A rational roadmap for SARS-CoV-2/COVID-19 pharmacotherapeutic research and development: IUPHAR Review 29

2.1 Cellular attachment and entry: replication, assembly and release

Coronavirus binds to cell surface proteins on target cells and, following proteinase priming of spike proteins on the virus surface, the virus is internalized into endosomal fractions that are subsequently acidified or accumulates through a non-endosomal route (Fehr & Perlman, 2015) (Figure 1). The endosomal route appears to involve clathrin (Inoue et al., 2007), but there are contradictory reports of the importance of the intracellular C-terminus of ACE2 in this mechanism (Haga et al., 2008; Inoue et al., 2007). A fusion domain permits insertion of a key protein (the spike glycoprotein of the virus, S, see below), which then allows mixing of the viral and cellular membranes and subsequent release of the coronaviral genome into the cytoplasm.

Following entry into the host cell cytoplasm and viral uncoating, the replicase gene of the viral RNA is translated. The genome of coronaviruses consists of a single, continuous and linear ssRNA, capped at the 5′ end and with a 3′-polyA tail (Fehr & Perlman, 2015). Translation occurs from ORF1a and 1b at the 5′ terminus, with a ribosomal frameshifting mechanism allowing the overlap between ORF1a and ORF1b to generate the two polyproteins pp1a and pp1ab (Fehr & Perlman, 2015; Perlman & Netland, 2009; Snijder et al., 2003; Thiel et al., 2003). In SARS-CoV-2, the polyproteins are long, 4,405 and 7,096 amino acids respectively. Encoded within the polyproteins of betacoronaviruses are two proteinases, papain-like proteinase (PL_pro) and chymotrypsin-like proteinase (3CL_pro). In SARS-CoV, PL_pro, derived from the polyproteins, has three endoproteinase target sites, which release nsp1–3 (Thiel et al., 2003). 3CL_pro has 11 cleavage sites to release the remaining non-structural proteins. In the coronavirus family, these proteinases process the polyproteins to generate 16 functional non-structural proteins identified as nsp1–16 (Anand, Ziebuhr, Wadhwani, Mesters, & Hilgenfeld, 2003; Cui et al., 2019; Kindler, Thiel, & Weber, 2016; Thiel et al., 2003; Ziebuhr et al., 2007).

Downstream of the ORF1a and 1b are genes encoding four structural proteins (spike, envelope, membrane and nucleocapsid) (Figure 2) and a short series (described as at least 13 in total, Srinivasan et al., 2020) of other proteins (see below). Once sufficient protein and RNA accumulate, coronavirus assembly takes place, centred on the structural proteins. The release of coronavirus particles involves the secretory pathway of the endoplasmic reticulum and Golgi apparatus and vesicular exocytosis (for review, see de Haan & Rottier, 2005; Fehr & Perlman, 2015) and it is likely, but as yet unconfirmed, that SARS-CoV-2 also adopts this mechanism.

To date, there is more evidence about the molecular detail involved in (and the possibilities to modify) viral recognition, entry and replication compared to uncoating, assembly and release, hence the attention paid here to the former three mechanisms.

3.1 The cell-surface anchor - ACE2

Among the coronaviruses, the spike protein interacts with proteinases to anchor on host cell surfaces. The cell-surface anchoring point for the alphacoronavirus HCoV-229E is aminopeptidase N (also known as CD13, Yeager et al., 1992). For the betacoronavirus MERS-CoV, dipeptidylpeptidase 4 (also known as CD26, Raj et al., 2013) is an anchor. Analysis of the co-crystal structure suggested that the SARS spike protein binds to the active site of ACE2 (Li, Li, Farzan, & Harrison, 2005). Binding of SARS-CoV spike to ACE2 seems to require cholesterol-rich rafts in the host cells (Glende et al., 2008). Recent evidence points to the spike protein of SARS-CoV-2 also binding to ACE2. Both SARS-CoV (Li et al., 2003) and SARS-CoV-2 (Hoffmann et al., 2020; Letko, Marzi, & Munster, 2020) have been described to require ACE2 to enter cells (Figure 1). A particular domain of the spike protein of SARS-CoV-2, a so-called receptor-binding domain (RBD), has been shown to facilitate binding to ACE2 (Hoffmann et al., 2020). The ACE2 peptidase active site is located remotely from the cell membrane (Li et al., 2005; Wrapp et al., 2020; Yan et al., 2020), into which the spike protein binds. The receptor-binding domain of the spike protein is located in the S1 ectodomain, approximately a third of the way along the protein. ACE2 is a carboxypeptidase, which means it removes the terminal amino acid from oligopeptides, and so it seems unlikely that the spike protein is a substrate for ACE2.

