Exploring antiviral and anti-inflammatory effects of thiol drugs in COVID-19
Abstract
The redox status of the cysteine-rich SARS-CoV-2 spike glycoprotein (SARS-2-S) is important for the binding of SARS-2-S to angiotensin-converting enzyme 2 (ACE2), suggesting that drugs with a functional thiol group (“thiol drugs”) may cleave cystines to disrupt SARS-CoV-2 cell entry. In addition, neutrophil-induced oxidative stress is a mechanism of COVID-19 lung injury, and the antioxidant and anti-inflammatory properties of thiol drugs, especially cysteamine, may limit this injury. To first explore the antiviral effects of thiol drugs in COVID-19, we used an ACE-2 binding assay and cell entry assays utilizing reporter pseudoviruses and authentic SARS-CoV-2 viruses. We found that multiple thiol drugs inhibit SARS-2-S binding to ACE2 and virus infection. The most potent drugs were effective in the low millimolar range, and IC50 values followed the order of their cystine cleavage rates and lower thiol pKa values. To determine if thiol drugs have antiviral effects in vivo and to explore any anti-inflammatory effects of thiol drugs in COVID-19, we tested the effects of cysteamine delivered intraperitoneally to hamsters infected with SARS-CoV-2. Cysteamine did not decrease lung viral infection, but it significantly decreased lung neutrophilic inflammation and alveolar hemorrhage. We speculate that the concentration of cysteamine achieved in the lungs with intraperitoneal delivery was insufficient for antiviral effects but sufficient for anti-inflammatory effects. We conclude that thiol drugs decrease SARS-CoV-2 lung inflammation and injury, and we provide rationale for future studies to test if direct (aerosol) delivery of thiol drugs to the airways might also result in antiviral effects.
INTRODUCTION
SARS-CoV-2 is a novel coronavirus that causes COVID-19, a multidimensional disease characterized predominantly by pneumonia that can progress to respiratory failure and death (1, 2). The envelope glycoproteins of SARS-CoV-2 form trimeric spikes (SARS-2-S) that bind angiotensin-converting enzyme 2 (ACE2) for viral cell entry (3). Coronavirus spike glycoproteins, together with the envelope glycoproteins of myxo‐ and paramyxoviruses, retroviruses, and filoviruses belong to class I viral fusion proteins that exhibit similar structural features and mechanistic strategies to fuse viral and cellular membranes, which is critical for infectious viral entry (4). The capacity of class I viral fusion proteins to mediate membrane fusion often depends on a precise thiol/disulfide balance in the viral fusion proteins (5–8). Natural and specific thiol/disulfide rearrangements in the fusion proteins can trigger conformational changes that promote virus entry (9–11), but the removal of disulfide bridges by chemical reduction or by mutation of cysteines can also disrupt virus binding to receptors and therefore prevent infection. For example, chemical reduction of the S1 domain of SARS-CoV decreases its binding to ACE2 and inhibits infection of Vero E6 cells by SARS-CoV pseudoviruses, and replacing cystine-forming cysteines with alanines in the receptor binding domain (RBD) of SARS-CoV spike (hereinafter SARS-1-S) prevents its binding to ACE2 (8). In addition, it has been reported that disulfide bonds are critical to the structure and function of SARS-CoV-2 spike glycoprotein (12–18). Thus, there is consistent literature that manipulation of the redox status of the cysteine-rich glycoproteins on the virus surface can influence or impair viral infectivity. We have earlier reported that mucin disulfide cross links can be targeted by drugs with at least one functional thiol group (“thiol drugs”) to have mucolytic effects (19). This led us to consider that thiol drugs might also be able to cleave cystines in the RBD of SARS-2-S to disrupt its binding to ACE2. The reactive or cystine cleaving form of a thiol is its deprotonated (thiolate) form (20), and the pKa or acid dissociation constant of a thiol group determines the fractions of the protonated (–SH) form and the deprotonated (–S−) forms at the pH of the working solution. Alkyl thiols are weakly acidic with pKa > 9, with a low fraction of thiolate present at physiologic pH of 7.4. Thiol compounds with a lower pKa (i.e., approaching 7.4) will have more thiolate species at physiologic conditions and may be more potent in inhibition of SARS-CoV-2 infection.
Apart from the virology of SARS-CoV-2, the exaggerated immune response postinfection is also important in the pathophysiology of COVID-19. COVID-19-related pneumonia is characterized by a high neutrophil to lymphocyte ratio and high levels of reactive oxygen species (ROS), which cause lung injury (21). Corticosteroids and cytokine inhibitors decrease SARS-CoV-2-related lung injury (22–25), and corticosteroids are especially effective, most likely because of their broad anti-inflammatory effects (26, 27). Thiol drugs also have broad antioxidant and anti-inflammatory effects related to their ability to scavenge ROS and interrupt ROS-mediated inflammatory cascades, including pathways for NF-κB activation and cytokine and chemokine production (28–33). They can also inhibit the activity of myeloperoxidase, a mediator of inflammation and oxidative stress (34, 35). Cysteamine is a thiol drug with an expanded range of anti-inflammatory activities related to the inhibition of transglutaminase 2 and somatostatin (36, 37). Indeed, cysteamine decreases aeroallergen-induced lung inflammation in mice (38, 39), suggesting that it may decrease SARS-CoV-2-induced lung inflammation as well.
In this study, we screened the efficacy of thiol drugs in SARS-2-S-mediated receptor binding and virus infection assays, and we explored the role of thiol pKa as a physicochemical property of thiol drug potency in these assays. In addition, in SARS-CoV-2-infected hamsters, we explored the effects of cysteamine on SARS-CoV-2 lung titers and measures of lung inflammation.
MATERIALS AND METHODS
Cells Lines, Plasmids, and Viruses
HEK293T/clone17 (CRL-11268) was obtained from American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Thermo Fisher Scientific). Vero cells stably overexpressing transmembrane protease, serine 2 (TMPRSS2) [Vero E6-TMPRSS2; provided courtesy of Stefan Pölhmann (3)] were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin and 5 µg/mL blastidicin (BSD; InvivoGen). MEXi (IBA LifeSciences) were in MEXi culture medium (IBA Lifesciences) at 37°C, 5% CO2, and 125 RPM as described by the manufacturer. Baby hamster kidney cell line (BHK-21/WI-2; obtained from Kerafast) was cultured in DMEM supplemented in 10% FBS and 1% penicillin/streptomycin. All cells were incubated at 37°C and 5% CO2.
The codon-optimized SARS-CoV-2 spike gene was subcloned from pCG SARS-CoV-2 spike [pCG1-SARS-2-S; provided courtesy of Stefan Pölhmann (3)] into the Epstein-Barr nuclear antigen 1 (EBNA-1) dependent expression vector pTT5 for high-level expression in MEXi 293E cells, whereas pCG SARS-CoV-2 B.1.617 [provided courtesy of Stefan Pölhmann (40)] was used to produce B.1.617 variant pseudoviruses (PVs). To boost cell surface expression of SARS-CoV-2 spike for efficient pseudotyping vesicular stomatitis virus (VSV), the C-terminal 21 amino acid containing the endoplasmic reticulum (ER)-retrieval signal (KxHxx) of spike was deleted. For generating B.1.1.529 BA.1 omicron PVs, pTwist-SARS-CoV-2 Δ18 B.1.1.529 was used [a gift from Alejandro Balazs (Addgene plasmid No. 179907)]. The plasmid for generating BA.1 variant has 18 rather than 21 amino-acid C-terminal deletion.
