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.