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

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 M with a greater than 80% maximal inhibition. Walrycin B (IC50 = 0.26 M), Hydroxocobalamin (IC50 = 3.29 M), Suramin sodium (IC50 = 6.50 M), Z-DEVD-FMK (IC50 = 6.81 M), and LLL-12 (IC50 = 9.84 M) are the most potent 3CLpro inhibitors with IC50s under 10 M. 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 known O_LISARS-CoV-2 3CLpro is a valid target for drug development. C_LI What this study adds O_LIIdentification of 27 inhibitors of SARS-CoV-2 3CLpro by a qHTS of 10,755 compounds consisting of approved and investigational drugs, and bioactive compounds. C_LI Clinical significance O_LISome 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. C_LI