In SARS-CoV-infected mouse lung, ACE2 protein expression was down-regulated compared to uninfected mice (Kuba et al., 2005). Following SARS-CoV spike protein administration to mice, angiotensin II was increased in the lungs (Kuba et al., 2005). These observations led to the suggestion that this was the molecular mechanism for the frequent development of acute respiratory distress syndrome (ARDS) during SARS-CoV infections (Imai et al., 2005; Kuba et al., 2005).

ACE2 has been reported to be released from plasma membranes by proteolysis, thought to be through the action of TNFα convertase (ADAM17, a disintegrin and metalloproteinase domain containing protein 17, Lambert et al., 2005) (Figure 1). The activity of ADAM17 can be increased by GPCR activation, including the AT₁ angiotensin receptor (Schafer, Marg, Gschwind, & Ullrich, 2004). ACE2 and ACE activities can be measured in human plasma (Herath et al., 2007; Lew et al., 2008; Ocaranza et al., 2006). Human plasma ACE2 activity is reported to be 'masked' by the presence of endogenous inhibitors (Lew et al., 2008), which do not yet appear to have been precisely defined. Blood ACE2 activity can be altered in pathology; for example, serum ACE2 was found to be decreased in patients following acute ischaemic stroke (Bennion et al., 2016).

The expression of ACE2 mRNA and enzyme activity in cardiac tissues were increased following repeated p.o. administration of the AT₁ receptor antagonist losartan, while p.o. administration of an ACE inhibitor lisinopril only increased cardiac mRNA expression, but not enzyme activity (Ferrario et al., 2005).

Studies using disruption of the ace2 gene in mice indicated an increase in circulating angiotensin II levels and a severe cardiac contractility defect, which could be 'rescued' with simultaneous genetic disruption of ACE (Crackower et al., 2002). An early investigation of ACE2 polymorphisms in man failed to show an association with hypertension (Benjafield, Wang, & Morris, 2004). A study of SARS victims and ACE2 polymorphisms failed to find a correlation with patient outcomes (Chiu et al., 2004).

3.2 The coronaviral spike protein

The spike protein is the largest viral structural protein (~1,200–1,400 amino acids) and is heavily glycosylated, forming extended trimeric structures providing the characteristic “crown” feature of coronaviruses (Belouzard, Millet, Licitra, & Whittaker, 2012) (see Figure 2). The ectodomain is divided into the S1 domain responsible for binding to ACE2, whereas the S2 domain is responsible for the fusion machinery. Following binding of the S1 domain to ACE2, a deformation of the pre-fusion trimer results (Wrapp et al., 2020). Surface plasmon resonance of the binding of human ACE2 to the immobilized SARS-CoV-2 indicated an affinity (K_D value) of 15 nM, an order of magnitude larger than SARS-CoV binding to ACE2 (Wrapp et al., 2020). Using a related label-free technique, biolayer interferometry, affinities of 5 and 1.2 nM for binding of SARS-CoV and SARS-CoV-2 spike protein, respectively, to human ACE2 has been reported (Walls et al., 2020).

Although a proteolytic cleavage site at the S1/S2 boundary of the SARS-CoV spike protein is best characterized, a second site upstream of the fusion peptide in the S2 domain, called S2′, has also been described (Belouzard, Chu, & Whittaker, 2009). This raises the possibility that multiple other proteases might be targeted to influence coronavirus activation (Millet & Whittaker, 2015). A key difference between the spike proteins in SARS-CoV and SARS-CoV-2 is the presence in the latter of a site at the S1/S2 boundary predicted to be sensitive to the proteinase furin, and which may be targeted during viral assembly and maturation (Walls et al., 2020).

The SARS-CoV S2 domain has a pair of α-helices, which may participate in coiled:coil structures during membrane fusion (Petit et al., 2005). The host complex of ZDHHC9 (Link to UniProt) with GOLGA7 (Link to UniProt), a palmitoyltransferase, which modifies the low MW G proteins NRAS and HRAS (Swarthout et al., 2005), also palmitoylates the cysteine-rich S2 endodomain of the SARS-CoV to facilitate membrane fusion (Petit et al., 2007).

Very recently, in a comparison of the S2 domains of SARS-CoV and SARS-Cov-2, an enhanced capacity of the novel virus' S2 domain for membrane fusion was observed and suggested to result from eight differing amino acids (Xia et al., 2020). Using a series of oligopeptides conjugated to lipid entities, high affinity (IC₅₀ values in the nanomolar range) inhibitors of cell fusion were identified.

