonded dimer energies and relative free energies of solvation using AM1-BCC charges in conjunction with the 1994 Cornell et al. forcefield for AMBER. Homo-dimer and hetero-dimer hydrogen-bond energies of a diverse set of org. mols. were reproduced to within 0.95 kcal/mol RMS deviation from the ab initio values, and for DNA dimers the energies were within 0.9 kcal/mol RMS deviation from ab initio values. The calcd. relative free energies of solvation for a diverse set of monofunctional isosteres were reproduced to within 0.69 kcal/mol of expt. In all these validation tests, AMBER with the AM1-BCC charge model maintained a correlation coeff. above 0.96. Thus, the parameters presented here for use with the AM1-BCC method present a fast, accurate, and robust alternative to HF/6-31G* ESP-fit charges for general use with the AMBER force field in computer simulations involving org. small mols.
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      58. 58
        Bayly, C. I.; Cieplak, P.; Cornell, W. D.; Kollman, P. A. A Well-Behaved Electrostatic Potential Based Method Using Charge Restraints for Deriving Atomic Charges: The RESP Model. J. Phys. Chem. 1993, 97, 10269– 10280,  DOI: 10.1021/j100142a004
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        A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: the RESP model
        Bayly, Christopher I.; Cieplak, Piotr; Cornell, Wendy; Kollman, Peter A.
        Journal of Physical Chemistry (1993), 97 (40), 10269-80CODEN: JPCHAX; ISSN:0022-3654.
        The authors present a new approach to generating electrostatic potential (ESP) derived charges for mols. The major strength of electrostatic potential derived charges is that they optimally reproduce the intermol. interaction properties of mols. with a simple two-body additive potential, provided, of course, that a suitably accurate level of quantum mech. calcn. is used to derive the ESP around the mol. Previously, the major weaknesses of these charges have been that they were not easily transferably between common functional groups in related mols., they have often been conformationally dependent, and the large charges that frequently occur can be problematic for simulating intramol. interactions. Introducing restraints in the form of a penalty function into the fitting process considerably reduces the above problems, with only a minor decrease in the quality of the fit to the quantum mech. ESP. Several other refinements in addn. to the restrained electrostatic potential (RESP) fit yield a general and algorithmic charge fitting procedure for generating atom-centered point charges. This approach can thus be recommended for general use in mol. mechanics, mol. dynamics, and free energy calcns. for any org. or bioorg. system.
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      59. 59
        Xia, S.; Liu, Q.; Wang, Q.; Sun, Z.; Su, S.; Du, L.; Ying, T.; Lu, L.; Jiang, S. Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Entry Inhibitors Targeting Spike Protein. Virus Res. 2014, 194, 200– 210,  DOI: 10.1016/j.virusres.2014.10.007
        59
        Middle East respiratory syndrome coronavirus (MERS-CoV) entry inhibitors targeting spike protein
        Xia, Shuai; Liu, Qi; Wang, Qian; Sun, Zhiwu; Su, Shan; Du, Lanying; Ying, Tianlei; Lu, Lu; Jiang, Shibo
        Virus Research (2014), 194 (), 200-210CODEN: VIREDF; ISSN:0168-1702. (Elsevier B.V.)
        A review. The recent outbreak of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) infection has led to more than 800 lab.-confirmed MERS cases with a high case fatality rate (∼35%), posing a serious threat to global public health and calling for the development of effective and safe therapeutic and prophylactic strategies to treat and prevent MERS-CoV infection. Here we discuss the most recent studies on the structure of the MERS-CoV spike protein and its role in virus binding and entry, and the development of MERS-CoV entry/fusion inhibitors targeting the S1 subunit, particularly the receptor-binding domain (RBD), and the S2 subunit, esp. the HR1 region, of the MERS-CoV spike protein. We then look ahead to future applications of these viral entry/fusion inhibitors, either alone or in combination with specific and nonspecific MERS-CoV replication inhibitors, for the treatment and prevention of MERS-CoV infection.