SARS-CoV-2 {Wuhan-1 [isolate USA-WA1/2020 (NR-52281)] and Delta (isolate hCoV-19/USA/MD-HP05647/2021 Lineage B.1.617.2)} were obtained from Biodefense and Emerging Infections Research Resources Repository (BEI resources) and passaged in Vero E6-TMPRSS2 cells. Confluent Vero E6 cells grown in T175 flasks were infected with SARS-CoV-2, and the culture supernatant was collected when widespread cytopathic effect (CPE) was observed. After filtration through 0.45 μm filters, the virus-containing culture supernatant was stored at −80°C in small aliquots.
Thiol Drugs and Thiol Content Determination
n-acetylcysteine (NAC) and 2-mercaptoethane sulfonate sodium salt (Mesna) were used as the commercially available pharmaceutical formulations, with NAC manufactured by American Reagent INC at 200 mg/mL, pH 6.0–7.5 and Mesna by Baxter at 100 mg/mL, pH 7.5–8.5. Cysteamine (MilliporeSigma), amifostine (MilliporeSigma), WR-1065 (MilliporeSigma), and penicillamine (MP Biomedicals) were provided as lyophilized powders that were solubilized as 500 mM concentrated stocks in water. Cysteamine and WR-1065 solutions were at pH 5. Amifostine solution was at pH 7, which was adjusted to pH 5 using 1 M hydrochloric acid. To ensure that amifostine does not auto-dephosphorylate to WR-1065, the solution was made fresh before the experiment each time. Bucillamine (MilliporeSigma) and tiopronin (Spectrum Chemicals) were lyophilized powders that were solubilized as 500 mM concentrated stocks in equimolar NaOH to increase the solubility, and the pH was adjusted to pH 5. Carbocysteine (MilliporeSigma) and succimer (MilliporeSigma) were solubilized as 250 mM concentrated stocks in 500 mM NaOH to increase solubility with pH adjusted to pH 5. Methyl 6-thio-6-deoxy-α-d-galactopyranoside (TDG; lyophilized powder synthesized according to Ref. 19) was solubilized as 350 mM stock in water. Free thiol content, and thus concentration of an active drug, was measured before every experiment using Ellman’s reagent, 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB; Abcam), with the molar extinction coefficient of 14,150 M–1· cm−1 at 412 nm (41). Active drug concentration measured by DTNB was within 85%–99% of nominal drug concentration. The stock solutions were stored at –20°C and discarded if the thiol content went below 85%. Drug concentrations reported in plate-binding and viral infection assays are based on active drug concentration in the stock solution. The drugs were diluted in PBS for the plate assay and cell culture medium containing pseudoviruses or authentic viruses in the in vitro assays to ensure that all drugs are at physiological pH during the experiments.
RBD to ACE2 Plate Based Binding Assay
Wells of amine-reactive maleic anhydride-derivatized plates (Thermo Fisher Scientific) were coated overnight with recombinant SARS-CoV-2 receptor binding domain (Accession No. QHD43416.1, ACRO Biosystems). The following day, the plates were washed and blocked with bovine serum albumin (BSA) for 1 h at 37°C. Wells were then incubated for an hour at 37°C with drug solutions diluted in PBS at concentrations ranging from 0 to 20 mM. Negative controls included wells with no RBD or no ACE2. After washing, biotinylated soluble recombinant ACE2 (ACRO Biosystems) was added and incubated at 37°C for 60 min. After washing, streptavidin-horseradish peroxidase (HRP; ACRO Biosystems) was added to wells for an hour at 37°C. The plates were washed and incubated with 3,3′,5,5′-tetramethylbenzidine (TMB; obtained from Sera Care). The reaction was stopped, and absorbance was read at 450 nm on a Biotek plate reader. Absorbance readings, after subtracting from negative control wells, were transformed to percent binding, with the wells containing no drug set as 100% binding. To measure the stability of binding of cysteamine, WR-1065, Mesna, and bucillamine, wells were incubated with either of the drugs at 5 mM for 1 h, followed by three washes. ACE2 was then added to the wells either immediately, after 60 min, or after 120 min. Wells waiting for ACE2 were filled with dilution buffer. This was followed by the same steps to assess ACE2 binding as described earlier. For all binding assays, four to six independent experiments were carried out for all drugs.
BODIPY FL l-Cystine Cleaving Assay
BODIPY FL l-cystine (Thermo Fisher Scientific) was reconstituted in methanol. In a black Maxi Sorp 96-well flat-bottomed plate (Nunc), 10 µM of BODIPY reagent prepared in PBS was mixed with 25 µM of the thiol drugs, and the change in fluorescence was kinetically measured with excitation at 490 nm and emission at 520 nm, at 1 min intervals, for 1 h at 37°C. Fluorescence reads, after subtracting the drug-free control reads, were plotted against time. The maximum slope (Max V) over a 10 min interval for all the thiol drugs from this plot was calculated and represented as relative fluorescence units/minute (RFU/min) to assess the cystine cleaving ability of the drugs. The experiment was repeated three times.
Production of Pseudoviruses
Pseudoviruses bearing SARS-2-S were generated as previously described using recombinant VSVΔG-luciferase-based viruses, which lack glycoprotein (G) gene and instead code for reporter gene firefly luciferase (42). Briefly, either MEXi or BHK-21/WI-2 cells were transfected with SARS-CoV-2 spike expression plasmid (pTT5 SARS-CoV-2 SΔ21), using polyethylenimine (PEI) or TransIT-2020 (Mirus Bio) as described by the manufacturer. Mock transfection served as the “no glycoprotein” control. 24 h after transfection, the cells were inoculated with VSVΔG-luc (VSV-G) at a multiplicity of infection (MOI) of 0.3–3. After 6 h of incubation, the cells were washed three times with PBS and resuspended in a culture medium containing 1% I1 anti-VSV-G hybridoma supernatant (ATCC CRL-2700). 24 h after infection, the culture supernatant was collected by centrifugation and filtered through a 0.45 μm syringe filter to clear off cellular debris. The supernatant containing viral particles was aliquoted and stored at –80°C until further use.
Establishment of HEK293T Cells Stably Expressing ACE2 and TMPRSS2 (HEK293T-ACE2-TMPRSS2)
ACE2 was cloned into pLKO5d.SFFV.dCas9-KRAB.P2A.BSD (a gift from Dirk Heckl, Addgene plasmid), and TMPRSS2 was cloned into pDUAL CLDN [green fluorescent protein (GFP); a gift from Joe Grove, Addgene plasmid]. All cloning steps were confirmed by Sanger sequencing. Lentiviral particles for delivery of lentiviral ACE2 and TMPRSS2 vectors were produced using transfection with PEI. HEK293T cells were transfected with three plasmids: lentiviral ACE2 or TMPRSS2 constructs, psPAX2, and pVSV-G. Forty-eight hours after transfection, cell supernatants containing the newly produced viral particles were centrifuged and subsequently filtered using 0.22 µm vacuum filter units (MilliporeSigma). The supernatants were then aliquoted and stored at −80°C. To establish HEK293T-ACE2-TMPRSS2 cells, HEK293T cells were transduced with lentiviral particles containing the ACE2 vector. Forty-eight hours after transduction, medium was replaced with blastidicin (BSD; InvivoGen) selection medium. After 5 days of selection, further expansion of cells stably expressing ACE2 was carried out. The process was then repeated to further transduce cells with TMPRSS2 lentiviral particles, and cells were cultured in antibiotic selection medium containing BSD and puromycin 48 h posttransduction. The expression of ACE2 and TMPRSS2 was confirmed by Western Blot and compared with nontransduced cells.
