The Discovery of Small Allosteric and Active Site Inhibitors of the SARS-CoV-2 Main Protease via Structure-Based Virtual Screening and Biological Evaluation

Abstract

The main protease enzyme (Mpro) of SARS-CoV-2 is one of the most promising targets for COVID-19 treatment. Accordingly, in this work, a structure-based virtual screening of 3.8 million ligand libraries was carried out. After rigorous filtering, docking, and post screening assessments, 78 compounds were selected for biological evaluation, 3 of which showed promising inhibition of the Mpro enzyme. The obtained hits (CB03, GR04, and GR20) had reasonable potencies with Ki values in the medium to high micromolar range. Interestingly, while our most potent hit, GR20, was suggested to act via a reversible covalent mechanism, GR04 was confirmed as a noncompetitive inhibitor that seems to be one of a kind when compared to the other allosteric inhibitors discovered so far. Moreover, all three compounds have small sizes (~300 Da) with interesting fittings in their relevant binding sites, and they possess lead-like characteristics that can introduce them as very attractive candidates for the future development of COVID-19 treatments.

SARS-CoV-2 Mpro Inhibitors: Achieved Diversity, Developing Resistance and Future Strategies

“…Despite a low free plasma concentration (Cmax = 0.55 mM) due to high binding affinity for albumin, apixabans free fraction was ~10-times higher than its SARS-CoV-2 M pro affinity, serving as a good basis for further anti-coronaviral pharmacological studies. A structure-based virtual screening of 3.8 million small-molecule ligands recently identified three hits that showed promising complexation with the M pro enzyme [95]. Among them, GR20 (Figure 16) showed with IC50 = 91.8 µM the best M pro inhibition.…”

“…The other antibody binds instead to an epitope that overlaps with the substrate binding site, thus suggesting NB1A2 as not only an allosteric but also a competitive inhibitor (PDB 7vfa). [89], AT-7519 [89], pelitinib [89], RS-102895 [89], ifenprodil [89], JMX0286 [92], apixaban [94], and GR20 [95] with proposed binding sites and antiviral efficacy. Binding sites refer to Figure 15.…”

The Discovery of Novel Small Oxindole‐Based Inhibitors Targeting the SARS‐CoV‐2 Main Protease (Mpro)

“…[47] Table 3 displays the estimated ligand efficiency values for non-competitive M pro inhibitors, calculated based on their experimental binding affinity. Smaller compounds with fewer than 38 heavy atoms, like GR04 [49] and CL02, had LE scores of 0.20 and 0.22 Kcal mol-1, respectively. However, despite their smaller size, both compounds fall short of the optimal LE value.…”