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      60. 60
        Du, L.; Zhao, G.; Yang, Y.; Qiu, H.; Wang, L.; Kou, Z.; Tao, X.; Yu, H.; Sun, S.; Tseng, C.-T. K.; Jiang, S.; Li, F.; Zhou, Y. A Conformation-Dependent Neutralizing Monoclonal Antibody Specifically Targeting Receptor-Binding Domain in Middle East Respiratory Syndrome Coronavirus Spike Protein. J. Virol. 2014, 88, 7045– 7053,  DOI: 10.1128/JVI.00433-14
        60
        A conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in Middle East respiratory syndrome coronavirus spike protein
        Du, Lanying; Zhao, Guangyu; Yang, Yang; Qiu, Hongjie; Wang, Lili; Kou, Zhihua; Tao, Xinrong; Yu, Hong; Sun, Shihui; Tseng, Chien-Te K.; Jiang, Shibo; Li, Fang; Zhou, Yusen
        Journal of Virology (2014), 88 (12), 7045-7053, 10 pp.CODEN: JOVIAM; ISSN:1098-5514. (American Society for Microbiology)
        Prophylactic and therapeutic strategies are urgently needed to combat infections caused by the newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV). Here, we have developed a neutralizing monoclonal antibody (MAb), designated Mersmab1, which potently blocks MERS-CoV entry into human cells. Biochem. assays reveal that Mersmab1 specifically binds to the receptor-binding domain (RBD) of the MERS-CoV spike protein and thereby competitively blocks the binding of the RBD to its cellular receptor, dipeptidyl peptidase 4 (DPP4). Furthermore, alanine scanning of the RBD has identified several residues at the DPP4-binding surface that serve as neutralizing epitopes for Mersmab1. These results suggest that if humanized, Mersmab1 could potentially function as a therapeutic antibody for treating and preventing MERS-CoV infections. Addnl., Mersmab1 may facilitate studies of the conformation and antigenicity of MERS-CoV RBD and thus will guide rational design of MERS-CoV subunit vaccines.
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      61. 61
        Li, Y.; Wan, Y.; Liu, P.; Zhao, J.; Lu, G.; Qi, J.; Wang, Q.; Lu, X.; Wu, Y.; Liu, W.; Zhang, B.; Yuen, K. Y.; Perlman, S.; Gao, G. F.; Yan, J. A Humanized Neutralizing Antibody against MERS-CoV Targeting the Receptor-Binding Domain of the Spike Protein. Cell Res. 2015, 25, 1237– 1249,  DOI: 10.1038/cr.2015.113
        61
        A humanized neutralizing antibody against MERS-CoV targeting the receptor-binding domain of the spike protein
        Li, Yan; Wan, Yuhua; Liu, Peipei; Zhao, Jincun; Lu, Guangwen; Qi, Jianxun; Wang, Qihui; Lu, Xuancheng; Wu, Ying; Liu, Wenjun; Zhang, Buchang; Yuen, Kwok-Yung; Perlman, Stanley; Gao, George F.; Yan, Jinghua
        Cell Research (2015), 25 (11), 1237-1249CODEN: CREEB6; ISSN:1001-0602. (NPG Nature Asia-Pacific)
        The newly-emerging Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe and fatal acute respiratory disease in humans. Despite global efforts, the potential for an assocd. pandemic in the future cannot be excluded. The development of effective counter-measures is urgent. MERS-CoV-specific anti-viral drugs or vaccines are not yet available. Using the spike receptor-binding domain of MERS-CoV (MERS-RBD) to immunize mice, we identified two neutralizing monoclonal antibodies (mAbs) 4C2 and 2E6. Both mAbs potently bind to MERS-RBD and block virus entry in vitro with high efficacy. We further investigated their mechanisms of neutralization by crystg. the complex between the Fab fragments and the RBD, and solved the structure of the 4C2 Fab/MERS-RBD complex. The structure showed that 4C2 recognizes an epitope that partially overlaps the receptor-binding footprint in MERS-RBD, thereby interfering with the virus/receptor interactions by both steric hindrance and interface-residue competition. 2E6 also blocks receptor binding, and competes with 4C2 for binding to MERS-RBD. Based on the structure, we further humanized 4C2 by preserving only the paratope residues and substituting the remaining amino acids with the counterparts from human Igs. The humanized 4C2 (4C2h) antibody sustained similar neutralizing activity and biochem. characteristics to the parental mouse antibody. Finally, we showed that 4C2h can significantly abate the virus titers in lungs of Ad5-hCD26-transduced mice infected with MERS-CoV, therefore representing a promising agent for prophylaxis and therapy in clin. settings.
