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SARS-CoV-2 Mpro inhibitor identification using a cellular gain-of-signal assay for high-throughput screening
Renee Delgado1, Jyoti Vishwakarma1, Seyed Arad Moghadasi2
1Department of Biochemistry and Structural Biology, University of Texas Health San Antonio, San Antonio, TX 78229, USA.
Abstract:
Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2, SARS2) is responsible for the COVID-19 pandemic and infections that continue to affect the lives of millions of people worldwide, especially those who are older and/or immunocompromised. The SARS2 main protease enzyme, Mpro (also called 3C-like protease, 3CLpro), is a bona fide drug target as evidenced by potent inhibition with nirmatrelvir and ensitrelvir, the active components of the drugs Paxlovid and Xocova, respectively. However, the existence of nirmatrelvir and ensitrelvir-resistant isolates underscores the need to develop next-generation drugs with different resistance profiles and/or distinct mechanisms of action. Here, we report the results of a high-throughput screen of 649,568 compounds using a cellular gain-of-signal assay. In this assay, Mpro inhibits expression of a luciferase reporter, and 8,777 small molecules were considered hits by causing a gain in luciferase activity 3x SD above the sample field activity (6.8% gain-of-signal relative to 100 µM GC376). Single concentration and dose-response gain-of-signal experiments confirmed 3,522/8,762 compounds as candidate inhibitors. In parallel, all initial high-throughput screening hits were tested in a peptide cleavage assay with purified Mpro and only 39/8,762 showed inhibition. Importantly, 19/39 compounds (49%) re-tested positive in both SARS2 assays, including two previously reported Mpro inhibitors, demonstrating the efficacy of the overall screening strategy. This approach led to the rediscovery of known Mpro inhibitors such as calpain inhibitor II, as well as to the discovery of novel compounds that provide chemical information for future drug development efforts.
Insights
Researchers screened over 600,000 compounds to find new inhibitors for the SARS-CoV-2 main protease (Mpro). This screen identified novel drug candidates to combat COVID-19, addressing resistance to existing treatments.
Area of Science:
- Biochemistry
- Drug Discovery
- Virology
Background:
- Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) causes COVID-19, a global health crisis.
- The SARS-CoV-2 main protease (Mpro) is a validated drug target, with nirmatrelvir and ensitrelvir demonstrating efficacy.
- Drug resistance necessitates the development of novel antiviral agents with distinct mechanisms or resistance profiles.
Purpose of the Study:
- To identify novel inhibitors of the SARS-CoV-2 main protease (Mpro) through a large-scale high-throughput screening campaign.
- To validate hits from a cellular assay using an enzymatic peptide cleavage assay.
- To discover new chemical entities for the development of next-generation COVID-19 therapeutics.
Main Methods:
- A high-throughput screen of 649,568 compounds was conducted using a cellular gain-of-signal assay where Mpro activity is linked to luciferase expression.
- Initial hits were confirmed through dose-response experiments in the cellular assay.
- Confirmed hits were subsequently tested in a biochemical assay measuring Mpro's inhibition of peptide cleavage.
Main Results:
- The initial screen identified 8,777 potential hits, with 3,522 confirmed as candidate inhibitors in the cellular assay.
- A parallel biochemical assay with purified Mpro identified 39 compounds that inhibited peptide cleavage.
- Of the 39 compounds, 19 (49%) validated in both assays, including known Mpro inhibitors and novel chemical structures.
Conclusions:
- The high-throughput screening strategy effectively identified validated inhibitors of the SARS-CoV-2 Mpro.
- The study rediscovered known inhibitors and discovered novel compounds, providing valuable chemical starting points for future drug development.
- These findings contribute to the ongoing effort to develop new antiviral drugs against SARS-CoV-2, particularly those effective against resistant strains.
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