Pseudovirus Transduction Experiments
For pseudovirus pretreatment, the pseudoviruses were preincubated with different concentrations (1.56–100 mM) of the thiol drugs for 2 h at 37°C, followed by 66-fold dilution with standard culture media. The cells were then transduced with these pretreated virions for 2 h at 37°C. After the incubation, the virions were removed, and cells were cultured in a standard culture medium. For cell pretreatment, the HEK293T-ACE2-TMPRSS2 cells were incubated with the different drug concentrations (0.02–1.5 mM) for 2 h at 37°C, 5% CO2. These concentrations reflect the 66-fold dilution of drugs when the virus/drug mix was incubated with the cells in the pseudovirus pretreatment experiment. After incubation, the media was removed, and the cells were transduced with untreated pseudoviruses for 2 h at 37°C. After the incubation, the virions were removed, and the cells were cultured in a standard culture medium. For both experimental conditions, at 18 h posttransduction, the cells were lysed and luciferase activity was measured using Promega luciferase assay system and Biotek Synergy H1 plate reader. Data were normalized to the viral particles without any viral envelope protein. For each experiment, luciferase reads of drug free group were set as 100%, and the relative transduction efficiencies in the presence of thiol-based drugs were calculated. Three to five independent experiments were carried out for each PV pretreatment and cell pretreatment strategies, with 6–12 replicates in each for all the drug concentrations.
Inhibition of Authentic SARS-CoV-2 Infection
For the optimized live virus experiments (Fig. 4, J and K and Fig. 5, H–M), SARS-CoV-2 (Wuhan-1 and Delta) of 1.5 × 103 TCID50/mL (50% Tissue Culture Infectious Dose/mL) was incubated with threefold serially diluted carbocysteine and cysteamine (0.007–150 mM) at 37°C for 2 h. Virus-drug mixtures were diluted 24-fold before addition to Vero E6-TMPRSS2 cell monolayer in 96-well plates. For each drug concentration, virus-drug mixtures were added to 10 replicate wells at 100 μL/well. The final titer of virus added to cells was 63.2 TCID50/mL. After 2 h of infection, virus-drug inoculum was replaced with fresh DMEM medium containing 2% FBS. Clear CPE developed after 7 days of incubation at 37°C with 5% CO2. The experiment was repeated thrice. Wells with clear CPE were counted positive, and percentage of positive wells for each concentration of tested drugs was plotted. The effect of thiol drugs on Vero E6-TMPRSS2 cells during the 2 h of SARS-CoV-2 infection was evaluated by the addition of 6.25 mM of each drug and 63.2 TCID50/mL SARS-CoV-2 simultaneously to Vero E6-TMPRSS2 cell monolayer in 96-well plates. This concentration reflects the highest concentration, post 24-fold dilution of drugs when virus/drug mix was incubated with the cells in the virus pretreatment experiment. After 2 h of infection, cells were washed and then cultured with fresh DMEM medium containing 2% FBS at 37°C with 5% CO2. Clear CPE developed 7 days after infection. Details about the preliminary experiment are in the online Supplemental material.
Quantification of Cell Viability
For all cell viability experiments, the experimental protocol was the same as the main experiment except for the step of pseudovirus or authentic virus infection. For cell viability measurement corresponding to pseudovirus experiments, HEK293T-ACE-TMPRSS2 cells were seeded in 96-well black plates 18 h before the experiment. The cells were then incubated with different concentrations (0.02–1.5 mM) of the thiol drugs for 2 h at 37°C, followed by removal of the drugs and incubation of cells with a standard culture medium for 18 h. The experiment was carried out thrice with five to six replicates in each for all the drug concentrations. These concentrations reflect the 66-fold dilution of drugs when pseudovirus/drug mix was incubated with the cells in the pseudovirus pretreatment setting. For the cell viability measurement corresponding to the live virus experiment (Fig. 4, J and K and Fig. 5, H–M), Vero E6-TMPRSS2 cells were incubated with different concentrations (0.0003–6.25 mM) of thiol drugs for 2 h, followed by removal of the drugs and incubation of cells with culture medium containing 2% FBS for 7 days. These concentrations reflect the 24-fold dilution of drugs when the virus/drug mix was incubated with the cells in the live virus infection setting. The cell viability experiment on Vero E6-TMPRSS2 cells was carried out thrice with six replicates in each for all the drug concentrations. For all cell viability experiments, after the respective incubations, the plates and their contents were equilibrated at room temperature for 30 min before the addition of equal volumes of CellTiter Glo2.0 reagent. Afterwards, the contents were mixed on a plate shaker to induce cell lysis. The plates were then incubated at room temperature for 10 min followed by a measurement of luminescence using Biotek plate reader. Luciferase reads of control-treated cells were set as 100%, and the relative viability of cells incubated in the presence of thiol drugs was calculated.
Studies in Syrian Hamsters
The hamster studies reported here were performed in an animal biosafety level 3 (ABSL3) facility at the Lovelace Biomedical Research Institute (LBRI) and conducted under protocols approved by the Institutional Animal Care and Use Committee (IACUC). Twenty Syrian hamsters (Mesocricetus auratus; 10 males and 10 females), with a target age of 6–10 wk old, and a body weight of 115–136 g (means ± SD = 126.1 g ± 6 g), were used in the study. The sample size is based on the published literature (43, 44) and the experience of the LBRI investigators in conducting drug efficacy studies for COVID-19 in hamsters. Animals were placed in groups using Provantis with stratified (body weight) randomization so that animals with similar body weights were included in the study. The animals were divided into two groups: receiving intraperitoneal dosing of the vehicle (water) and receiving cysteamine hydrochloride (147 mg/kg; equal to 100 mg/kg cysteamine free base). The 100 mg/kg dose of cysteamine (MilliporeSigma) was administered to hamsters based on the conversion of human daily dose (2 g/day) to animal dose based on body surface area using FDA guidance (45). The animals were dosed twice daily for 5 days, as shown in Fig. 7A, with the first dose administered 2 h before viral inoculation. All animals were inoculated intranasally with SARS-CoV-2 (isolate USA-WA1/2020 or Wuhan-1) at 2.94 × 104 TCID50/animal. Efficacy of the drug was determined by measuring viral load by RT-qPCR and lung inflammation, as measured by lung weight gain, total and differential cell counts in the bronchoalveolar lavage (BAL) fluid, and histopathological analysis of the lung sections. Blinding to groups was not possible for this study. No data were excluded; two BAL samples from cysteamine-treated group were not analyzed due to a technical error in collecting the BAL.
