Dataset Information

Expression, purification, and biophysical characterization of recombinant MERS-CoV main (M^pro) protease.

ABSTRACT: MERS-CoV main protease (M^pro) is essential for the maturation of the coronavirus; therefore, considered a potential drug target. Detailed conformational information is essential to developing antiviral therapeutics. However, the conformation of MERS-CoV M^pro under different conditions is poorly characterized. In this study, MERS-CoV M^pro was recombinantly produced in E.coli and characterized its structural stability with respect to changes in pH and temperatures. The intrinsic and extrinsic fluorescence measurements revealed that MERS-CoV M^pro tertiary structure was exposed to the polar environment due to the unfolding of the tertiary structure. However, the secondary structure of MERS-CoV M^pro was gained at low pH because of charge-charge repulsion. Furthermore, differential scanning fluorometry studies of M^pro showed a single thermal transition at all pHs except at pH 2.0; no transitions were observed. The data from the spectroscopic studies suggest that the MERS-CoV M^pro forms a molten globule-like state at pH 2.0. Insilico studies showed that the covid-19 M^pro shows 96.08% and 50.65% similarity to that of SARS-CoV M^pro and MERS-CoV M^pro, respectively. This study provides a basic understanding of the thermodynamic and structural properties of MERS-CoV M^pro.

SUBMITTER: Almutairi GO

PROVIDER: S-EPMC9017057 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Expression, purification, and biophysical characterization of recombinant MERS-CoV main (Mpro) protease.

Almutairi Ghada Obeid GO Malik Ajamaluddin A Alonazi Mona M Khan Javed Masood JM Alhomida Abdullah S AS Khan Mohd Shahnawaz MS Alenad Amal M AM Altwaijry Nojood N Alafaleq Nouf Omar NO

International journal of biological macromolecules 20220419 Pt A

MERS-CoV main protease (Mpro) is essential for the maturation of the coronavirus; therefore, considered a potential drug target. Detailed conformational information is essential to developing antiviral therapeutics. However, the conformation of MERS-CoV Mpro under different conditions is poorly characterized. In this study, MERS-CoV Mpro was recombinantly produced in E.coli and characterized its structural stability with respect to changes in pH and temperatures. ...[more]

PMID: 35452699

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Project description:Ibuzatrelvir (1) was recently disclosed and patented by Pfizer for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It has received fast-track status from the USA Food and Drug Administration (FDA) and has entered phase III clinical trials as a possible replacement for Paxlovid. Like nirmatrelvir (2) in Paxlovid, this orally active drug candidate is designed to target viral main proteases (Mpro) through reversible covalent interaction of its nitrile warhead with the active site thiol of the chymotrypsin-like cysteine protease (3CL protease). Inhibition of Mpro hinders the processing of the proteins essential for viral replication in vivo. However, ibuzatrelvir apparently does not require ritonavir (3), which is coadministered in Paxlovid to block human oxidative metabolism of nirmatrelvir. Here, we report the crystal structure of the complex of ibuzatrelvir with the active site of SARS-CoV-2 Mpro at 2.0 Å resolution. In addition, we show that ibuzatrelvir also potently inhibits the Mpro of Middle East respiratory syndrome-related coronavirus (MERS-CoV), which is fortunately not widespread but can be dangerously lethal (∼36% mortality). Co-crystal structures show that the binding mode of the drug to both active sites is similar and that the trifluoromethyl group of the inhibitor fits precisely into a critical S2 substrate binding pocket of the main proteases. However, our results also provide a rationale for the differences in potency of ibuzatrelvir for these two proteases due to minor differences in the substrate preferences leading to a weaker H-bond network in MERS-CoV Mpro. In addition, we examined the reversibility of compound binding to both proteases, which is an important parameter in reducing off-target effects as well as the potential immunogenicity. The crystal structures of the ibuzatrelvir complexes with Mpro of SARS-CoV-2 and of MERS-CoV will further assist drug design for coronaviral infections in humans and animals.

Dataset Information

Expression, purification, and biophysical characterization of recombinant MERS-CoV main (M^pro) protease.

Publications

Expression, purification, and biophysical characterization of recombinant MERS-CoV main (M<sup>pro</sup>) protease.

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