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Discovery of a Novel Inhibitor of Coronavirus 3CL Protease for the Potential Treatment of COVID-19

Abstract COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. The designed phosphate prodrug PF-07304814 is metabolized to PF-00835321 which is a potent inhibitor in vitro of the coronavirus family 3CL pro, with selectivity over human host protease targets. Furthermore, PF-00835231 exhibits potent in vitro antiviral activity against SARS-CoV-2 as a single agent and it is additive/synergistic in combination with remdesivir. We present the ADME, safety, in vitro, and in vivo antiviral activity data that supports the clinical evaluation of this compound as a potential COVID-19 treatment.

Introduction In December 2019, COVID-19 was identified as a new, potentially fatal, respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Unlike previous coronavirus outbreaks that receded relatively quickly, the resultant COVID-19 pandemic spread across the globe. As of December 2020, over 75 million people have been infected and over 1.6 million people have died globally with no approved drugs available to treat the disease3. The RNA-dependent RNA polymerase (RdRp) inhibitor remdesivir is currently undergoing clinical investigation for the treatment of SARS-CoV-2 and was granted emergency use authorization by the U.S. Food and Drug Administration (FDA) in May 2020. To date, the results of trials of remdesivir have been mixed5 despite being granted full authorization in October 2020. Monoclonal antibodies that target the viral spike protein have also been successful in treating disease when applied at the early stage of infection7. Though the availability of highly efficacious vaccines provides great hope for the eradication of COVID-19, it is expected that it will still take time for cases to subside globally and does not address the potential threat of future coronaviruses. Thus, other classes of antivirals that exhibit single agent efficacy or that are complementary to remdesivir for use in combination regimens are essential to meet this substantial unmet need. SARS-CoV-2 produces two large viral polyproteins, pp1a and pp1ab, which are processed by two virally encoded cysteine proteases, the main protease, also called 3C-like protease (3CL protease or 3CLpro) and the papain-like protease. Mutagenesis experiments with other coronaviruses have demonstrated that the activity of the 3CLpro is essential for viral replication8,9. 3CLpro proteolytically processes the virus p1a/p1ab polyproteins at more than 10 junctions to generate a series of non-structural proteins critical for virus replication and transcription, including RdRp, the helicase, and the 3CLpro itself10. No close human analogs of the coronavirus 3CLpro are known, suggesting that selective 3CLpro inhibitors should avoid unwanted polypharmacology11. The essential functional importance of proteases in virus replication has led to the clinical success of protease inhibitors in combating both human immunodeficiency virus (HIV) and hepatitis C virus (HCV)12–14. This together with the opportunity for selectivity, makes 3CLpro an attractive antiviral drug target15. Following the severe acute respiratory syndrome (SARS) outbreak in 2002-2003 we identified a potential small molecule protease inhibitor (PF-00835231) for the treatment of SARS-CoV-1, using structure-based drug design16. Due to the SARS pandemic being brought under control in July 2003 following public health measures which incorporated patient isolation and travel restrictions, this project was discontinued due to the lack of a path forward to demonstrate clinical efficacy. Given that the SARS-CoV-1 and SARS-CoV-2 3CLpro sequences share 96% identity overall and 100% identity in the active site1,17,18, following the emergence of SARS-CoV-2, PF-00835231 was identified as a potential SARS-CoV-2 3CLpro inhibitor for the treatment of COVID-19 disease16. Subsequent antiviral data presented here indicate that PF-00835231 has similar potency against either SARS-CoV-1 or SARS-CoV-2 (Table S1).

Results and Discussion

PF-00835231 exhibits tight and specific binding to SARS-CoV-2 3CL in vitro A thermal-shift assay was used to evaluate the direct binding between PF-00835231 and SARS-CoV-2 3CLpro. The melting temperature of SARS-CoV-2 3CLpro was shifted by 14.6℃ upon binding of PF-00835231, from 55.9±0.11℃ (n=16) to 70.5±0.12℃ (n=8). These data support tight and specific binding of PF-00835231 to SARS-CoV-2 3CLpro (Fig. 1) as was shown previously by X-ray co-crystal structure and evaluation of Ki 16.