Bronchoalveolar Lavage Collection and Processing
BAL was performed on the right lung lobe. The left lobe was clamped off and the right lung lobes were lavaged with sterile saline. Half of the BAL collected was UV irradiated for sterilization out of the ABSL3 and used for differential analysis. Irradiation was carried out at LBRI using a protocol adapted from the article by Darnell et al. (46), which shows that UV-C light > 5 min (4,016 µW/cm2, where µW = 10−6 J/s) inactivated SARS-CoV. In our study, the BAL samples were irradiated by placing them in a UV crosslinker for 10 min at a constant energy of 5,000 µJ/cm2. The inactivation of the viruses with such treatment has already been confirmed at LBRI by infecting VeroE6 cells with 100% inactivated samples and testing cytopathic effects at up to 5 days postinfection. The UV-treated aliquot was centrifuged at 1,000 g, 2°C–8°C, ≥10 min. The cell pellet was resuspended in the appropriate amount of resuspension buffer, and red blood cell lysis buffer was used on samples as necessary. The total cell count was counted using a Nexcelom automated cell counter. A total of 50,000 cells per slide were used to prepare microscope slides by cytocentrifugation. The cells on slides were fixed and stained using Modified Wright’s or Wright-Giemsa Stain. Differential counts on at least 200 nucleated cells per slide were conducted using morphological criteria to classify cells into neutrophils, macrophages, lymphocytes, and eosinophils.
Histopathology
At scheduled necropsy, animals were euthanized by intraperitoneal injection with an overdose of a barbiturate-based sedative. The right lobes of the lungs were used for BAL collection and processed to measure viral loads. The left lobes of the lungs were collected, examined, and weighed, and representative samples were preserved for histopathology. Lung lobes were instilled via major airway(s) with 10% neutral buffered formalin (NBF) to approximate physiologic full lung volume at 25 cm hydrostatic pressure; the major airway(s) used for instillation were closed, and the lobes were immersed in NBF for fixation. Tissues were trimmed and processed routinely, paraffin-embedded, sectioned at 4 µm, and stained with hematoxylin and eosin for microscopic examination. Histopathologic examination was conducted on the left lung lobes with findings for a given tissue graded subjectively and semiquantitatively by a single board-certified veterinary pathologist on a scale of 1–5 (1 = minimal, 2 = mild, 3 = moderate, 4 = marked, 5 = severe). The Provantis v10.2.3.1 (Instem LSS Ltd., Staffordshire, England) computer software/database was used for necropsy and histopathology data acquisition, reporting, and analysis.
Viral Titers Using RT-qPCR
Lung samples were homogenized in TRIzol using a TissueLyser and centrifuged at 4,000 g for 5 min. From the supernatants, RNA was isolated using the Direct Zol-96 RNA Kit (Zymo Research), according to the manufacturer’s instructions.
SARS-CoV-2 viral RNA was quantified by a qPCR assay targeting the SARS-CoV-2 nucleocapsid phosphoprotein gene (N gene). Genome copies per gram (g) equivalents were calculated from a standard curve generated from RNA standards of known copy concentration. All samples were run in triplicate. The SARS-CoV-2 N gene primers and probe sequences are as follows: SARS-CoV-2 forward: 5′- TTACAAACATTGGCCGCAAA-3′; SARS-CoV-2 reverse: 5′- GCGCGACATTCCGAAGAA-3′; SARS-CoV-2 probe: 6FAM- ACAATTTGCCCCCAGCGCTTCAG-BHQ-1.
Amplification and detection were performed using a suitable real-time thermal cycler under the following cycling conditions: 50°C for 5 min, 95°C for 20 s, and 40 cycles of 95°C for 3 s, and 60°C for 30 s.
Statistical Analyses
For analyzing the statistical significance of difference in loss of binding for each drug in the plate binding assay, area under the curve (AUC) was plotted, and ordinary one-way ANOVA followed by Dunnett’s post hoc analysis was performed. For the in vitro pseudovirus and authentic virus assays, IC50 of the drugs was determined using the nonlinear regression fitting with a variable slope (details in the online Supplemental material). For the in vivo data, statistical significance was analyzed by two tailed, unpaired t test between cysteamine treated and the control groups. All statistical analyses were performed using GraphPad Prism software (v. 9.2).
RESULTS
Cystine Bridges in the S1 Domain of SARS-2-S and Point Mutations in SARS-CoV-2 Variants
In examining literature data for cystine bridges in the S1 domain of SARS-CoV-2 spike (SARS-2-S) and by comparing the amino acid alignment of the RBDs in SARS-2-S and SARS-CoV spike (SARS-1-S), we generated a schematic of the 10 cystine bridges in the S1 domain of SARS-2-S (Fig. 1A) and the four conserved cystines between SARS-1-S and SARS-2-S RBD (Fig. 1B). The conserved Cys467-Cys474 in SARS-1-S and Cys480-Cys488 in SARS-2-S constrain the ACE2 binding domains, and previous studies with SARS-1-S RBD have shown that mutagenesis of either conserved cysteine leads to loss of ACE-2 binding (8). To further explore if Cys480-Cys488 in SARS-2-S might be vulnerable to chemical cleavage, we used protein modeling software to render the SARS-2-S RBD based on the Protein Data Bank (PDB) entry 6M0J (Fig. 1C). Space filling images and receptor distance calculations were performed using indicated PDB entries with UCSF Chimera (47). The rendering shows that Cys480-Cys488 is very near the RBD surface (Fig. 1C) and could be accessible to cleavage by thiol drugs. Apart from Cys480-Cys488 cystine, cleavage of the other six cystines in the RBD could also allosterically modify the binding interface in ways that decrease its binding to ACE2. Studies in SARS-CoV have shown that cysteine residues flanking the S2 domain, including Cys822 and Cys833, are important for membrane fusion of SARS-CoV (48). Amino acid alignment of the spike protein of SARS-CoV and SARS-CoV-2 shows that these cysteine residues are conserved in the spike protein of SARS-CoV-2 (Cys 840 and Cys 851; data not shown), which raises the possibility that thiol drugs could inhibit membrane fusion in addition to inhibitory effects on receptor binding. SARS-CoV-2 variants are classified as variants of interest (VOI) or variants of concern (VOC) based on their increased transmissibility and/or evasion from natural or therapeutic antibodies (49), and Fig. 1D shows the point mutations in the RBD of SARS-CoV-2 VOIs and VOCs.
Figure 1.Cystine mapping and conservation of cystines in RBD of SARS-1-S and SARS-2-S. A: cystine map for SARS-2-S domain S1 (UniProt: P0DTC2). Ten cystine linkages are denoted by dashed lines with amino acid residue number above. The dark gray region is the RBD, and the lighter gray box highlights the ACE2 binding motif, a cluster of amino acids that make contact with ACE2. B: amino acid alignment of SARS-2-S RBD domain (aa 319-541, PDB: 6M0J) and SARS-1-S RBD domain (aa 306-517, PDB: 3SCI). Residues that are shared are highlighted by black boxes, and residues that represent a similar amino acid class replacement are bound by gray boxes. The solid lines link cystine-forming cysteines. The solid red line and red numbers highlight the conserved critical cystine bridge in the RBDs for both viruses. *Amino acids that are within 4 Å of ACE2 in their respective solved structures. C: a surface rendering of SARS-2-RBD (PDB: 6M0J) oriented with the ACE2 binding region (blue) facing forward. D: amino acid sequence of SARS-2-S RBD highlighting the RBD mutations identified in the circulating SARS-CoV-2 variants. Amino acids are noted with single letter code and sequence number. The conserved RBD cystine (C480-C488) is highlighted in red lines. The amino acid mutations are highlighted in red with the variant designations in blue. BA.1 and BA.2 represent the subvariants of B.1.1.529 Omicron variant. ACE2, angiotensin-converting enzyme 2; RBD, receptor binding domain.
