Structure-Based Identification of Naphthoquinones and Derivatives as Novel Inhibitors of Main Protease M^pro and Papain-like Protease PL^pro of SARS-CoV-2

Abstract

The worldwide COVID-19 pandemic caused by the coronavirus SARS-CoV-2 urgently demands novel direct antiviral treatments. The main protease (M pro ) and papain-like protease (PL pro ) are attractive drug targets among coronaviruses due to their essential role in processing the polyproteins translated from the viral RNA. In this study, we virtually screened 688 naphthoquinoidal compounds and derivatives against M pro of SARS-CoV-2. Twenty-four derivatives were selected and evaluated in biochemical assays against M pro using a novel fluorogenic substrate. In parallel, these compounds were also assayed with SARS-CoV-2 PL pro . Four compounds inhibited M pro with half-maximal inhibitory concentration (IC 50 ) values between 0.41 μM and 9.0 μM. In addition, three compounds inhibited PL pro with IC 50 ranging from 1.9 μM to 3.3 μM. To verify the specificity of M pro and PL pro inhibitors, our experiments included an assessment of common causes of false positives such as aggregation, high compound fluorescence, and inhibition by enzyme oxidation. Altogether, we confirmed novel classes of specific M pro and PL pro inhibitors. Molecular dynamics simulations suggest stable binding modes for M pro inhibitors with frequent interactions with residues in the S1 and S2 pockets of the active site. For two PL pro inhibitors, interactions occur in the S3 and S4 pockets. In summary, our structure-based computational and biochemical approach identified novel naphthoquinonal scaffolds that can be further explored as SARS-CoV-2 antivirals.

AI-Driven Discovery of SARS-CoV-2 Main Protease Fragment-like Inhibitors with Antiviral Activity In Vitro

“…The first protocol was carried out to explore the ligand’s ability to interact with the most important subsites (S1 and S2) of the active site, since these are the most common binding regions of known inhibitors. 66 The unrestricted protocol was also carried out to fully explore MPro’s active site and allows ligands to bind freely with other subsites as a means to obtain different insights on their binding modes.…”

“…Due to the small size of the ligands studied, we performed two types of docking protocols: one restricting the binding site to the catalytic site (restricted protocol) and the other probing the full substrate binding site (unrestricted protocol). The first protocol was carried out to explore the ligand’s ability to interact with the most important subsites (S1 and S2) of the active site, since these are the most common binding regions of known inhibitors . The unrestricted protocol was also carried out to fully explore MPro’s active site and allows ligands to bind freely with other subsites as a means to obtain different insights on their binding modes.…”

Unraveling the catalytic mechanism of SARS-CoV-2 papain-like protease with allosteric modulation of C270 mutation using multiscale computational approaches

“…It is interesting that water molecules have been detected in the active site of SARS-CoV-2 3CL pro . A recent study using the ProBiS H 2 O approach to investigate water molecules within 72 3CL pro crystal structures observed four conserved water molecules, 52 one of which is near H41 and may mediate formation of hydrogen bonds with H41 and D187. 16 To evaluate the interactions of H41 with this water molecule, we ran conventional MD simulations on apo SARS-CoV-2 3CL pro containing either the neutral or ion pair form of the catalytic dyad.…”

COVID-19 therapeutics: Small-molecule drug development targeting SARS-CoV-2 main protease

“…Similarly, Santos et al virtually screened 688 naphthoquinone compounds and derivatives against M pro and PL pro . 68 Among the finally selected candidates, four inhibited M pro with IC 50 values between 0.41 and 9.0 μM ( Figure S4 E in the supplemental information online), and three inhibited PL pro (IC 50 1.9–3.3 μM). Given the quinone aggregation properties, experiments to exclude aggregation, high compound fluorescence, and inhibition by enzyme oxidation confirmed their specificity against the enzymes.…”