Key result
Acrylamide fragment inhibitors covalently bound to the catalytic cysteines of EV71 3Cpro and SARS-CoV-2 Mpro, inhibiting their activity (IC50 10-68 µM) and inducing profound conformational distortions that disrupt Mpro dimerization.
Why the study?
RNA virus proteases with structurally conserved catalytic cysteines present an opportunity for irreversible inhibition using electrophilic small molecules.
Acrylamide fragments can irreversibly inhibit EV71 3Cpro and SARS-CoV-2 Mpro by covalently binding to the catalytic cysteine and inducing allosteric conformational distortions.
Requires validation in infection models; leaves open therapeutic potential of covalent allosteric Mpro inhibitors.
RNA viruses are critically dependent upon virally encoded proteases that cleave the viral polyproteins into functional mature proteins. Many of these proteases are structurally conserved with an essential catalytic cysteine and this offers the opportunity to irreversibly inhibit these enzymes with electrophilic small molecules. Here we describe the successful application of quantitative irreversible tethering (qIT) to identify acrylamide fragments that selectively target the active site cysteine of the 3C protease (3C pro ) of Enterovirus 71, the causative agent of hand, foot and mouth disease in humans, altering the substrate binding region. Further, we effectively re-purpose these hits towards the main protease (M pro ) of SARS-CoV-2 which shares the 3C-like fold as well as similar catalytic-triad. We demonstrate that the hit fragments covalently link to the catalytic cysteine of M pro to inhibit its activity. In addition, we provide the first demonstration that targeting the active site cysteine of M pro can also have profound allosteric effects, distorting secondary structures required for formation of the active dimeric unit of M pro . These new data provide novel mechanistic insights into the design of EV71 3C pro and SARS-CoV-2 M pro inhibitors and identify acrylamide-tagged pharmacophores for elaboration into more selective agents of therapeutic potential.
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Qin et al. (2020) studied Enterovirus 71 and SARS-CoV-2 (in vitro). Acrylamide fragment inhibitors vs. Unliganded protease or Glutathione (GSH) was evaluated on Protease inhibition (IC50) and covalent modification. Acrylamide fragment inhibitors covalently bound to the catalytic cysteines of EV71 3Cpro and SARS-CoV-2 Mpro, inhibiting their activity (IC50 10-68 µM) and inducing profound conformational distortions that disrupt Mpro dimerization.
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