Thiol Drugs Cleave Cystines in the RBD of SARS-2-S to Disrupt Binding to ACE2
To test if thiol drugs cleave cystines in the RBD of SARS-2-S to disrupt its binding to ACE2, we exposed the RBD of the ancestral SARS-CoV-2 Wuhan-1 isolate (RBDWuhan-1) to eight thiol drugs and quantified ACE2 binding affinity in a plate-based binding assay. Carbocysteine and amifostine were included as negative controls because carbocysteine is a sulfur-containing drug lacking a free thiol warhead, and amifostine is a phosphorothioate prodrug whose dephosphorylated metabolite (WR-1065) is the active form of the drug (Fig. 2). A commercially available ACE2-SARS-2-S RBD binding assay was optimized by covalently coupling RBDWuhan-1 to plates functionalized with primary amine-reactive maleic anhydride, and ACE2 binding was quantified after RBDWuhan-1 exposure to drugs for 60 min (Fig. 3A). We found that all of the thiol drugs inhibited binding of RBDWuhan-1 to ACE2 in a dose-dependent manner. Penicillamine and succimer had relatively weak inhibitory effects (Supplemental Fig. S1), but 2-mercaptoethane sulfonate sodium salt (Mesna), bucillamine, cysteamine, and WR-1065 had much stronger effects (Fig. 3, B and C), which was retained for 2 h (Fig. 3D).
Figure 2.Currently approved thiol drug compounds or drugs that generate an active thiol-containing drug metabolite. The chemical name along with the chemical structures, thiol pKa values (from published literature and PubChem), routes of administration, clinical indications, and clinical doses are provided. Two thiol pKa values are provided for bucillamine and succimer because these drugs have two thiol groups. The clinical doses provided for NAC and Mesna are those used for inhalation route. Two compounds, amifostine and erdosteine, are prodrugs that can convert to active metabolites. Carbocysteine is not a thiol drug but it is a sulfur containing drug that we use in our experiments as a negative control because it does not have a free thiol warhead. Not shown here are three thiol containing drugs (captopril, zofenopril, and racecadotril) in which primary mechanisms of action is not through reactions with the thiol group. Mesna, 2-mercaptoethane sulfonate sodium salt; NAC, n-acetylcysteine.
Figure 3.Binding of SARS-CoV-2 RBD to ACE2 is inhibited by thiol drugs. A: schematic representation of the SARS-CoV-2 RBD to ACE2 binding assay. B: percentage of binding of RBDWuhan-1 to ACE2 in the presence of the drugs (n = 4–6 independent experiments). C: histograms denoting areas under the curve (AUC) analyses for binding studies in B, in which the AUC is the percent binding of ACE2 to RBD in the presence of each drug. The reference AUC is determined from the percent binding of ACE to RBD in the absence of drug. The dashed line represents 50% of reference AUC. D: binding of RBDWuhan-1 to ACE2 at 1 and 2 h after drug exposure and washout (n = 4–5 independent experiments). Statistical significance for C was analyzed by one-way ANOVA followed by Dunnett’s post hoc analysis. Data are means ± SE. Significance indicates differences from reference AUC. **P ≤ 0.01, ****P ≤ 0.0001. ACE2, angiotensin-converting enzyme 2; HRP, horseradish peroxidase; RBD, receptor binding domain.
Thiol Drugs Inhibit Infection with Wuhan-1 Pseudovirus and Wuhan-1 Authentic Virus
Vesicular stomatitis virus (VSV) derived pseudovirus (PV) particles carrying SARS-2-S on their surface lack attachment glycoprotein (G) of VSV and instead code for reporter gene firefly luciferase (42). We tested if thiol drugs could inhibit infection of cells with SARS-2-PV particles bearing spike from ancestral strain (Wuhan-1). We utilized HEK293T cells that were engineered to stably express human ACE2 and transmembrane protease, serine 2 (TMPRSS2, a priming serine protease for SARS-CoV-2; HEK293T-ACE2-TMPRSS2; 3). To determine the effect of thiol drugs on the virus, we pretreated the pseudoviruses with thiol drugs for 2 h, diluted the drug/virus mix, and infected the cells. To ensure that the effect seen is because of the drug action on the virus and not the host cells, we 1) diluted the drug/virus mix before infecting the cells, 2) included a control experiment where we pretreated the cells with the diluted thiol drugs for 2 h, followed by removal of the drugs and transduction with untreated pseudoviruses (Supplemental Fig. S2). We found that none of the drugs significantly affected cell viability, and pretreatment of Wuhan1-PV with carbocysteine and amifostine controls did not inhibit virus entry into the cells (Fig. 4, A and B). In contrast, pretreatment of Wuhan1-PV with thiol drugs significantly inhibited virus entry in a dose-dependent manner (Fig. 4, C–H). Cysteamine and WR-1065 were much more effective than n-acetyl cysteine (NAC) and tiopronin (Fig. 4I). Thiol drugs had only small and inconsistent effects on PV infectivity when the HEK293T-ACE2-TMPRSS2 cells were pretreated with thiol drugs and then infected with untreated Wuhan1-PV (Supplemental Fig. S3, A–H). Together, these data show that thiol drugs act mainly on the viral spike protein and not on any host cell proteins. To confirm that data with Wuhan1-PV extend to authentic Wuhan-1 virus, we initially carried out dose-ranging experiments to preliminarily test the effects of the most potent thiol drugs (Mesna, bucillamine, WR-1065, and cysteamine) on SARS-CoV-2 (Wuhan-1) infection in Vero E6 cells. We found that cysteamine and WR-1065 were very effective at decreasing virus-induced cytopathic effects (Supplemental Fig. S4, A–E). In a subsequent dose-optimized experiment, we tested the effects of cysteamine on SARS-CoV-2 Wuhan-1 virus infection of Vero E6 cells stably expressing TMPRSS2 (Vero E6-TMPRSS2). We found that cysteamine significantly decreased virus-induced cytopathic effects, whereas carbocysteine did not (Fig. 4, J and K). For both preliminary and optimized experiments, inhibition of infection was minimal when the respective cells were treated with the diluted thiol drugs and infected with SARS-CoV-2 Wuhan-1 virus (Supplemental Fig. S4, F and G).