3.3 Interfering with the ACE2:spike protein interaction

Given that the spike protein binds to the active site of ACE2 (Li et al., 2005), in theory, any alteration in the availability of the active site should influence the binding of the spike protein and, hence, interfere with SARS-CoV-2 infection. One option would be to provide an excess of an endogenous peptide substrate, or more conventionally to apply a selective enzyme inhibitor.

3.4 The cell-surface priming mechanism - TMPRSS2

TMPRSS2 is a single transmembrane domain protein with an extracellular serine protease domain, which appears to cleave substrates preferentially at basic residues (arg/lys), with a calcium-binding LDL receptor class A domain (Paoloni-Giacobino, Chen, Peitsch, Rossier, & Antonarakis, 1997). The TMPRSS2 gene encodes a cell-surface proteinase (transmembrane serine protease 2, TMPRSS2) and is located at chromosomal locus 21q22.3 in close proximity to ERG, a gene encoding an ETS transcription factor (Link to UniProt, Paoloni-Giacobino et al., 1997) (ERG fusion with EWS leads to Ewing's sarcoma). Fusion of the TMPRSS2 and ERG (or the related ETV1) genes has been reported to occur in the majority of prostate cancers and is suggested to lead to an androgen-dependent amplification of ETS-regulated genes (Tomlins et al., 2005). TMPRSS2 expression is androgen-regulated (Chen et al., 2019; Lin et al., 1999). It is expressed highly in prostate cancer (Lin et al., 1999; Lucas et al., 2008) (for review, see Tanabe & List, 2017) and loss of TMPRSS2 in the prostate is associated with reduced metastatic potential (Lucas et al., 2014). In aggressive versions of prostate cancer, TMPRSS2 undergoes autocatalytic proteolysis at Arg²⁵⁵-Ile²⁵⁶ (Afar et al., 2001), where the two chains may remain in combination due to interchain disulphide bridges (Chen et al., 2010) or the catalytic moiety may be secreted (Chen et al., 2010). In LNCaP human prostate cancer cells, the PPARα (NR1C1) agonist fenofibrate was able to mitigate against the androgen receptor agonist-evoked increase in TMPRSS2 expression (Zhao et al., 2013).

Following binding of the S protein to ACE2, TMPRSS2 “primes” the spike protein to facilitate entry of the virus into the target cell (Hoffmann et al., 2020; Matsuyama et al., 2020). Pathogenesis of two strains of influenza virus has been reported to be markedly diminished by gene disruption of tmprss2 in mice (Hatesuer et al., 2013; Tarnow et al., 2014), inferring that targeting this enzyme may have antiviral potential.

3.5 Potential ancillary proteins for virus entry - B⁰AT1/SLC6A19 and B⁰AT3/SLC6A18

The SLC6 family of transporters includes the well-characterized NET, SERT and DAT monoamine transporters, as well as the less well-exploited neutral amino acid transporter subfamily. B⁰AT1/SLC6A19 and B⁰AT3/SLC6A18 allow sodium- and chloride-dependent accumulation of neutral, aliphatic amino acids at the apical membranes of epithelial cells in the small intestine (B⁰AT1/SLC6A19) and kidney (B⁰AT1/SLC6A19 and B⁰AT3/SLC6A18) (for review, see Broer & Gether, 2012). B⁰AT3/SLC6A18 is also highly expressed in the GI tract and gall bladder (Protein Atlas). The cell-surface expression of these neutral amino acid transporters is dependent on co-expression of ACE2 (Fairweather, Broer, O'Mara, & Broer, 2012; Kowalczuk et al., 2008), aminopeptidase N (Fairweather et al., 2012) or collectrin (an adaptor protein, which has high homology to the transmembrane region of ACE2, Camargo et al., 2009, Link to UniProt), in an apparently tissue-dependent manner (Kuba, Imai, Ohto-Nakanishi, & Penninger, 2010). A recent cryo-EM structure suggested that ACE2 and B⁰AT1/SLC6A19 form a heterodimer, which pairs up through interfaces between the two ACE2 partners (Figure 1), with the receptor-binding domain of SARS-CoV-2 spike protein binding to the peptidase active site of ACE2 (Yan et al., 2020) suggesting that B⁰AT1/SLC6A19 may facilitate entry of the novel coronavirus. In the small intestine, absorptive epithelial cells were identified to co-express mRNAs encoding for ACE2 and TMPRSS2 (Ziegler et al., 2020). Although it is not yet tested, it would be attractive to speculate that the co-localized expression of these targets may play a role in the faecal:oral transmission of coronavirus (Yeo, Kaushal, & Yeo, 2020).

