ABSTRACT
Background: The unprecedented scale of the COVID-19 pandemic and rapid evolution of SARS-CoV-2 variants underscores the need for broadly active inhibitors with a high barrier to resistance. The coronavirus main protease (Mpro) is an essential viral enzyme required for viral polyprotein processing and is highly conserved across human coronaviruses. Pomotrelvir (PBI-0451) is a novel Mpro inhibitor currently completing phase 2 clinical trial. Here we describe the mechanism of action, broad activity against SARS-CoV-2 clinical isolates, combination studies with other SARS-CoV-2 inhibitors and favorable resistance profile of pomotrelvir. Method(s): The kinetic parameters of pomotrelvir Mpro inhibition and its interaction with nirmaltrevir were determined in a kinetic protease assay. The IC50s of pomotrelvir on mutant Mpro proteins were measured in an endpoint Mpro assay. Combination studies of pomotrelvir with remdesivir and molnupiravir were carried out in A549-hACE2 cells infected with SARS-CoV-2 NLuc virus. Activity against SARS-CoV-2 clinical variants was assessed by infection of A549-ACE2-TMPRSS2 cells followed by immunostaining of the viral nucleocapsid protein. Result(s): Pomotrelvir is a potent competitive inhibitor of SARS-CoV-2 Mpro (Ki =2.7 nM). Binding of pomotrelvir and the Mpro inhibitor nirmatrelvir to the active site is mutually exclusive. In the SARS-CoV-2 NLuc assay, pomotrelvir is additive when combined with remdesivir or molnupiravir, two nucleoside analogs targeting viral RNA synthesis. When the effect of Mpro substitutions previously selected in a resistance study of pomotrelvir were analyzed in an enzyme assay, only Mpro-N133H showed a significant increase in IC50 (45-fold). The catalytic efficiency of Mpro-N133H is reduced by 10-fold and the recombinant virus SARSCoV-2 (WA1) -N133H is not viable, suggesting that N133H has lower replicative fitness. Lastly, pomotrelvir exhibits broad activity against all SARS-CoV-2 clinical isolates tested to date, including five omicron variants. Conclusion(s): PBI-0451 is a potent competitive inhibitor of SARS-CoV-2 Mpro and is broadly active against SARS-CoV-2 clinical isolates including omicron variants. Results from inhibitor interaction studies support the potential combination of pomotrelvir with remdesivir and molnupiravir but not nirmatrelvir. Enzymatic characterization of in vitro selected pomotrelvir resistant variants indicates they either confer no resistance or have reduced fitness.
ABSTRACT
Background: The 3CL protease (3CLpro) of coronaviruses (CoV) is responsible for essential & early steps of viral replication. Early treatment of SARS-CoV2 infection with a 3CLpro inhibitor has shown to substantially reduce the rate of hospitalization & death from COVID-19. There is a need for a protease inhibitor that can be used as a stand-alone agent to treat and prevent SARS-CoV-2 infection globally, in the setting of remote testing & healthcare delivery, and as unsupervised outpatient use by a significant number of people who take other medications. Methods: PBI-0451 was assessed in cultures of inducible pluripotent stem cell-derived alveolar type II (iPS-AT2) cells, in nonclinical PK and toxicity studies, and an ongoing randomized, double-blind first-in-human (FIH) study evaluating the tolerability, safety, and PK of single and multiple doses administered as an oral suspension to healthy adult subjects. The effect of food and the potential for a drug-drug interaction (DDI) with ritonavir were also explored. Results: PBI-0451 potently inhibited SARS-CoV-2 replication in iPS-AT2 cells with multi-log reductions in viral titer and mean (SD) IC50 & EC90 values of 32 (25) & 106 (90) nM, respectively. No clinically relevant adverse effects of PBI-0451 were observed in 14-day GLP toxicity studies in mice and dogs, including on the cardiovascular, CNS, or respiratory systems. PBI-0451 was not genotoxic in Ames and micronucleus tests. In the ongoing FIH study to date, study treatments were generally well tolerated with no study drug or study discontinuations. No Grade 2, 3, 4, or severe adverse events were reported. Preliminary single-dose concentration-time profile of PBI-0451 following administration with food demonstrated a 2-compartment PK profile with a median terminal elimination t1/2 ranging from 11-14 hours. PBI-0451 demonstrated good oral bioavailability and a linear increase in exposure over a 10-fold dose range when administered with food, achieving concentrations >1-, 3-& 10-fold the plasma protein binding-adjusted EC90 value (374 ng/mL) against SARS-CoV-2 at doses of 100, 300 & 1050 mg, respectively. The PK of PBI-0451 was unaffected by coadministration with ritonavir. Conclusion: PBI-0451 has shown favorable nonclinical properties and early clinical safety & PK that supports its continued evaluation as a stand-alone agent. Ongoing multiple-dose evaluation will further elucidate its clinical profile and inform the dose & dosing regimen selection for potential Phase II/III studies.