Discovery of a Novel Inhibitor of Coronavirus 3CL Protease for the Potential Treatment of COVID-19

To explore the notion that PF‑00835231 could have pan-coronavirus activity, PF-00835231 was evaluated against 3CLpro from a variety of other coronaviruses representing alpha, beta and gamma groups of Coronaviridae, using biochemical Förster Resonance Energy Transfer (FRET) protease activity assays. PF-00835231 demonstrated potent inhibitory activity against all tested coronavirus 3CLpro including members of alpha-coronaviruses (NL63-CoV, PEDV, FIPV), beta-coronaviruses (HKU4-CoV, HKU5-CoV, HKU9-CoV, MHV-CoV, OC43-CoV, HKU1-CoV), and gamma-coronavirus (IBV-CoV), with Ki values, ranging from 30 pM to 4 nM (Table 1). The demonstrated activity is consistent with a potential therapeutic use against emerging coronaviruses. This inhibitory activity is restricted to coronavirus 3CLpros as PF-00835231 was inactive against a panel of human proteases and HIV protease (Table S2). PF-00835231 showed detectable activity against human cathepsin B but 1000-fold weaker (6.9 nM vs 6 μM) activity compared to 3CLpro (Table S2). Thereby, these data collectively support PF-00835231 is a selective in vitro protease inhibitor with broad coronavirus activity.

n vitro cellular antiviral activity of PF-00835231 against SARS-CoV-2

The antiviral activity of PF-00835231 against SARS-CoV-2 in cell culture was evaluated with a cytopathic effect (CPE) assay using either VeroE6 kidney cells enriched for angiotensin-converting enzyme 2 (ACE2) (VeroE6-enACE2) receptor or VeroE6 cells constitutively expressing EGFP (VeroE6-EGFP). These cell lines were infected with the SARS-CoV-2 Washington strain 1 (WA1 - EPI_ISL_404895) or the Belgium/GHB-03021/2020 strain (GHB-03021 - EPI_ISL_407976)19, respectively, which have identical 3CLpro amino acid sequences. PF-00835231 exhibited viral CPE EC50 values of 39.7μM and 88.9μM, respectively (EC50, Fig. 2). However, Vero cells express high levels of the efflux transporter P-glycoprotein (P-gp) (also known as MDR1 or ABCB1), of which PF-00835231 is a known substrate16 suggesting that the intracellular concentration of PF-00835231 was lower than it initially appeared. Therefore, to evaluate the full potency of PF-00835231, the assays were repeated in the presence of a P-gp efflux inhibitor, CP-10035620. PF-00835231 exhibited a 117- to 173-fold increase in activity in the presence of 2 μM P-gp inhibitor, with EC50 values of 0.23μM in VeroE6–enACE2 cells and 0.76μM in the VeroE6-EGFP cells (Fig. 2). The P-gp inhibitor alone had no antiviral or cytotoxic activity at these concentrations and did not cause cytotoxicity in the presence the protease inhibitor. The use of VeroE6 cells by many in the field to evaluate inhibitor activity could be problematic, since the true activity of some compounds could be masked by efflux of inhibitors from these cells (Fig. 2B). Consistent with many viral protease inhibitors21, there was a steep response to increasing doses of PF-00835231, with a ~2-3 fold difference between EC50 and EC90 in both cell types (EC90 = 0.48μM in VeroE6-enACE2 cells and EC90= 1.6μM in VeroE6-EGFP cells in the presence of the P-gp inhibitor). As expected, when lung cell lines were tested for antiviral potency in the presence and absence of P-gp inhibitor (A549-ACE222 and MRC5), no statistical difference in antiviral potency was observed (Fig. 2A). Additionally, antiviral activities in both VeroE6 cell lines with 2μM P-gp inhibitor are similar to those observed in more physiologically relevant human lung cell culture systems, including A549-ACE2 and polarized human airway epithelial cells22, where P-gp expression is lower. These data support the potential for single agent antiviral activity. As will be presented later, despite seeing apparent antiviral activity for PF-07304814, a prodrug of PF-00835231, in cell-based assays (Fig. 2A) this activity is likely due to conversion of PF-07304814 to PF-00835231 in the assay.

Discovery of a Novel Inhibitor of Coronavirus 3CL Protease for the Potential Treatment of COVID-19

Credited to BioRxiv


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