4.1 Viral uncoating

Once inside the cell, the endosomal cysteine proteases cathepsin B and cathepsin L have been described to process SARS-CoV (Simmons et al., 2005) and this appears to be maintained for SARS-CoV-2 (Hoffmann et al., 2020), although the significance of such intracellular proteinase activity is unclear. Potent inhibitors for these two proteinases have been reported as pharmacological probes, but there are no licensed drugs identified to target them.

Following entry into the cell, many viruses accumulate in acidified lysosome-like vesicles and so weak bases (including ammonium chloride and chloroquine), which target the lysosome have been used in vitro to target this mechanism. Ammonium chloride (at 20 mM) has been described as a non-specific inhibitor of viral replication in vitro, targeting viral uncoating (Mizzen, Hilton, Cheley, & Anderson, 1985) and, at 50 mM, ammonium chloride inhibited cell entry of both SARS-CoV and SARS-CoV-2 (Hoffmann et al., 2020). Chloroquine was also observed to reduce infection of L cells by mouse hepatitis virus 3 (Krzystyniak & Dupuy, 1984).

4.2 Viral replication

Following entry into the cell, the virus subverts nucleotide, protein, lipid, and carbohydrate turnover of the host cell to produce multiple copies of itself. The viral RNA is translated into multiple proteins to produce the replication machinery. As protein translation from the viral genome occurs, the two polyproteins are the first to be synthesized, with the two intrinsic proteases able to cleave the polyproteins into their constituent products.

4.2.1.1 The papain-like proteinase of the virus (PL_pro)

The more N-terminally located PL_pro is the larger (~2,000 aa) of the two proteins (for review, see Baez-Santos, St John, & Mesecar, 2015; Lei, Kusov, & Hilgenfeld, 2018) and in SARS-CoV is a membrane-associated, polyfunctional entity (Harcourt et al., 2004). Sequence modelling of SARS-CoV-2 PL_pro suggested the presence of 6 TM domains towards the C-terminus, with the majority of the protein extending into the cell cytoplasm (Angeletti et al., 2020). In other coronaviruses, the enzyme is also capable of hydrolysing ubiquitin from protein substrates (Barretto et al., 2005; Ratia et al., 2006), as well as removing the ubiquitin-like protein IFN-stimulated gene 15 (ISG, Link to UniProt) from ISG-conjugated proteins (Yang et al., 2014). Using the orthologous proteinase from the mouse hepatitis coronavirus, analysis of three distinct structural domains suggested that the PL_pro domain coincided with the deubiquitinylating and deISGylating functions (Chen et al., 2015). In SARS-CoV, the PL_pro also contains an ADP-ribose-1″-phosphatase functional phosphatase domain directed at ADP-ribose-1″-phosphates, although the functional significance of the hydrolase activity may be less impactful than the capacity to bind ADP-ribose, at least for the enzyme from HCoV-229E (Putics, Filipowicz, Hall, Gorbalenya, & Ziebuhr, 2005). This domain is thought to contribute to processing of the viral subgenomic RNAs and the suppression of the innate immune system through reducing IFN production (Lei et al., 2018).

Investigating the peptidase activity of SARS-CoV PL_pro suggested a preference for larger proteins (ubiquitinated or ISGylated) rather than simpler fluorescent-tagged oligopeptide substrates (Baez-Santos, Mielech, Deng, Baker, & Mesecar, 2014; Lindner et al., 2005; Lindner et al., 2007; Ratia, Kilianski, Baez-Santos, Baker, & Mesecar, 2014) making screening more complicated.

4.2.1.2 The chymotrypsin-like proteinase, 3C-like proteinase of the virus (3CL_pro)

The smaller proteinase from SARS-CoV-2 is 3CL_pro (sometimes called the main prote(in)ase, M_pro). The use of in silico docking models of SARS-CoV-2 3CL_pro has led to suggestions that particular existing antiviral agents, including velpatasvir and ledipasvir (licensed agents for treating hepatitis C when combined with sofosbuvir in the United Kingdom), should be screened for functional activity (Chen, Yiu, & Wong, 2020). A recent screen of ~10,000 compounds including approved drugs, candidate drugs and natural products used a substrate derived from the N-terminal autocleavage site of the SARS-CoV-2 3CL_pro, which was modified (methylcoumarinylacetyl-AVLQSGFR-Lys (Dnp)-Lys-NH₂) to allow a FRET-based assay (Jin et al., 2020). The same substrate was used in a screen of the equivalent enzyme from another coronavirus, HCoV-HKU1, which transferred to humans (Zhao et al., 2008).

