Screening, Synthesis and Biochemical Characterization of SARS-CoV-2 Protease Inhibitors

The severe acute respiratory syndrome-causing coronavirus 2 (SARS-CoV-2) papain-like protease (PL^pro) and main protease (M^pro) play an important role in viral replication events and are important targets for anti-coronavirus drug discovery. In search of these protease inhibitors, we screened a library of 1300 compounds using a fluorescence thermal shift assay (FTSA) and identified 53 hits that thermally stabilized or destabilized PL^pro. The hit compounds structurally belonged to two classes of small molecules: thiazole derivatives and symmetrical disulfide compounds. Compound dissociation constants (K_d) were determined using an enzymatic inhibition method. Seven aromatic disulfide compounds were identified as efficient PL^pro inhibitors with K_d values in the micromolar range. Two disulfides displayed six-fold higher potency for PL^pro (K_d = 0.5 µM) than for M^pro. The disulfide derivatives bound covalently to both proteases, as confirmed through mass spectrometry. The identified compounds can serve as lead compounds for further chemical optimization toward anti-COVID-19 drugs.