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Allosteric inhibition of the SARS-CoV-2 main protease - insights from mass spectrometry-based assays
Tarick J El-Baba; Corinne A Lutomski; Anastassia L Kantsadi; Tika R Malla; Tobias John; Victor Mikhailov; Jani R Bolla; Christopher J Schofield; Nicole Zitzmann; Ioannis Vakonakis; Carol V Robinson.
Afiliación
  • Tarick J El-Baba; Physical and Theoretical Chemistry Laboratory University of Oxford South Parks Rd. OX1 3QZ Oxford UK
  • Corinne A Lutomski; Physical and Theoretical Chemistry Laboratory University of Oxford South Parks Rd. OX1 3QZ Oxford UK
  • Anastassia L Kantsadi; Department of Biochemistry University of Oxford South Parks Rd. OX1 3QU, Oxford UK
  • Tika R Malla; Chemistry Research Laboratory University of Oxford 12 Mansfield Rd, OX1 3TA, Oxford UK
  • Tobias John; Chemistry Research Laboratory University of Oxford 12 Mansfield Rd, OX1 3TA, Oxford UK
  • Victor Mikhailov; Chemistry Research Laboratory University of Oxford 12 Mansfield Rd, OX1 3TA, Oxford UK
  • Jani R Bolla; Physical and Theoretical Chemistry Laboratory University of Oxford South Parks Rd. OX1 3QZ Oxford UK
  • Christopher J Schofield; Chemistry Research Laboratory University of Oxford 12 Mansfield Rd, OX1 3TA, Oxford UK
  • Nicole Zitzmann; Department of Biochemistry University of Oxford South Parks Rd. OX1 3QU, Oxford UK
  • Ioannis Vakonakis; Department of Biochemistry University of Oxford South Parks Rd. OX1 3QU, Oxford UK
  • Carol V Robinson; Physical and Theoretical Chemistry Laboratory University of Oxford South Parks Rd. OX1 3QZ Oxford UK
Preprint en En | PREPRINT-BIORXIV | ID: ppbiorxiv-226761
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ABSTRACT
Following translation of the SARS-CoV-2 RNA genome into two viral polypeptides, the main protease Mpro cleaves at eleven sites to release non-structural proteins required for viral replication. MPro is an attractive target for antiviral therapies to combat the coronavirus-2019 disease (COVID-19). Here, we have used native mass spectrometry (MS) to characterize the functional unit of Mpro. Analysis of the monomer-dimer equilibria reveals a dissociation constant of Kd = 0.14 {+/-} 0.03 M, revealing MPro has a strong preference to dimerize in solution. Developing an MS-based kinetic assay we then characterized substrate turnover rates by following temporal changes in the enzyme-substrate complexes, which are effectively "flash-frozen" as they transition from solution to the gas phase. We screened small molecules, that bind distant from the active site, for their ability to modulate activity. These compounds, including one proposed to disrupt the catalytically active dimer, slow the rate of substrate processing by ~35%. This information was readily obtained and, together with analysis of the x-ray crystal structures of these enzyme-small molecule complexes, provides a starting point for the development of more potent molecules that allosterically regulate MPro activity.
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Texto completo: 1 Colección: 09-preprints Base de datos: PREPRINT-BIORXIV Idioma: En Año: 2020 Tipo del documento: Preprint
Texto completo
Añadir a Mi BVS
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XML
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Texto completo: 1 Colección: 09-preprints Base de datos: PREPRINT-BIORXIV Idioma: En Año: 2020 Tipo del documento: Preprint