A number of inhibitors of the SARS-CoV 3CL_pro proteinase have been described (Goetz et al., 2007; Lu et al., 2006; Yang et al., 2006), without progressing into the clinic. Recently, an in silico approach using orthologues of the SARS-CoV 3CL_pro from other coronaviruses and enteroviruses allowed production and testing in vitro of a series of α-ketoamides (Zhang et al., 2020). One compound (11r) exhibited submicromolar potency against SARS-CoV 3CL_pro in a cell-free FRET-based assay and micromolar potency in a cell infection assay with SARS-CoV (Zhang et al., 2020).

In a very recent report, the SARS-CoV-2 3CL_pro expressed in HEK293 cells was found to interact with histone deacetylase 2 (HDAC2) by affinity purification/MS (Gordon, Jang, et al., 2020). A number of approved drugs target HDAC2 in the treatment of various T cell lymphomas, including romidepsin, belinostat and vorinostat with nanomolar potency (Bradner et al., 2010).

4.2.2.1 Protein:protein interactions in recombinant expression

In a very recent report, a series of tagged recombinant proteins from SARS-CoV-2 were expressed in HEK293 cells and then protein partners were identified by affinity purification/MS (Gordon, Jang, et al., 2020). For nsp12 (RNA-dependent RNA polymerase) and nsp14 (3′-5′-exonuclease) of SARS-CoV-2, interactions with receptor interacting protein kinase 1 (RIPK1) and inosine monophosphate dehydrogenase 2 (IMPDH2) respectively, were identified. For these two targets, there are established approved drugs. Thus, ponatinib, which is used to treat acute myelogenous leukaemia or chronic myelogenous leukaemia (Philadelphia chromosome), targets multiple protein kinases, inhibiting RIPK1 with an IC₅₀ value of 12 nM (Najjar et al., 2015). Mycophenolic acid and ribavirin are IMPDH2 inhibitors with IC₅₀ values of 20 nM (Nelson, Eugui, Wang, & Allison, 1990) and 1-3 μM (Wittine et al., 2012) ranges, respectively, with clinical uses in organ transplantation and antiviral therapy, respectively.

Reservations about the use of ribavirin have already been noted above. Mycophenolic acid as a monotherapy was examined in a MER-CoV-infected non-human primate model, where the authors concluded it actually worsened the condition (Chan et al., 2015).

Nsp13 (helicase) and nsp15 (endoribonuclease) have been described to bind to centrosome-associated protein 250 (CEP250) and RNF41 (also known as NRDP1, Link to UniProt) respectively, in a report of recombinant expression (Gordon, Jang, et al., 2020). CEP250 is suggested to influence centrosome cohesion during interphase (de Castro-Miro et al., 2016) and to be elevated in peripheral T cell lymphomas (Cooper et al., 2011). The functional relevance of nsp13 interaction with CEP250 is not yet clear. RNF41 is an E3 ubiquitin ligase, which polyubiquitinates myeloid differentiating primary response gene 88 (MyD88, link to UniProt), an adaptor protein for Toll-like receptors, which allows activation of TBK1 and IRF3 (see below) and thereby increases type I IFN production (Wang et al., 2009).

