Evaluation of SARS-CoV-2 Main Protease Inhibitors Using a Novel Cell-Based Assay

Abstract

As an essential enzyme of SARS-CoV-2, main protease (M Pro ) triggers acute toxicity to its human cell host, an effect that can be alleviated by an M Pro inhibitor. Using this toxicity alleviation, we developed an effective method that allows a bulk analysis of the cellular potency of M Pro inhibitors. This novel assay is advantageous over an antiviral assay in providing precise cellular M Pro inhibition information to assess an M Pro inhibitor. We used this assay to analyze 30 known M Pro inhibitors. Contrary to their strong antiviral effects and up to 10 μM, 11a, calpain inhibitor II, calpain XII, ebselen, bepridil, chloroquine, and hydroxychloroquine showed relatively weak to undetectable cellular M Pro inhibition potency implicating their roles in interfering with key steps other than just the M Pro catalysis in the SARS-CoV-2 life cycle. Our results also revealed that MPI5, MPI6, MPI7, and MPI8 have high cellular and antiviral potency. As the one with the highest cellular and antiviral potency among all tested compounds, MPI8 has a remarkable cellular M Pro inhibition IC 50 value of 31 nM that matches closely to its strong antiviral effect with an EC 50 value of 30 nM. Therefore, we cautiously suggest exploring MPI8 further for COVID-19 preclinical tests.

A multi-pronged evaluation of aldehyde-based tripeptidyl main protease inhibitors as SARS-CoV-2 antivirals

“…In previous studies, we developed MPI1-10 and characterized both their M Pro inhibition potency and their antiviral potency [ 13 , 14 ]. As a tripeptidyl aldehyde, MPI8 shows the highest antiviral potency with an EC 50 value of 30 nM to neutralize SARS-CoV-2 (USA-WA1/2020) in Vero E6 cells [ 31 ]. MPI3, another tripeptidyl aldehyde, doesn't have high antiviral potency.…”

“…M Pro is acutely toxic to a human cell host. Using this unique characteristic, we developed a cell-based analysis to characterize cellular M Pro inhibition potency for compounds [ 31 ]. A similar assay was also used by others to analyze cellular potency of M Pro inhibitors [ 33 , 34 ].…”

Progress and Challenges in Targeting the SARS-CoV-2 Papain-like Protease

“…Other cell-based assays, including the GFP ER translocation assay, the bioluminescence resonance energy transfer (BRET) assay, and the cell cytotoxicity assay, can be similarly engineered for PL pro . 75 − 77 …”

Fluorine in Medicinal Chemistry: In Perspective to COVID-19

“…Coronavirus disease 2019 (COVID-19) is a contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, Scheme 1 ). 1 The disease has spread worldwide, and the threat to global health is ongoing since the first known case was identified in December 2019. As of 10 April 2022, SARS-CoV-2 has caused a combined total of more than 494588000 confirmed infections and more than 6170000 reported deaths in 213 different countries.…”

“…TMPRSS2, CtsL, and furin are three host proteases that prime Spike for viral entry and new virion packaging. Reproduced with permission from ref ( 1 ). Copyright 2022 American Chemical Society.…”