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      62. 62
        Coughlin, M.; Lou, G.; Martinez, O.; Masterman, S. K.; Olsen, O. A.; Moksa, A. A.; Farzan, M.; Babcook, J. S.; Prabhakar, B. S. Generation and Characterization of Human Monoclonal Neutralizing Antibodies with Distinct Binding and Sequence Features against SARS Coronavirus Using XenoMouse®. Virology 2007, 361, 93– 102,  DOI: 10.1016/j.virol.2006.09.029
        62
        Generation and characterization of human monoclonal neutralizing antibodies with distinct binding and sequence features against SARS coronavirus using XenoMouse
        Coughlin, Melissa; Lou, Gin; Martinez, Osvaldo; Masterman, Stephanie K.; Olsen, Ole A.; Moksa, Angelica A.; Farzan, Michael; Babcook, John S.; Prabhakar, Bellur S.
        Virology (2007), 361 (1), 93-102CODEN: VIRLAX; ISSN:0042-6822. (Elsevier)
        Passive therapy with neutralizing human monoclonal antibodies (mAbs) could be an effective therapy against severe acute respiratory syndrome coronavirus (SARS-CoV). Utilizing the human Ig transgenic mouse, XenoMouse, the authors produced fully human SARS-CoV spike (S) protein specific antibodies. Antibodies were examd. for reactivity against a recombinant S1 protein, to which 200 antibodies reacted. Twenty-seven antibodies neutralized 200TCID50 SARS-CoV (Urbani). Addnl., 57 neutralizing antibodies were found that are likely specific to S2. Mapping of the binding region was achieved with several S1 recombinant proteins. Most S1 reactive neutralizing mAbs bound to the RBD, aa 318-510. However, two S1 specific mAbs reacted with a domain upstream of the RBD between aa 12 and 261. Ig gene sequence analyses suggested at least 8 different binding specificities. Unique human mAbs could be used as a cocktail that would simultaneously target several neutralizing epitopes and prevent emergence of escape mutants.
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      63. 63
        Lei, C.; Qian, K.; Li, T.; Zhang, S.; Fu, W.; Ding, M.; Hu, S. Neutralization of SARS-CoV-2 Spike Pseudotyped Virus by Recombinant ACE2-Ig. Nat. Commun. 2020, 11, 1– 5,  DOI: 10.1038/s41467-020-16048-4
        There is no corresponding record for this reference.
      64. 64
        Zhang, G.; Pomplun, S.; Loftis, A. R.; Loas, A.; Pentelute, B. L. The First-in-Class Peptide Binder to the SARS-CoV-2 Spike Protein. bioRxiv 2020, 2020.03.19.999318.
        There is no corresponding record for this reference.
      65. 65
        Xia, S.; Liu, M.; Wang, C.; Xu, W.; Lan, Q.; Feng, S.; Qi, F.; Bao, L.; Du, L.; Liu, S.; Qin, C.; Sun, F.; Shi, Z.; Zhu, Y.; Jiang, S.; Lu, L. Inhibition of SARS-CoV-2 (Previously 2019-NCoV) Infection by a Highly Potent Pan-Coronavirus Fusion Inhibitor Targeting Its Spike Protein That Harbors a High Capacity to Mediate Membrane Fusion. Cell Res. 2020, 30, 343– 355,  DOI: 10.1038/s41422-020-0305-x
        65
        Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion
        Xia, Shuai; Liu, Meiqin; Wang, Chao; Xu, Wei; Lan, Qiaoshuai; Feng, Siliang; Qi, Feifei; Bao, Linlin; Du, Lanying; Liu, Shuwen; Qin, Chuan; Sun, Fei; Shi, Zhengli; Zhu, Yun; Jiang, Shibo; Lu, Lu
        Cell Research (2020), 30 (4), 343-355CODEN: CREEB6; ISSN:1001-0602. (Nature Research)
        The recent outbreak of coronavirus disease (COVID-19) caused by SARS-CoV-2 infection in Wuhan, China has posed a serious threat to global public health. To develop specific anti-coronavirus therapeutics and prophylactics, the mol. mechanism that underlies viral infection must first be defined. Therefore, we herein established a SARS-CoV-2 spike (S) protein-mediated cell-cell fusion assay and found that SARS-CoV-2 showed a superior plasma membrane fusion capacity compared to that of SARS-CoV. We solved the X-ray crystal structure of six-helical bundle (6-HB) core of the HR1 and HR2 domains in the SARS-CoV-2 S protein S2 subunit, revealing that several mutated amino acid residues in the HR1 domain may be assocd. with enhanced interactions with the HR2 domain. We previously developed a pan-coronavirus fusion inhibitor, EK1, which targeted the HR1 domain and could inhibit infection by divergent human coronaviruses tested, including SARS-CoV and MERS-CoV. Here we generated a series of lipopeptides derived from EK1 and found that EK1C4 was the most potent fusion inhibitor against SARS-CoV-2 S protein-mediated membrane fusion and pseudovirus infection with IC50s of 1.3 and 15.8 nM, about 241- and 149-fold more potent than the original EK1 peptide, resp. EK1C4 was also highly effective against membrane fusion and infection of other human coronavirus pseudoviruses tested, including SARS-CoV and MERS-CoV, as well as SARSr-CoVs, and potently inhibited the replication of 5 live human coronaviruses examd., including SARS-CoV-2. Intranasal application of EK1C4 before or after challenge with HCoV-OC43 protected mice from infection, suggesting that EK1C4 could be used for prevention and treatment of infection by the currently circulating SARS-CoV-2 and other emerging SARSr-CoVs.