5 THE OTHER VIRAL STRUCTURAL PROTEINS

6 INTERACTIONS WITH THE HOST INNATE IMMUNE SYSTEM

SARS-CoV produces proteins that interfere with IFN pathways (nsp1, nsp3, nsp16, ORF3b, ORF6, ORF9b, M and N proteins Wong, Lui, & Jin, 2016) and NLRP3 inflammasome activators (E, ORF3a, and ORF8b), which are closely related to orthologues found in SARS-CoV-2. Fung, Yuen, Ye, Chan, and Jin (2020) have recently reviewed the molecular aspects whereby SARS-CoV and, by inference SARS-CoV-2, evade immune surveillance, activate the inflammasome and cause pyroptosis. Other coronaviruses may give an indication as to how this is happening. HCoV-229E rapidly kills dendritic cells, while monocytes are much more resistant. The rapid invasion of and replication in dendritic cells kills them within a few hours of infection (Mesel-Lemoine et al., 2012). Dendritic cells are sentinel cells in the respiratory tract and plasmacytoid dendritic cells are a crucial antiviral defence via IFN production and by modifying antibody production. Thus, these viruses can impair control of viral dissemination and the formation of long-lasting immune memory. Penetration of SARS-CoV-2 infection deep into the lungs and eventually the alveolae, results in the “cytokine storm”, which accompanies pneumonia and lung fibrosis and is probably a major determinant of the necessity for intubation and also mortality (Shi, Wang, et al., 2020). It is currently not known what specific factor/s differentiate the patients who develop this, although mortality among younger health workers may indicate that initial viral load may play a role. Immunological agents, which can prevent or control the “cytokine storm” could therefore have a major effect on necessity to intubate and mortality. Tocilizumab is a monoclonal antibody targeting IL-6 receptors, as a means to interfere with the effects of chronic autoimmune disorders such as rheumatoid arthritis. The Chinese Clinical Trials Registry has two studies that are designed to evaluate tocilizumab efficacy in patients with severe COVID-19 pneumonia (Registration Numbers ChiCTR2000029765 and ChiCTR2000030442). Similarly, anakinra, which is a slightly modified version of an endogenous antagonist of IL-1 receptors, is being investigated in clinical trials in multiple locations in patients with COVID-19 infection (NCT04324021, NCT04330638, and NCT02735707).

It has been reported that in stage III of COVID-19, a critical point with a high viral load and severe respiratory involvement, lungs of patients appear with “ground-glass” patches in CT scans, while autopsy reports indicate that the lungs are filled with a “clear liquid jelly” (Shi, Wang, et al., 2020; Xu et al., 2020), similar to an observation in drowning victims. On the hypothesis that inflammation-driven hyaluronan production (via hyaluronan synthase 2 [HAS2] Link to UniProt) and associated water retention may be critical. A recent study proposed therapy via administration of recombinant hyaluronidase or inhibitors of HAS2 (Shi, Wang, et al., 2020).

The interaction between the virus and the innate immune system is complex and multifactorial, with temporal intricacies. It is beyond the scope of this review to identify all the multiple components and so we discuss here those pathways we consider most tractable.

6.1 Viral nucleotides and MDA5/MAVS/IFN production

The positive sense RNA of coronaviruses is translated to produce the replication machinery, which allows complementary negative sense RNA to be synthesized, which itself is the template for the synthesis of positive strand RNA. As a consequence, double-stranded RNA is produced, which act as a pathogen-associated molecular pattern (PAMP) targeting MDA5 (IFN induced with helicase C domain I, also known as melanoma differentiation antigen 5, Kato et al., 2006) from the RIG-1-like receptor family of cytoplasmic pattern recognition receptors (for reviews, see Schlee, 2013; Bryant et al., 2015). MDA5 differs from RIG-1 (DexD/H-box helicase 58, also known as retinoic acid-inducible gene 1) in recognizing longer dsRNA (Goubau et al., 2014; Kato et al., 2006), and it has been proposed that this differentiates the sensing of positive-stranded viruses by MDA5 compared to negative strand virus sensing by RIG-I (Goubau, Deddouche, & Reise Sousa, 2013; Kato et al., 2006). RIG-1-like receptors have an N-terminal caspase activation and recruitment domain (CARD), which shows ligand-dependent interaction with CARDs from other proteins, such as mitochondrial antiviral signalling protein (MAVS, Link to UniProt). MAVS activates IKK family kinases, such as TANK binding kinase (TBK1) and IKK-ε, leading to the phosphorylation of IFN regulatory factors, such as IRF3 (Link to UniProt) and IRF7 (Link to UniProt). This induces the transcription of Type I IFN genes, such as IFN-β and CCL5 (also known as RANTES) (Doyle et al., 2002; Fitzgerald et al., 2003; Sharma et al., 2003). MAVS present in peroxisomes is also able to recruit short-acting, IFN-independent defence factors (Dixit et al., 2010).

The ORF9b protein from SARS-CoV has also been reported to target mitochondrial MAVS to limit the IFN response, as well as triggering proteolysis of dynamin-like protein 1 (Link to UniProt) thereby prompting the formation of mitochondria-associate autophagosomes claimed to create “havoc” in energy production in infected cells (Shi et al., 2014). In a very recent study, ORF9b of SARS-CoV-2 has been reported to interact with translocases of outer membrane 70 (Tom70, Link to UniProt) when expressed in HEK293 cells (Gordon, Jang, et al., 2020) Tom70 activates mitochondrial IRF3 (Liu, Wei, Shi, Shan, & Wang, 2010) and so this is a potential locus for pharmacological intervention, but as yet with no inhibitors described in the literature.

