Identification of SARS-CoV-2 3CL Protease Inhibitors by a Quantitative High-throughput Screening

Abstract

Background and PurposeThe COVID-19 caused by SARS-CoV-2 has emphasized the urgent need for therapeutic development. Drug repurposing screening is the most practical and rapid approach for discovery of such therapeutics. The 3CLpro, or main protease (Mpro) of SARS-CoV-2 is a valid drug target as it is a viral enzyme with an essential role in viral replication, and cleavage specificity that is distinct from host proteases.Experimental ApproachWe employed and miniaturized a fluorogenic 3CLpro enzyme assay in which 3CLpro cleaves a quenched peptide substrate and releases a fluorescent fragment, resulting an increase in fluorescence signal. By using this SARS-CoV-2 3CLpro assay, we conducted a qHTS of 10,755 compounds consisting of approved and investigational drugs, and bioactive compounds, at 4 compound concentrations. The confirmed 3CLpro inhibitors were also tested in a SARS-CoV-2 cytopathic effect assay to determine their effects on rescuing of cell death caused by the virus infection.Key ResultsTwenty-seven small molecule inhibitors of SARS-CoV-2 3CLpro have been identified with IC50s ranging from 0.26 to 27.1 μM with a greater than 80% maximal inhibition. Walrycin B (IC50 = 0.26 μM), Hydroxocobalamin (IC50 = 3.29 μM), Suramin sodium (IC50 = 6.50 μM), Z-DEVD-FMK (IC50 = 6.81 μM), and LLL-12 (IC50 = 9.84 μM) are the most potent 3CLpro inhibitors with IC50s under 10 μM. The activities of anti-SARS-CoV-2 viral infection were confirmed in 11 of 27 compounds.Conclusion and ImplicationsSome of the newly identified inhibitors of SARS-CoV-2 3CLpro may be used in combination therapy with other drugs for synergistic effect to treat COVID-19 patients. The other inhibitors found in this study can provide starting points for medicinal chemistry optimizations.Bullet point summaryWhat is already knownSARS-CoV-2 3CLpro is a valid target for drug development.What this study addsIdentification of 27 inhibitors of SARS-CoV-2 3CLpro by a qHTS of 10,755 compounds consisting of approved and investigational drugs, and bioactive compounds.Clinical significanceSome of the newly identified 3CLpro inhibitors can be evaluated in drug combination therapy for synergistic effect to treat COVID-19 patients, while the others can serve as starting points for medicinal chemistry optimization to improve potency and drug like properties for drug development.

Drug repurposing screen identifies masitinib as a 3CLpro inhibitor that blocks replication of SARS-CoV-2in vitro

“…This cleavage is mediated by two virally encoded proteases: the main viral protease, known as Mpro, 3CLpro or non-structural protein 5 (nsp5) and a second protease known as the papain-like protease, PLpro, a domain within nsp3 3 . There is much interest in developing de-novo inhibitors to target these proteases [5][6][7][8][9][10] but the full clinical development of such drugs is a time-consuming process, usually requiring several years.…”

A simplified cell-based assay to identify coronavirus 3CL protease inhibitors

“…While multiple assays exist to evaluate protease inhibitors, an assay of the nature we envisioned has clear advantages, as it requires minimal upfront cost or effort, is accessible to many biomedical research labs, does not involve the use of live virus, and requires no specialized reporter to read out protease activity. In contrast, in vitro protease assays using purified protein have formed the backbone of inhibitor screening, but require upfront efforts to isolate the pure protease and are not conducted under physiologic cellular conditions 13,14 . In addition, if one desires to identify broad-acting coronavirus inhibitors, one must purify and identify experimental conditions suitable for testing each protease in vitro.…”

Targeted redesign of suramin analogs for novel antimicrobial lead development

“…Additionally, our studies identified the potential to target SARS-CoV-2 nucleocapsid with suramin analogs and proposed analogs possessing enhanced affinity and specificity. This approach could also be applied to the 3CL protease which was recently identified as another possible suramin binding target (40). Our phylogenetic and evolutionary analyses also suggest that the suramin binding pockets are evolutionary conserved in related coronavirus RdRp and nucleocapsid proteins which could make these suramin analogs more broadly applicable therapeutics against this group of viral pathogens.…”