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      66. 66
        Janin, J.; Chothia, C. The Structure of Protein-Protein Recognition Sites. J. Biol. Chem. 1990, 265, 16027– 16030
        66
        The structure of protein-protein recognition sites
        Janin, Joel; Chothia, Cyrus
        Journal of Biological Chemistry (1990), 265 (27), 16027-30CODEN: JBCHA3; ISSN:0021-9258.
        A review, with 67 refs., on the basis of protein-protein recognition. The general features of the recognition sites are discussed. The nature of the conformational changes that occur on the assocn. of proteins and the implications of these structural results for the kinetics and thermodn. of assocn. are evaluated.
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      67. 67
        Spinello, A.; Saltalamacchia, A.; Magistrato, A. Is the Rigidity of SARS-CoV-2 Spike Receptor-Binding Motif the Hallmark for Its Enhanced Infectivity? An Answer from All-Atoms Simulations. ChemRxiv . 2020, Preprint.  DOI: 10.26434/Chemrxiv.12091260.V3 .
        There is no corresponding record for this reference.
      68. 68
        Laha, S.; Chakraborty, J.; Das, S.; Manna, S. K.; Biswas, S.; Chatterjee, R. Characterizations of SARS-CoV-2 Mutational Profile, Spike Protein Stability and Viral Transmission. Infect., Genet. Evol. 2020, 85, 85,  DOI: 10.1016/j.meegid.2020.104445
        There is no corresponding record for this reference.
      69. 69
        Senathilake, K. S.; Samarakoon, S. R.; Tennekoon, K. H. Virtual Screening of Inhibitors against Spike Glycoprotein of SARS-CoV-2: A Drug Repurposing Approach. Preprint. 2020,  DOI: 10.20944/Preprints202003.0042.V2 .
        There is no corresponding record for this reference.
      70. 70
        Evaluating the Use of Polymyxin B Cartridge Hemoperfusion for Patients With Septic Shock and COVID 19 https://clinicaltrials.gov/ct2/show/NCT04352985 (accessed Jun 1, 2020).
        There is no corresponding record for this reference.
      71. 71
        Sajib, A. Repurposing of Approved Drugs with Potential to Block SARS-CoV-2 Surface Glycoprotein Interaction with Host Receptor. Preprint. 2020,  DOI: 10.20944/Preprints202004.0369.V1 .
        There is no corresponding record for this reference.
      72. 72
        Belouzard, S.; Millet, J. K.; Licitra, B. N.; Whittaker, G. R. Mechanisms of Coronavirus Cell Entry Mediated by the Viral Spike Protein. Viruses 2012, 4, 1011– 1033,  DOI: 10.3390/v4061011
        72
        Mechanisms of coronavirus cell entry mediated by the viral spike protein
        Belouzard, Sandrine; Millet, Jean K.; Licitra, Beth N.; Whittaker, Gary R.
        Viruses (2012), 4 (), 1011-1033CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)
        A review. Coronaviruses are enveloped pos.-stranded RNA viruses that replicate in the cytoplasm. To deliver their nucleocapsid into the host cell, they rely on the fusion of their envelope with the host cell membrane. The spike glycoprotein (S) mediates virus entry and is a primary determinant of cell tropism and pathogenesis. It is classified as a class I fusion protein, and is responsible for binding to the receptor on the host cell as well as mediating the fusion of host and viral membranes - A process driven by major conformational changes of the S protein. This review discusses coronavirus entry mechanisms focusing on the different triggers used by coronaviruses to initiate the conformational change of the S protein: receptor binding, low pH exposure and proteolytic activation. We also highlight commonalities between coronavirus S proteins and other class I viral fusion proteins, as well as distinctive features that confer distinct tropism, pathogenicity and host interspecies transmission characteristics to coronaviruses.