Figure 4.Thiol drugs inhibit ancestral SARS-CoV-2 in vitro. Pseudovirus (PV) entry efficiency after pseudovirus exposure to carbocysteine (sulfide drug, negative control; A), amifostine (parent drug of WR-1065, negative control; B), thiol drugs NAC (C), tiopronin (D), Mesna (E), bucillamine (F), WR-1065 (G), and cysteamine (H) before transduction into HEK293T-ACE2-TMPRSS2 cells (n = 3–4 independent experiments). The effects of drugs on cell viability were quantified using Cell Titer Glo 2.0 with lower drug dose exposures, reflecting 66-fold dilution of drugs when pseudovirus/drug mixture was incubated with cells (n = 3 independent experiments). I: IC50 values of the different thiol drugs in the PV assay. Cytopathic effects (CPE) quantified by visual inspection when authentic SARS-CoV-2 is exposed to carbocysteine (negative control; J) and cysteamine (thiol drug; K) before infection in Vero E6-TMPRSS2 cells (n = 3 independent experiments). The effects of drugs on VeroE6-TMPRSS2 cell viability were quantified with exposure of cells to lower drug doses, reflecting the 24-fold dilution of drugs when virus/drug mixture was incubated with cells (n = 3 independent experiments). The X-axes are scaled to log10—the lower X-axis refers to the concentration of drugs on the PV or authentic virus, and the upper X-axis refers to concentration of drugs on the cells post dilution. Percentage changes are with respect to no drug control which is set as 100%. IC50 of the drugs was determined using the nonlinear regression fitting with a variable slope. Data are means ± SD. Mesna, 2-mercaptoethane sulfonate sodium salt; NAC, n-acetylcysteine; TMPRSS2, transmembrane protease, serine 2.
Thiol Drugs Inhibit Entry Mediated by SARS-2-S from Multiple Variants
We tested the most potent thiol drugs in SARS-2-PV assays with spikes from B.1.617.2 (Delta variant), the related B.1.617.1 (Kappa variant), and the recent variant of concern circulating globally—B.1.1.529 BA.1 (Omicron variant). In addition to Mesna, bucillamine, cysteamine, and WR-1065, we included a novel thiol saccharide compound [methyl 6-thio-6-deoxy-α-d-galactopyranoside (TDG)], previously proposed by us as a novel mucolytic because of its favorable properties as an inhaled drug (19). We found that cysteamine and WR-1065 were the most potent inhibitors of the entry of the PVs (Fig. 5, A–G). Pretreatment of cells followed by transduction with untreated PV had no significant effect (Supplemental Fig. S5, A–F). The IC50 values for thiol drugs to inhibit Delta variant entry were higher than those for Kappa and Omicron BA.1 (Fig. 5, A–F). With Omicron BA.1 variant, the IC50 values were lower for Mesna and bucillamine; TDG, cysteamine, and WR-1065 had similar inhibitory effects on Omicron BA.1 and Kappa variant, with cysteamine and WR-1065 being most potent having IC50 values in low millimolar range. Overall, the order of potency was similar in all PV assays (Fig. 5G).
Figure 5.Thiol drugs inhibit Delta SARS-CoV-2 in vitro. Pseudovirus (PV) entry efficiency when the B.1.617.1, B.1.617.2, and B.1.1.529 BA.1 pseudoviruses were exposed to carbocysteine (sulfide drug, negative control; A), thiol drugs Mesna (B), bucillamine (C), TDG (D), cysteamine (E), and WR-1065 (F) before transduction into HEK293T-ACE2-TMPRSS2 cells (n = 3–5 independent experiments). The X-axes are scaled to log10. Percentage changes are with respect to no drug control which is set as 100%. IC50 of the drugs was determined using the nonlinear regression fitting with a variable slope. Data are means ± SD. G: IC50 values of the thiol drugs in the PV transduction assay and their respective thiol pKa values. Bucillamine has 2 pKa values corresponding to the two thiol groups in its structure. Data are means ± SE. Cytopathic effects (CPE) quantified by visual inspection when authentic Delta SARS-CoV-2 is exposed to carbocysteine (sulfide drug, negative control; H), thiol drugs Mesna (I), bucillamine (J), TDG (K), cysteamine (L), and WR-1065 (M) before infection in Vero E6-TMPRSS2 cells (n = 3 independent experiments). The effects of drugs on VeroE6-TMPRSS2 cell viability were quantified with exposure of cells to lower drug doses, reflecting the 24-fold dilution of drugs when virus/drug mixture was incubated with cells (n = 3 independent experiments). The X-axes are scaled to log10—the lower X-axis refers to the concentration of drugs on the authentic virus, and the upper X-axis refers to concentration of drugs on the cells post dilution. Percentage changes are with respect to no drug control which is set as 100%. IC50 of the drugs was determined using the nonlinear regression fitting with a variable slope. Data are means ± SD. Mesna, 2-mercaptoethane sulfonate sodium salt; TDG, methyl 6-thio-6-deoxy-α-d-galactopyranoside; TMPRSS2, transmembrane protease, serine 2.
To confirm that data with Delta-PV extend to authentic Delta virus, we tested the effect of thiol drugs on authentic Delta virus infection in Vero E6-TMPRSS2 cells. We found that the drugs inhibited cell infection with an order of drug potency similar to the PV assay, but the IC50 values were lower with the authentic Delta virus than with the Delta pseudovirus (Fig. 5, H–M). The drugs had minimal effects on the inhibition of infection when the cells were treated with the diluted thiol drugs and infected with Delta SARS-CoV-2 (Supplemental Fig. S5G).
Because cysteamine potently inhibited ancestral, Delta and Omicron BA.1 variants, we also tested its ability to inhibit entry of PVs for D614G, P.1., B.1.1.7, B.1.429, and B.1.351. As shown in Supplemental Fig. S6, A–C, cysteamine was most potent against the Wuhan-1, Wuhan-1-D614G, Kappa, and Omicron BA.1 variants. The entry of the PV variants was not affected when the cells were pretreated with cysteamine before transduction with untreated PV (Supplemental Fig. S6, D and E).
pKa Is A Determinant of Thiol Drug Efficacy in SARS-2-PV Assays
We noted that the potency of the thiol drugs was inversely related to their thiol pKa values (Fig. 5G), suggesting that a key physicochemical property of an optimal thiol drug to inhibit SARS-CoV-2 entry is a lower thiol pKa that is approaching the physiological pH. To further build support for this concept, we compared the efficacy of cysteine derivatives with a low pKa [e.g., cysteine methyl ester (CME), pKa = 7.0] (50) and a high pKa (e.g., NAC, pKa = 9.5) in a B.1.617.1 PV assay. We found that the IC50 for CME was 25-fold lower than the IC50 for NAC and that l-cysteine (pKa = 8.4) (50) had intermediate potency (Fig. 6, A–C, and Supplemental Fig. S7, A–C). These data indicate that the mechanism of action of thiol drugs as SARS-CoV-2 inhibitors is the cleavage of disulfide bridges in SARS-2-S. To demonstrate that the cystine cleaving potency of these different thiol drugs relates to their thiol pKa, we leveraged the BODIPY FL l-cystine reagent, which fluoresces when thiol-specific exchange leads to mixed disulfide formation (Fig. 6D). We observed similar order of potency in the BODIPY assay as compared with the RBD-ACE2 plate-binding assay, but the thiol drugs were effective at lower concentrations in the BODIPY assay. Several factors could explain this difference. First, the BODIPY reagent has one cystine, whereas the RBD molecule has four. Second, the BODIPY assay measures kinetics of cystine cleavage by thiol reagents in solution, whereas the RBD-ACE2 assay measures the effect of a thiol drug at a single timepoint on surface-bound cystines in the RBD. Third, in the RBD-ACE2 binding assay, the RBD cleaved by the thiol drugs is hindered in its binding to biotinylated ACE2, which is indirectly measured using a streptavidin-HRP system. Importantly, we found that the cystine cleavage rates of thiol drugs in the BODIPY assay were inversely related to their pKa values (Fig. 6, E and F) and mirrored the potency order seen in the SARS-2-PV assays. An outlier was TDG, which has a pKa of 9.6 but fivefold higher potency than compounds with similarly high pKa values (NAC, Mesna). pKa is therefore not the only property that determines the potency of a thiol drug; interactions between the thiol and RBD interface, hydrophilic or hydrophobic variations in each cystine microenvironment, or steric factors may also affect potency.
