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.