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      73. 73
        Liu, L.; Chopra, P.; Li, X.; Wolfert, M. A.; Tompkins, S. M.; Boons, G.-J. SARS-CoV-2 Spike Protein Binds Heparan Sulfate in a Length- and Sequence-Dependent Manner. bioRxiv Prepr. Serv. Biol. 2020, 2020.05.10.087288.
        There is no corresponding record for this reference.
      74. 74
        Kim, S. Y.; Jin, W.; Sood, A.; Montgomery, D.; Grant, O.; Fuster, M.; Fu, L.; Dordick, J.; Woods, R.; Zhang, F.; Linhardt, R. Glycosaminoglycan Binding Motif at S1/S2 Proteolytic Cleavage Site on Spike Glycoprotein May Facilitate Novel Coronavirus (SARS-CoV-2) Host Cell Entry. bioRxiv 2020, 2020.04.14.041459.
        There is no corresponding record for this reference.
      75. 75
        Mycroft-West Su, D.; Elli, S.; Guimond, S. E.; Miller, G. J.; Turnbull, J. E.; Yates, E. A.; Guerrini, M.; Fernig, D. G.; Lima, M. A.; de Skidmore, M. A. The 2019 Coronavirus (SARS-CoV-2) Surface Protein (Spike) S1 Receptor Binding Domain Undergoes Conformational Change upon Heparin Binding. bioRxiv 2020, No. April, 2020.02.29.971093.
        There is no corresponding record for this reference.
      76. 76
        Nguyen, T. D.; Bottreau, E.; Aynaud, J. M. Effet Du Désoxycholate, de l’amphotéricine B et de La Fongizone Sur Le Coronavirus de La Gastroentérite Transmissible. Ann. Inst. Pasteur/Virol. 1987, 138, 331– 336,  DOI: 10.1016/S0769-2617(87)80019-9
        76
        Effect of deoxycholate, amphotericin B and fungizone on transmissible gastroenteritis coronavirus
        Nguyen, T. D.; Bottreau, E.; Aynaud, J. M.
        Annales de l'Institut Pasteur/Virology (1987), 138 (3), 331-6CODEN: AIPVEU; ISSN:0769-2617.
        At a concn. of 2 μg/mL, neither amphotericin B nor deoxycholate had an inactivating effect upon transmissible gastroenteritis coronavirus infectivity. However, amphotericin B stimulated plaque formation in agarose and facilitated the entry of viral RNA into swine testis cells. The combination of amphotericin B + deoxycholate inactivated virus infectivity and induced a decrease in plaque diam. Finally, in the presence of these agents, the prodn. of infectious virus and interferon was unchanged.
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      77. 77
        Abobaker, A. Can Iron Chelation as an Adjunct Treatment of COVID-19 Improve the Clinical Outcome? Eur. J. Clin. Pharmacol. Springer, June 30, 2020; pp 1– 2.
        There is no corresponding record for this reference.
      78. 78
        Katie, Heiser; McLean, P. F.; Davis, C. T.; Fogelson, B.; Gordon, H. B.; Jacobson, P.; Hurst, B.; Miller, B.; Alfa, R. W.; Earnshaw, B. A.; Victors, M. L.; Chong, Y. T.; Haque, I. S.; Low, A. S.; Gibson, C. C. Identification of Potential Treatments for COVID-19 through Artificial Intelligence Enabled Phenomic Analysis of Human Cells Infected with SARS-CoV-2. Preprint. bioRxiv . 2020,  DOI: 10.1101/2020.04.21.054387 .
        There is no corresponding record for this reference.
      79. 79
        Chen, Y.; Li, Y.; Wang, X.; Zou, P. Montelukast, an Anti-Asthmatic Drug, Inhibits Zika Virus Infection by Disrupting Viral Integrity. Front. Microbiol. 2020, 10, 3079,  DOI: 10.3389/fmicb.2019.03079
        There is no corresponding record for this reference.