A number of other coronavirus proteins have been identified to influence the IRF3 pathway to restrict IFN production. This includes the MERS-CoV PL_pro proteinase (Yang et al., 2014), as well as the ORF6 and nucleocapsid proteins from SARS-CoV (Kopecky-Bromberg, Martinez-Sobrido, Frieman, Baric, & Palese, 2007). The ORF6 protein of SARS-CoV has also been described to reduce the activity of a series of karyopherin-dependent host transcription factors (Sims et al., 2013). Karyopherin is an importin, which traffics proteins between the cytoplasm and the nucleus (for review, see Kosyna & Depping, 2018; Guo, Fare, & Shorter, 2019).

Clearly, the induction and suppression of IFN production are central to numerous human diseases and have been extensively studied; the 'trick' to treat COVID-19 will be to identify a novel angle for therapeutic exploitation.

6.2 Nsp1

Working with SARS-CoV (not SARS-CoV-2), Pfefferle et al. (2011) used yeast two-hybrid screens to identify interactions between the viral and human proteomes. They identified an interesting interaction between viral Nsp1 and a group of host peptidyl-prolyl cis-trans-isomerases (PPIA, PPIG, PPIH and FKBP1A, FKBP1B), all of which modulate the calcineurin/NFAT pathway important in immune activation (reviewed by Hogan, Chen, Nardone, & Rao, 2003). The nsp1 protein acts on these to activate NFAT signalling and immune activation. Cyclosporine A, an inhibitor of this pathway, has been used for several decades to control transplant rejection and some autoimmune diseases and, in a simple in vitro assay, cyclosporine inhibited SARS-CoV transcription/replication in (non-immune-system) cells (Pfefferle et al., 2011). SARS-CoV-2 has an nsp1 protein closely related to that of SARS-CoV (Dong et al., 2020; Srinivasan et al., 2020), though its effect on the NFAT pathway seems not to have been reported. Nevertheless, cyclosporine has been shown to inhibit SARS-CoV2 in an in vitro Vero cell-based assay in a recent report (Jeon et al., 2020). It has therefore been suggested as a drug target (see, for example, Li & De Clercq, 2020). It may seem paradoxical to suggest an inhibitor of immune activation as a treatment for viral disease, but for the subgroup of patients that might suffer cytokine storms (Mehta et al., 2020), the double-action might be useful.

6.3 ORF3a, ORF6, ORF8 and other viral proteins

The ORF3a protein of SARS-CoV appears to bind calcium in a cytoplasmic domain (Minakshi, Padhan, Rehman, Hassan, & Ahmad, 2014) and to elicit a response from the innate immune system by enhancing the ubiquitination of apoptosis-associated speck-like protein containing a CARD (Asc, Link to UniProt), which in turn activates the NLRP3 inflammasome and caspase 1 (Siu et al., 2019). The potential for targeting Asc and the NLRP3 inflammasome for therapeutic benefit in inflammatory conditions has recently been reviewed (Mangan et al., 2018), although there are no inhibitors in the clinic as yet.

In SARS-CoV, the Orf8a and Orf8b genes became separated as the disease progressed by a 29-nucleotide deletion (Chinese SARS Molecular Epidemiology Consortium, 2004; Oostra, de Haan, & Rottier, 2007). The Orf8a gene of SARS-CoV encodes a short (31 aa, 1 TM, Link to UniProt) protein, which forms a cation channel of predicted pentameric structure (Chen et al., 2011). In SARS-CoV-2 and a bat-derived coronavirus, in contrast to the SARS-CoV-2 genome, Orf8 encodes a continuous 121 aa ORF8 protein (Cagliani, Forni, Clerici, & Sironi, 2020). Given that sequence analysis of different strains of SARS-CoV-2 suggests that the Orf8 locus displays only limited evidence of positive selection (Cagliani et al., 2020), it seems germane to investigate the profile of ORF8 protein in more depth. Sequence comparisons led to prediction of secondary structure composed of an α-helix and a β-sheet containing six strands (Chan et al., 2020), but there appears not to be any literature as to whether this entity is a functional ion channel.

In a very recent report, the ORF14 protein (Link to UniProt) of SARS-CoV-2 has been reported to interact with NOD-like receptor X1 (NLRX1), proteinase-activated receptor 2 (PAR2/F2RL1), and NEDD4 family-interacting protein 2 (NDFIP2, Link to UniProt), among other proteins of the IκB/NF-κB pathway, when expressed in HEK293 cells (Gordon, Jang, et al., 2020). At the moment, there are no approved drugs targeting PAR2, although AZ3451 (Link to GtoP) acts as a negative allosteric modulator with pIC₅₀ values of 5–23 nM (Cheng et al., 2017).

