Structure-Based Design of Potent Peptidomimetic Inhibitors Covalently Targeting SARS-CoV-2 Papain-like Protease

The papain-like protease (PL^pro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a critical role in the proteolytic processing of viral polyproteins and the dysregulation of the host immune response, providing a promising therapeutic target. Here, we report the structure-guide design of novel peptidomimetic inhibitors covalently targeting SARS-CoV-2 PL^pro. The resulting inhibitors demonstrate submicromolar potency in the enzymatic assay (IC₅₀ = 0.23 μM) and significant inhibition of SARS-CoV-2 PL^pro in the HEK293T cells using a cell-based protease assay (EC₅₀ = 3.61 μM). Moreover, an X-ray crystal structure of SARS-CoV-2 PL^pro in complex with compound 2 confirms the covalent binding of the inhibitor to the catalytic residue cysteine 111 (C111) and emphasizes the importance of interactions with tyrosine 268 (Y268). Together, our findings reveal a new scaffold of SARS-CoV-2 PL^pro inhibitors and provide an attractive starting point for further optimization.