Evaluation of in vitro antiviral activity of SARS-CoV-2 Mpro inhibitor pomotrelvir and cross-resistance to nirmatrelvir resistance substitutions

Abstract

The unprecedented scale of the COVID-19 pandemic and the rapid evolution of SARS-CoV-2 variants underscore the need for broadly active inhibitors with a high barrier to resistance. The coronavirus main protease (M^pro) is an essential cysteine protease required for viral polyprotein processing and is highly conserved across human coronaviruses. Pomotrelvir is a novel M^pro inhibitor that has recently completed a phase 2 clinical trial. In this report, we demonstrated that pomotrelvir is a potent competitive inhibitor of SARS-CoV-2 M^pro with high selectivity against human proteases. In the enzyme assay, pomotrelvir is also active against M^pro proteins derived from human coronaviruses CoV-229E, CoV-OC43, CoV-HKU1, CoV-NL63, MERS, and SARS-CoV. In cell-based SARS-CoV-2 replicon and SARS-CoV-2 infection assays, pomotrelvir has shown potent inhibitory activity and is broadly active against SARS-CoV-2 clinical isolates including Omicron variants. Many resistance substitutions of the M^pro inhibitor nirmatrelvir confer cross-resistance to pomotrelvir, consistent with the finding from our enzymatic analysis that pomotrelvir and nirmatrelvir compete for the same binding site. In a SARS-CoV-2 infection assay, pomotrelvir is additive when combined with remdesivir or molnupiravir, two nucleoside analogs targeting viral RNA synthesis. In conclusion, our results from the in vitro characterization of pomotrelvir antiviral activity support its further clinical development as an alternative COVID-19 therapeutic option.

Keywords: Mpro inhibitor; PBI-0451; SARS-CoV-2; antiviral resistance; coronavirus; pomotrelvir.

Fig 1

Structural analysis of pomotrelvir. (A) Chemical structure of pomotrelvir. (B) Crystal structure of pomotrelvir bound to SARS-CoV-2 M^pro. (C) X-ray diffraction data collection and refinement statistics.

Fig 2

Characterization of pomotrelvir (PBI-0451) inhibition of SARS-CoV-2 M^pro in enzyme assays. (A) Pomotrelvir was tested in the presence of 3 nM SARS-CoV-2 M^pro and 1 µM of substrate peptide (FAMTSAVLQSGFRK-NH2); cleavage was measured by microfluidic electrophoresis using Caliper’s LabChip 3000. Progress curve was generated by plotting conversion% of substrate over incubation time. (B) The K _i for pomotrelvir was obtained by a global fit with the Morrison correction (with 2.3–300 μM substrate); an alpha factor of 10.4 (>>1) was consistent with a competitive inhibition model.

Fig 3

Binding exclusivity study of pomotrelvir (PBI-0451) and nirmatrelvir. The initial velocity was determined in the presence of pomotrelvir (0–20 nM) and nirmatrelvir (0–8 nM). The binding mode was assessed by the Yonetani–Theorell equation. The binding exclusivity coefficient (γ) was >>1 as shown by the near-parallel lines under various nirmatrelvir concentrations, demonstrating that the two inhibitors share the same binding site.