There is a limited insight into the roles or potential exploitability of the remaining range of other viral proteins (nsp2; nsp9; nsp11, proteins Orf3b; ORF6; ORF7a; ORF7b; ORF10).

7 ANIMAL MODELS OF SARS-COV-2 INFECTION

The spike glycoproteins in SARS-CoV and MERS-CoV are crucial for host specificity and jumping between species, for example, from bats to humans (Lu, Wang, & Gao, 2015) and from dromedary camels to humans (MERS-CoV) and also the recent crossover of a HKU2-related coronavirus to pigs as a swine acute diarrhoea syndrome (SADS-CoV) (Zhou et al., 2018). SADS-CoV appears to influence the innate immune system by reducing IFN-β production evoked through IPS-1 and RIG-I pathways, but not through IRF3, TBK1 and IKKε (Zhou et al., 2020).

ACE2, as an anchoring point for the spike glycoprotein, is present throughout the animal kingdom, but small structural differences are critical for interaction with the spike protein (Li, Qiao, & Zhang, 2020; Luan, Lu, Jin, & Zhang, 2020). Key sequences of the spike protein from SARS-CoV and SARS-CoV-2 are responsible for binding to ACE2. Luan et al. (2020) found that the key residues in S protein, from SARS-CoV and SARS-CoV-2, recognized in ACE2 from dog, cat, pangolin and Circetidae mammals (simulated through homology modelling) were broadly similar. Mouse ACE2 is inefficient in prompting entry of both SARS-CoV and SARS-CoV-2 (Fung et al., 2020). Cats and dogs suffer from their own specific coronavirus infections (e.g. canine respiratory coronavirus and feline coronavirus) without significant crossover to humans. A very recent report has suggested that cats and ferrets are sensitive to SARS-CoV-2, but dogs, pigs, chickens and ducks are much less sensitive (Shi, Wen, Zhong, Yang, Wang, Huang, Liu, He, Shuai, Sun, & Zhao, 2020). Ferrets, which have previously been used as models for respiratory tract infections, retained the SARS-CoV-2 virus in the respiratory tract, while the infection was transmitted between cats by aerosol (which may have implications for confinement); infected cats subsequently produced antibodies (Shi, Wen, et al., 2020).

The Syrian hamster has been used as a model for SARS-CoV (de Wit et al., 2013; Roberts et al., 2005; Roberts et al., 2006) and studies with mice and Syrian hamsters are ongoing with SARS-CoV-2. In a small study where three juvenile (3–5 years old) and two mature (15 years old) rhesus macaques were infected intratracheally with the SARS-CoV-2 virus, all the monkeys showed symptoms of inflammation and interstitial pneumonia, with a greater apparent severity in the older animals (Yu et al., 2020).

Thus, while there is intensive research in animal models, a clearly validated model is still not apparent.

8 INTER-INDIVIDUAL VARIATIONS IN SUSCEPTIBILITY

Some of these questions are more tractable since the SARS and MERS outbreaks because of the strides being made in sophisticated molecular biological techniques (e.g. NextGen Sequencing). An additional distinction compared to the previous outbreaks is the major increase in patient numbers associated with COVID-19, allowing greater comparisons to be made in many more geographical locations.

Inevitably, other questions will form as greater detail becomes available.

9.1 Nomenclature of targets and ligands

Sequencing analysis of the novel virus has identified a high level of similarity with the virus identified to cause the severe acute respiratory syndrome (SARS) outbreak in China in 2002/03/04, which was known as the SARS coronavirus or SARS-CoV. Provisionally named as 2019-nCoV, the virus has been renamed SARS-CoV-2 (Coronaviridae Study Group of the International Committee on Taxonomy of Viruses, 2020). For the purposes of this document, the virus is described as SARS-CoV-2, while the infectious disease is named as COVID-19 (World Health Organization, 2020). One of the positive aspects of the emergence of SARS-CoV-2 and COVID-19 is the rapidity at which aspects like genome sequencing (for example, Lu et al., 2020; Wu et al., 2020) and 3D structures (for example, Yan et al., 2020) have been described.

Protein targets and drugs in the current review follow nomenclature as presented on the GuidetoPHARMACOLOGY.org website (Alexander, Ball & Tsoleridis. SARS-CoV-2 proteins, accessed on 2020-04-24) and the Concise Guide to PHARMACOLOGY 2019/20 (Alexander et al., 2019).