Inhibitors Targeting SARS-CoV-2 Papain-Like Protease: Screening, Design, Synthesis, and Biological Evaluation

Despite the availability of vaccines and antiviral drugs, SARS-CoV-2 still presents a global health threat, emphasizing the need for new therapeutic targets beyond the main protease and RNA-dependent RNA polymerase. The papain-like protease (PL^pro) is a highly conserved and essential viral enzyme that drives polyprotein processing and suppresses host innate immune responses through its deubiquitinating and deISGylating activities. This study employed two approaches to identify new PL^pro inhibitors: (i) the design and synthesis of a range of non-covalent PL^pro inhibitors based on a lead compound, GRL0617. (ii) Screening the in-house library for new covalent PL^pro inhibitors. Biochemical assays demonstrated effective inhibition of PL^pro activity, particularly with the non-covalent inhibitor 2t (TPG-2t, IC₅₀ = 0.634 µM) and with potential covalent inhibitors such as 13e (IC₅₀ = 22.0 µM) and 13f (TPG-13f, IC₅₀ = 5.0 µM). 2t exhibited antiviral activity (EC₅₀ = 2.89 µM) without causing cellular toxicity. Molecular modeling suggests that 2t can establish stable binding interactions within the PL^pro active site. Therefore, inhibitor 2t could be a promising candidate for future antiviral development.