Dataset Information

Efficiency Improvements and Discovery of New Substrates for a SARS-CoV-2 Main Protease FRET Assay.

ABSTRACT: The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has a huge impact on the world. Although several vaccines have recently reached the market, the development of specific antiviral drugs against SARS-CoV-2 is an important additional strategy in fighting the pandemic. One of the most promising pharmacological targets is the viral main protease (M^pro). Here, we present an optimized biochemical assay procedure for SARS-CoV-2 M^pro. We have comprehensively investigated the influence of different buffer components and conditions on the assay performance and characterized Förster resonance energy transfer (FRET) substrates with a preference for 2-Abz/Tyr(3-NO₂) FRET pairs. The substrates 2-AbzSAVLQSGTyr(3-NO₂)R-OH, a truncated version of the established DABCYL/EDANS FRET substrate, and 2-AbzVVTLQSGTyr(3-NO₂)R-OH are promising candidates for screening and inhibitor characterization. In the latter substrate, the incorporation of Val at position P5 improved the catalytic efficiency. Based on the obtained results, we present here a reproducible, reliable assay protocol using highly affordable buffer components.

SUBMITTER: Drazic T

PROVIDER: S-EPMC8458682 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature

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Efficiency Improvements and Discovery of New Substrates for a SARS-CoV-2 Main Protease FRET Assay.

Dražić Tonko T Kühl Nikos N Leuthold Mila M MM Behnam Mira A M MAM Klein Christian D CD

SLAS discovery : advancing life sciences R & D 20210619 9

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has a huge impact on the world. Although several vaccines have recently reached the market, the development of specific antiviral drugs against SARS-CoV-2 is an important additional strategy in fighting the pandemic. One of the most promising pharmacological targets is the viral main protease (M<sup>pro</sup>). Here, we present an optimized biochemical assay procedure for SARS-CoV-2 M<sup>pro</sup>. We have comprehensively investigated the i ...[more]

PMID: 34151620

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Project description:SARS-CoV-2 is the etiological pathogen of the COVID-19 pandemic, which led to more than 6.5 million deaths since the beginning of the outbreak in December 2019. The unprecedented disruption of social life and public health caused by COVID-19 calls for fast-track development of diagnostic kits, vaccines, and antiviral drugs. Small molecule antivirals are essential complements of vaccines and can be used for the treatment of SARS-CoV-2 infections. Currently, there are three FDA-approved antiviral drugs, remdesivir, molnupiravir, and paxlovid. Given the moderate clinical efficacy of remdesivir and molnupiravir, the drug-drug interaction of paxlovid, and the emergence of SARS-CoV-2 variants with potential drug-resistant mutations, there is a pressing need for additional antivirals to combat current and future coronavirus outbreaks.In this Account, we describe our efforts in developing covalent and noncovalent main protease (Mpro) inhibitors and the identification of nirmatrelvir-resistant mutants. We initially discovered GC376, calpain inhibitors II and XII, and boceprevir as dual inhibitors of Mpro and host cathepsin L from a screening of a protease inhibitor library. Given the controversy of targeting cathepsin L, we subsequently shifted the focus to designing Mpro-specific inhibitors. Specifically, guided by the X-ray crystal structures of these initial hits, we designed noncovalent Mpro inhibitors such as Jun8-76-3R that are highly selective toward Mpro over host cathepsin L. Using the same scaffold, we also designed covalent Mpro inhibitors with novel cysteine reactive warheads containing di- and trihaloacetamides, which similarly had high target specificity. In parallel to our drug discovery efforts, we developed the cell-based FlipGFP Mpro assay to characterize the cellular target engagement of our rationally designed Mpro inhibitors. The FlipGFP assay was also applied to validate the structurally disparate Mpro inhibitors reported in the literature. Lastly, we introduce recent progress in identifying naturally occurring Mpro mutants that are resistant to nirmatrelvir from genome mining of the nsp5 sequences deposited in the GISAID database. Collectively, the covalent and noncovalent Mpro inhibitors and the nirmatrelvir-resistant hot spot residues from our studies provide insightful guidance for future work aimed at developing orally bioavailable Mpro inhibitors that do not have overlapping resistance profile with nirmatrelvir.

Dataset Information

Efficiency Improvements and Discovery of New Substrates for a SARS-CoV-2 Main Protease FRET Assay.

Publications

Efficiency Improvements and Discovery of New Substrates for a SARS-CoV-2 Main Protease FRET Assay.

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