Figure 6.Thiol pKa influences the cystine cleaving rates of the thiol drugs and their efficacy in B.1.617.1 PV transduction assay. PV entry efficiency when B.1.617.1 pseudoviruses were treated with three cysteine derivates n-acetyl cysteine (A), l-cysteine (B), and l-cysteine methyl ester (CME; C) having variable thiol pKa (n = 3–6 independent experiments). The effects of cysteine derivatives on cell viability were quantified using Cell Titer Glo 2.0 with lower drug dose exposures, reflecting 66-fold dilution of drugs when pseudovirus/drug mixture was incubated with cells (n = 3 independent experiments). Data are means ± SD. IC50 of the drugs was determined using the nonlinear regression fitting with a variable slope. D: schematic representation of the BODIPY cystine assay. E: rate of BODIPY FL l-cystine cleavage when exposed to thiol drugs or controls (carbocysteine and amifostine; n = 3 independent experiments). Dotted lines indicate SE. F: maximum slope (Max V) for the reactivity of thiol drugs with the BODIPY FL cystine reagent (based on data in E). Numbers on graph (E) correspond with numbers designated to thiol compounds in F. Data in F are means ± SE. Pseudovirus, PV.
Cysteamine Reduces Lung Inflammation in Hamsters Infected with SARS-CoV-2
Golden Syrian hamsters (Mesocricetus auratus) infected with SARS-CoV-2 develop diffuse alveolar destruction, airway and alveolar infiltration with inflammatory cells (macrophages, lymphocytes, and neutrophils), and alveolar hemorrhage (43, 44, 51–53). The hamster model is, therefore, a valuable model to test if drugs have therapeutic potential against SARS-CoV-2 infection. We tested if cysteamine reduces viral load or lung inflammation in the hamster model of SARS CoV-2 infection. We chose cysteamine as the thiol drug to test in these in vivo experiments for two reasons. First, cysteamine was among the most potent thiol drugs in our in vitro antiviral assays described earlier. Second, cysteamine has a broad range of antioxidant and anti-inflammatory activities (29, 30, 37), and it reduces aeroallergen-induced lung inflammation in mice (38, 39).
Cysteamine was administered intraperitoneally (ip) to hamsters at a dose of 100 mg/kg to approximate the doses used clinically to treat cystinosis (54, 55; Fig. 7A). The first dose was given 2 h before intranasal viral inoculation of SARS-CoV-2 (Wuhan-1, 2.94E + 04 TCID50/animal) with subsequent doses given twice daily for 5 days (Fig. 7A). We found that multiple measures of lung inflammation were lower in cysteamine-treated animals. Lung weights in cysteamine-treated hamsters were significantly lower than in the control group (Fig. 7C, Supplemental Fig. S8), and total protein in BAL was also lower (Fig. 7D). Total cell counts in BAL from cysteamine-treated animals were also lower than in control-treated animals with effects driven by decreases in neutrophils and lymphocytes (Fig. 7, E–I). In addition, histopathology scores for mixed cell lung inflammation, composed primarily of macrophages and neutrophils, in lung tissue from cysteamine-treated animals were significantly lower than in the control group (Fig. 7J). Furthermore, alveolar hemorrhage scores from cysteamine-treated animals were significantly lower than in the control group (Fig. 7, K and L). In contrast, viral N2 gene transcripts in lung tissue from cysteamine-treated animals were not significantly lower than in controls (Fig. 7B).
Figure 7.Effects of cysteamine on a Syrian hamster model of SARS-CoV-2 infection. A: study design for assessing the effect of cysteamine in Syrian hamster model of COVID-19. Cysteamine (100 mg/kg) was administered twice daily via intraperitoneal injection for 5 days, with the first dose given 2 h before the virus inoculation on Day 0. B: viral RNA levels in the lungs of animals treated with cysteamine relative to the vehicle control group. C: lung weights, normalized to the terminal body weights, of the animals. Total protein (D) and total cell counts (E) in the BAL fluid of hamsters treated with cysteamine with respect to the vehicle controls. Differential leukocyte counts in the BAL fluid of animals, showing neutrophil (F), macrophage (G), lymphocyte (H), and eosinophil (I) counts in treated and vehicle control groups. Histopathology scores for mixed cell (macrophages and neutrophils) inflammation in the peribronchovascular and the centriacinar regions of the lung (J) and alveolar hemorrhage (K) in the lungs of animals treated with cysteamine relative to vehicle control group. L: representative images of the lung sections of the animals highlighting the extent of alveolar hemorrhage in the animals from the two groups. Scale bars for ×4 images equal 200 µm; scale bars for ×10 images equal 500 µm. Control, intraperitoneal vehicle control group. CYS, cysteamine treated group. Each group had n = 10 animals. Two BAL samples from the cysteamine group could not be analyzed due to a technical error. Data are means ± SE. Statistical significance was analyzed by two tailed, unpaired t test between cysteamine treated (CYS) and respective control group (control). BAL, bronchoalveolar lavage.
DISCUSSION
We explored if thiol drugs have antiviral or anti-inflammatory effects that could be beneficial in treating SARS-CoV-2 lung injury. Although in vitro assays showed that thiol drugs, especially cysteamine, can inhibit SARS-CoV-2 infection, in vivo experiments with cysteamine in hamsters did not show an antiviral effect. In contrast, cysteamine significantly decreased multiple measures of lung injury in SARS-CoV-2-infected hamsters.
We hypothesized that thiol drugs cleave critical cystines in the RBD of SARS-2-S to disrupt binding to ACE2, and we found support for this hypothesis in multiple assay systems. For example, pretreatment of pseudovirus and the authentic virus led to inhibition of viral infection in cells, whereas pretreatment of the cells did not prevent infection. Thus, the antiviral effect of the thiol drugs is via their action on the spike protein and not on any cellular protein. In a therapeutic scenario, thiol drugs could inhibit the propagation of SARS-CoV-2 viruses released after primary infection to have antiviral effects. In the PV entry assays, the potency of cysteamine as a viral entry inhibitor was variable with multiple variants, with IC50 values in low millimolar range, and lowest for the Wuhan-1, Wuhan-1-D614G, B.1.617.1 (Kappa), and B.1.1.529 (Omicron BA.1) variants. Our data do not reveal the reasons for this variability, which may relate to mutation-driven differences in the accessibility of cystine bridges. In general, though, the conservation of cystines in the spike protein of SARS-CoV-2 variants shows the importance of these cystines in the binding of the viral spike protein to the target cells.
We also noticed that low thiol pKa leading to high cystine cleaving activity was a key physicochemical property of the most potent thiol drugs. The ability of thiol drugs to cleave cystines is a function of the amount of drug in the deprotonated (thiolate) form, and a pKa that approaches 7.4 results in higher fraction of active thiolate anion to act as a nucleophile in thiol-disulfide exchange reactions (20, 56). The relatively low pKa values of cysteamine and WR-1065 (8.2 and 7.7, respectively) and cysteine methyl ester (pKa of 7.0) is the most likely explanation for their potency as SARS-CoV-2 entry inhibitors.
