Abstract
The main protease (Mpro) of SARS-CoV-2 is central to viral maturation and is a promising drug target, but little is known about structural aspects of how it binds to its 11 natural cleavage sites. We used biophysical and crystallographic data and an array of biomolecular simulation techniques, including automated docking, molecular dynamics (MD) and interactive MD in virtual reality, QM/MM, and linear-scaling DFT, to investigate the molecular features underlying recognition of the natural Mpro substrates. We extensively analysed the subsite interactions of modelled 11-residue cleavage site peptides, crystallographic ligands, and docked COVID Moonshot-designed covalent inhibitors. Our modelling studies reveal remarkable consistency in the hydrogen bonding patterns of the natural Mpro substrates, particularly on the N-terminal side of the scissile bond. They highlight the critical role of interactions beyond the immediate active site in recognition and catalysis, in particular plasticity at the S2 site. Building on our initial Mpro-substrate models, we used predictive saturation variation scanning (PreSaVS) to design peptides with improved affinity. Non-denaturing mass spectrometry and other biophysical analyses confirm these new and effective ‘peptibitors’ inhibit Mpro competitively. Our combined results provide new insights and highlight opportunities for the development of Mpro inhibitors as anti-COVID-19 drugs.
Highlights
Introduction(Mpro; 3 chymotrypsin-like or 3CL proteinase, 3C-like protease, 3CLpro; or non-structural protein 5, Nsp[5])
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of coronavirus disease 2019 (COVID19) that caused the World Health Organization to declare a global pandemic in March 2020
The main protease (Mpro) of SARS-CoV-2 is central to viral maturation and is a promising drug target, but little is known about structural aspects of how it binds to its 11 natural cleavage sites
Summary
(Mpro; 3 chymotrypsin-like or 3CL proteinase, 3C-like protease, 3CLpro; or non-structural protein 5, Nsp[5]). Computational and mechanistic studies on SARS-CoV Mpro[26,27,28,29] and SARS-CoV-2 Mpro[30,31,32] suggest that in the resting state His-41 and Cys-145 are likely neutral and that the protonation states of nearby histidines (e.g. His-163, 164, and 172) affect the structure of the catalytic machinery— it has been suggested in SARS-CoV Mpro that the protonation state of the catalytic dyad may change in the presence of an inhibitor or substrate.[33] A different picture has been obtained from neutron crystallographic studies, which indicate that an ion pair form of the dyad is favoured at pH 6.6.34 While neutron crystallography, in principle, enables the direct determination of hydrogen atom positions, questions remain about how pH and the presence of active site-bound ligands in uence the precise—and likely dynamic—protonation state(s) of the dyad. Important questions remain regarding Mpro catalysis, including to what extent the active site protonation state, solvent accessibility, induced t, and substrate sequence in uence activity The lack of this knowledge makes it difficult to carry out effective computational studies on Mpro catalysis and inhibition. The results are freely available via GitHub (https://github.com/gmm/SARS-CoV-2-Modelling)
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