Gain-of-Signal Assays for Probing Inhibition of SARS-CoV-2 Mpro/3CLpro in Living Cells

Seyed Arad Moghadasi, Morgan Esler, Yuka Otsuka, Jordan Becker, Sofia N. Moraes, Constance B. Anderson, Srinivas Chamakuri, Christopher Belica, Chloe Wick, Daniel A. Harki, Damian W. Young, Louis Scampavia, Timothy P. Spicer, Ke Shi, Hideki Aihara, William L Brown, Reuben Harris

Research output: Contribution to journalArticlepeer-review

Abstract

The main protease, Mpro, of SARS-CoV-2 is required to cleave the viral polyprotein into precise functional units for virus replication and pathogenesis. Here, we report quantitative reporters for Mpro function in living cells in which protease inhibition by genetic or chemical methods results in robust signal readouts by fluorescence (enhanced green fluorescent protein [eGFP]) or bioluminescence (firefly luciferase). These gain-of-signal systems are scalable to high-throughput platforms for quantitative discrimination between Mpro mutants and/or inhibitor potencies as evidenced by validation of several reported inhibitors. Additional utility is shown by single Mpro amino acid variants and structural information combining to demonstrate that both inhibitor conformational dynamics and amino acid differences are able to influence inhibitor potency. We further show that a recent variant of concern (Omicron) has an unchanged response to a clinically approved drug, nirmatrelvir, whereas proteases from divergent coronavirus species show differential susceptibility. Together, we demonstrate that these gain-of-signal systems serve as robust, facile, and scalable assays for live cell quantification of Mpro inhibition, which will help expedite the development of next-generation antivirals and enable the rapid testing of emerging variants. IMPORTANCE The main protease, Mpro, of SARS-CoV-2 is an essential viral protein required for the earliest steps of infection. It is therefore an attractive target for antiviral drug development. Here, we report the development and implementation of two complementary cell-based systems for quantification of Mpro inhibition by genetic or chemical approaches. The first is fluorescence based (eGFP), and the second is luminescence based (firefly luciferase). Importantly, both systems rely upon gain-of-signal readouts such that stronger inhibitors yield higher fluorescent or luminescent signal. The high versatility and utility of these systems are demonstrated by characterizing Mpro mutants and natural variants, including Omicron, as well as a panel of existing inhibitors. These systems rapidly, safely, and sensitively identify Mpro variants with altered susceptibilities to inhibition, triage-nonspecific, or off-target molecules and validate bona fide inhibitors, with the most potent thus far being the first-in-class drug nirmatrelvir.

Original languageEnglish (US)
JournalmBio
Volume13
Issue number3
DOIs
StatePublished - Jun 2022

Bibliographical note

Funding Information:
These studies were supported in part by grants to RSH from the National Institute for Allergy and Infectious Diseases (R37-AI064046) and the National Cancer Institute (P01-CA234228) and a grant to H.A. from the National Institute of General Medical Sciences (R35-GM118047). J.T.B. received salary support from the National Institute for Allergy and Infectious Diseases (F32-AI147813). This work was supported in part by a grant to the University of Minnesota (S.N.M. and R.S.H.) from the Howard Hughes Medical Institute through the James H. Gilliam Fellowships for Advanced Study program. NE-CAT of the Advanced Photon Source is supported by P30-GM124165. The Pilatus 6 M detector on 24-ID-C beamline is funded by an NIH-ORIP HEI grant (S10-RR029205). R.S.H. is the Margaret Harvey Schering Land Grant Chair for Cancer Research, a Distinguished University McKnight Professor, and an Investigator of the Howard Hughes Medical Institute. We have no competing interests to declare.

Funding Information:
These studies were supported in part by grants to RSH from the National Institute for Allergy and Infectious Diseases (R37-AI064046) and the National Cancer Institute (P01-CA234228) and a grant to H.A. from the National Institute of General Medical Sciences (R35-GM118047). J.T.B. received salary support from the National Institute for Allergy and Infectious Diseases (F32-AI147813). This work was supported in part by a grant to the University of Minnesota (S.N.M. and R.S.H.) from the Howard Hughes Medical Institute through the James H. Gilliam Fellowships for Advanced Study program. NE-CAT of the Advanced Photon Source is supported by P30-GM124165. The Pilatus 6 M detector on 24-ID-C beamline is funded by an NIH-ORIP HEI grant (S10-RR029205). R.S.H. is the Margaret Harvey Schering Land Grant Chair for Cancer Research, a Distinguished University McKnight Professor, and an Investigator of the Howard Hughes Medical Institute.

Publisher Copyright:
© 2022 Moghadasi et al.

Keywords

  • SARS-CoV-2 (SARS2)
  • coronavirus
  • gain-of-signal cell-based systems
  • main protease (M/ 3CL)
  • viral protease inhibitors

PubMed: MeSH publication types

  • Journal Article
  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

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