Despite the efficacy of cysteamine in our SARS-CoV-2 binding and infection assays in vitro, we did not observe antiviral effects in SARS-CoV-2-infected hamsters. A likely reason is that intraperitoneal delivery of cysteamine did not achieve the required millimolar concentrations in epithelial cells in the upper airways where viral titers are highest in the early stages of infection (57, 58). We did not measure cysteamine levels in the blood or BAL in this study, but there are published studies in children with cystinosis and in mouse models that indicate expected cysteamine levels after oral or systemic delivery. For example, a 15 mg/kg dose of orally ingested cysteamine bitartrate in children results in peak plasma concentrations between 0.03 and 0.07 mM (59). And a 120 mg/kg dose of cysteamine hydrochloride given intraperitoneally to mice results in peak plasma concentration of 0.6 mM within 5 min after administration followed by a rapid decline (60). These studies support an interpretation that intraperitoneal delivery of cysteamine is very unlikely to achieve millimolar concentrations in the upper airways. Because of this, direct administration of cysteamine to the upper or lower airways via nasal spray or aerosol delivery may be required to achieve higher drug concentrations in the airways and lungs. Other antiviral drugs, including remdesivir, are being formulated for inhaled route of administration in COVID-19 (58, 61, 62), and a phase-2 clinical trial with inhaled interferonβ-1a (IFNβ-1a) has also shown promise (63). Only a subset of currently approved thiol drugs (Mesna and NAC) are available as high-dose liquid formulations that can be delivered by nebulizer (Fig. 2), and these drugs have high pKa values (>9) and were among the least potent thiol drugs in vitro inhibition assays. The intermediate potency we found for TDG as a viral entry inhibitor is noteworthy. Previously proposed as a novel mucolytic because of its favorable properties as an inhaled drug (19), it may be possible to design thiol-saccharide drugs with lower thiol pKa values than TDG. Strategies like introduction of electron-withdrawing groups to the saccharide scaffold to reduce thiol pKa may be used to synthesize compounds with potency similar to or better than cysteamine and better suited to inhaled delivery.
Cysteamine significantly decreased SARS-CoV-2-related lung inflammation and lung injury in hamsters. Specifically, cysteamine treatment was associated with decreases in lung neutrophils, total protein, and alveolar hemorrhage. Although we did not investigate the specific anti-inflammatory mechanisms for cysteamine efficacy in the SARS-CoV-2-infected hamsters, the positive therapeutic effects are plausibly explained by its ability to scavenge reactive oxygen species (ROS), interrupt ROS-mediated inflammatory cascades (24–29), replenish intracellular glutathione (64), and inhibit the proinflammatory protein transglutaminase 2 (36; Supplemental Fig. S9). Such broad effects have also been hypothesized for the action of other thiol drugs like NAC in COVID-19 (65). In addition, SARS-CoV-2 infection has also been shown to reduce the levels of total cellular thiols and glutathione (66). Thiol drugs like cysteamine, by replenishing glutathione, can also help to restore the intracellular redox balance. Cysteamine is used most frequently as a drug to treat cystinosis, a lysosomal storage disease characterized by cystine accumulation, and it is approved in doses as high as 2 g daily for life in these patients (67). The beneficial anti-inflammatory effects of cysteamine in our hamster infection studies may extend to other 10 thiol drugs that are used as medicines (Fig. 2) because all of these thiol drugs can scavenge reactive oxygen species (ROS) and inhibit ROS-mediated inflammation in redox reactions that occur independently of their abilities to cleave cystine. Indeed, orally administered NAC is currently being tested in clinical trials in COVID-19 (NCT04419025, NCT04374461, and NCT04703036) as is bucillamine, a thiol drug with two thiol warheads and efficacy in rheumatoid arthritis (68, 69; NCT04504734). It is possible, however, that intravenous administration of thiol drugs will be needed for anti-inflammatory effects since the dose used in our hamster study is unlikely to be achieved by oral administration.
In summary, we show that cysteamine and multiple other thiol drugs can cleave cystines in the SARS-CoV-2 spike protein to act as entry inhibitors, but they do not have this antiviral effect when delivered systemically in vivo. We speculate that the concentration of cysteamine achieved in the lungs with intraperitoneal delivery was insufficient for antiviral effects but sufficient for anti-inflammatory effects. Our work demonstrates that cysteamine (and possibly other thiol drugs) can decrease SARS-CoV-2-related lung inflammation and injury to have beneficial effects in COVID-19 pneumonia. We also provide a rationale for future studies to test if direct (aerosol) delivery of thiol drugs to the airways results in antiviral effects in vivo.
DATA AVAILABILITY
The data that support the findings of this study will be made available upon reasonable request from the corresponding author.
SUPPLEMENTAL DATA
Supplemental Figs. S1–S9: https://doi.org/10.6084/m9.figshare.20097236.v1.
GRANTS
This work was funded by an intramural grant from UCSF—The COVID-19 Rapid Response Pilot Grant Initiative Funding Collaborative (to J.V.F.), a research grant from Revive Therapeutics (to J.V.F.), and National Institutes of Health Grant P01 HL128191 (to J.V.F.).
DISCLOSURES
J. V. Fahy, S. Oscarson, I. Gitlin, and W. W. Raymond are inventors on patent applications related to the use of thiol drugs as treatments for COVID-19. None of the other authors has any conflicts of interest, financial or otherwise, to disclose.
AUTHOR CONTRIBUTIONS
K.K., W.W.R., A.D.W., E.D.G., J.M.C., J.J., S.K.P., G.S., and J.V.F. conceived and designed research; K.K., W.W.R., J.J., A.R.C., A.D.W., J.M.C., S.M.B., R.M., H.S.S., S.F., T.D., M.H. performed experiments; K.K., W.W.R., J.J., A.R.C., I.G. M.T., A.D.W., E.G.B., S.M.B., G.S. and J.V.F. analyzed data; K.K., W.W.R., J.J., I.G., A.M.H., S.O., S.K.P., G.S., and J.V.F. interpreted results of experiments; K.K., W.W.R., A.R.C., and I.G. prepared figures; K.K. and J.V.F. drafted manuscript; K.K., W.W.R., J.J., A.R.C., I.G., A.M.H., S.O.,S.P., S.K.P., G.S., and J.V.F. edited and revised manuscript; K.K., W.W.R., J.J., A.R.C., I.G., M.T., A.D.W., E.G.B., J.M.C., S.M.B., R.M., H.S.S.,S.F., T.D., M.H., A.M.H., S.O., S.P., S.K.P., G.S., and J.V.F., approved final version of manuscript.
ACKNOWLEDGMENTS
The authors thank the technical staff at Lovelace Biomedical Research Institute for work infecting hamsters with SARS-CoV-2 in the ABSL3 facility. The authors also thank Christine Gralapp for assistance in drawing Figs. 3A and 7A and the online graphical abstract. The following reagent was deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH): Wuhan-1 SARS-CoV-2, Isolate USA-WA1/2020, NR-52281, and Delta SARS-CoV-2, isolate hCoV-19/USA/MD-HP05647/2021.
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