      80. 80
        Vasanthakumar, N. Can Beta-Adrenergic Blockers Be Used in the Treatment of COVID-19? Medical hypotheses. NLM (Medline) May 5, 2020; p 109809.
        There is no corresponding record for this reference.
      81. 81
        Lamiable, A.; Thévenet, P.; Rey, J.; Vavrusa, M.; Derreumaux, P.; Tufféry, P. PEP-FOLD3: Faster de Novo Structure Prediction for Linear Peptides in Solution and in Complex. Nucleic Acids Res. 2016, 44, W449– W454,  DOI: 10.1093/nar/gkw329
        81
        PEP-FOLD3: faster denovo structure prediction for linear peptides in solution and in complex
        Lamiable, Alexis; Thevenet, Pierre; Rey, Julien; Vavrusa, Marek; Derreumaux, Philippe; Tuffery, Pierre
        Nucleic Acids Research (2016), 44 (W1), W449-W454CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
        Structure detn. of linear peptides of 5-50 amino acids in aq. soln. and interacting with proteins is a key aspect in structural biol. PEPFOLD3 is a novel computational framework, that allows both (i) de novo free or biased prediction for linear peptides between 5 and 50 amino acids, and (ii) the generation of native-like conformations of peptides interacting with a protein when the interaction site is known in advance. PEP-FOLD3 is fast, and usually returns solns. in a few minutes. Testing PEP-FOLD3 on 56 peptides in aq. soln. led to exptl.-like conformations for 80% of the targets. Using a benchmark of 61 peptide - protein targets starting from the unbound form of the protein receptor, PEP-FOLD3 was able to generate peptide poses deviating on av. by 3.3% from the exptl. conformation and return a native-like pose in the first 10 clusters for 52% of the targets.
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      82. 82
        Mascart-Lemone, F.; Huygen, K.; Clumeck, N.; Brenez, D.; Bolla, K.; Duchateau, J. Stimulation of Cellualr Function by Thymopentin (TP-5) in Three AIDS Patients. The Lancet; Elsevier, September 24, 1983; pp 735– 736.
        There is no corresponding record for this reference.
      83. 83
        Clumeck, N.; Cran, S.; Van de Perre, P.; Mascart-Lemone, F.; Duchateau, J.; Bolla, K. Thymopentin Treatment in Aids and Pre-Aids Patients. Surv. Immunol. Res. 1985, 4, 58– 62
        83
        Thymopentin treatment in AIDS and pre-AIDS patients
        Clumeck N; Cran S; Van de Perre P; Mascart-Lemone F; Duchateau J; Bolla K
        Survey of immunologic research (1985), 4 Suppl 1 (), 58-62 ISSN:0252-9564.
        Three pilot studies testing thymopentin in AIDS patients are presented. One study included 5 patients with the full-blown syndrome, all treated with 50 mg thymopentin 3 times a week by intravenous slow infusion; no immunologically nor clinically positive results were observed, indicating that the T cell pool in such patients is severely depleted. Six other patients with the prodromal stage of AIDS were treated 1 month with 50 mg thymopentin administered as an intravenous bolus injection 3 times weekly and thereafter for another month with same dose regimen as intravenous slow infusions. The patients on infusion therapy experienced statistically significant immunological improvements; these positive findings were paralleled with an improvement of the patients' clinical condition. These positive responses persisted for an average of 8 months. In another group of 5 pre-AIDS patients thymopentin was administered via the subcutaneous route using 15 mg 3 times weekly; only 1 patient revealed immunological and clinical improvement. In summary, only patients with the pre-AIDS syndrome are likely to benefit from immunomodulation therapy with thymopentin, and the mode of administration seems to be crucial.
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      The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jproteome.0c00383.

      • Figure S1. RMSD of the RBD-hACE2 complex backbone atoms from the X-ray structure during the MD simulation. Table S1. Difference in the binding free energies between the mutated and the native RBD-hACE2 complex obtained from the alanine scanning. Table S2. Compounds selected by docking on Mpro (6LU7 model) that failed during the MD/Nwat-MMGBSA rescoring step. Table S3. Compounds selected by docking on Mpro (QHD43415 homology model) that failed during the MD/Nwat-MMGBSA rescoring step. Table S4. Hydrogen bonds between RBD and hACE2 during the last half of the 20 ns MD simulation. Figure S2. Superposition of thymopentin docked to RBD BS2 and the PEPFOLD3 structure prediction. (PDF)


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