Dataset Information

A proteomics-MM/PBSA dual approach for the analysis of SARS-CoV-2 main protease substrate peptide specificity.

ABSTRACT: The main protease M^pro of SARS-CoV-2 is a well-studied major drug target. Additionally, it has been linked to this virus' pathogenicity, possibly through off-target effects. It is also an interesting diagnostic target. To obtain more data on possible substrates as well as to assess the enzyme's primary specificity a two-step approach was introduced. First, Terminal Amine Isobaric Labeling of Substrates (TAILS) was employed to identify novel M^pro cleavage sites in a mouse lung proteome library. In a second step, using a structural homology model, the MM/PBSA variant MM/GBSA (Molecular Mechanics Poisson-Boltzmann/Generalized Born Surface Area) free binding energy calculations were carried out to determine relevant interacting amino acids. As a result, 58 unique cleavage sites were detected, including six that displayed glutamine at the P1 position. Furthermore, modeling results indicated that M^pro has a far higher potential promiscuity towards substrates than expected. The combination of proteomics and MM/PBSA modeling analysis can thus be useful for elucidating the specificity of M^pro, and thus open novel perspectives for the development of future peptidomimetic drugs against COVID-19, as well as diagnostic tools.

SUBMITTER: Gallo G

PROVIDER: S-EPMC9134770 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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A proteomics-MM/PBSA dual approach for the analysis of SARS-CoV-2 main protease substrate peptide specificity.

Gallo Gloria G Barcick Uilla U Coelho Camila C Salardani Murilo M Camacho Maurício F MF Cajado-Carvalho Daniela D Loures Flávio V FV Serrano Solange M T SMT Hardy Leon L Zelanis André A Würtele Martin M

Peptides 20220526

The main protease M<sup>pro</sup> of SARS-CoV-2 is a well-studied major drug target. Additionally, it has been linked to this virus' pathogenicity, possibly through off-target effects. It is also an interesting diagnostic target. To obtain more data on possible substrates as well as to assess the enzyme's primary specificity a two-step approach was introduced. First, Terminal Amine Isobaric Labeling of Substrates (TAILS) was employed to identify novel M<sup>pro</sup> cleavage sites in a mouse lu ...[more]

PMID: 35644302

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Project description:SARS coronavirus main protease (SARS-CoV M(pro)) is essential for the replication of the virus and regarded as a major antiviral drug target. The enzyme is a cysteine protease, with a catalytic dyad (Cys-145/His-41) in the active site. Aldehyde inhibitors can bind reversibly to the active-site sulfhydryl of SARS-CoV M(pro). Previous studies using peptidic substrates and inhibitors showed that the substrate specificity of SARS-CoV M(pro) requires glutamine in the P1 position and a large hydrophobic residue in the P2 position. We determined four crystal structures of SARS-CoV M(pro) in complex with pentapeptide aldehydes (Ac-ESTLQ-H, Ac-NSFSQ-H, Ac-DSFDQ-H, and Ac-NSTSQ-H). Kinetic data showed that all of these aldehydes exhibit inhibitory activity towards SARS-CoV M(pro), with K(i) values in the ?M range. Surprisingly, the X-ray structures revealed that the hydrophobic S2 pocket of the enzyme can accommodate serine and even aspartic-acid side-chains in the P2 positions of the inhibitors. Consequently, we reassessed the substrate specificity of the enzyme by testing the cleavage of 20 different tetradecapeptide substrates with varying amino-acid residues in the P2 position. The cleavage efficiency for the substrate with serine in the P2 position was 160-times lower than that for the original substrate (P2=Leu); furthermore, the substrate with aspartic acid in the P2 position was not cleaved at all. We also determined a crystal structure of SARS-CoV M(pro) in complex with aldehyde Cm-FF-H, which has its P1-phenylalanine residue bound to the relatively hydrophilic S1 pocket of the enzyme and yet exhibits a high inhibitory activity against SARS-CoV M(pro), with K(i)=2.24±0.58 ?M. These results show that the stringent substrate specificity of the SARS-CoV M(pro) with respect to the P1 and P2 positions can be overruled by the highly electrophilic character of the aldehyde warhead, thereby constituting a deviation from the dogma that peptidic inhibitors need to correspond to the observed cleavage specificity of the target protease.

Dataset Information

A proteomics-MM/PBSA dual approach for the analysis of SARS-CoV-2 main protease substrate peptide specificity.

Publications

A proteomics-MM/PBSA dual approach for the analysis of SARS-CoV-2 main protease substrate peptide specificity.

Similar Datasets

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