Identification of inhibitors and inhibitory mechanisms for sars-cov-2 papain-like protease

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in late 2019 in Wuhan, China, and rapidly spread worldwide. The continuous emergence of SARS-CoV-2 variants necessitates the investigation of viral proteins as potential drug targets. While the main protease (Mpro) has been extensively studied, papain-like protease (PLpro) remains relatively underexplored despite its critical roles in viral replication and the inhibition of host innate immune responses. This study aims to identify and characterize potential inhibitors of SARS-CoV-2 PLpro using a combination of in vitro protease activity assays, enzyme kinetics, molecular docking, molecular dynamics simulations, and structural characterization. Recombinant PLpro was expressed in E.coli Tuner (DE3) cells under various IPTG concentrations and temperatures, with optimal conditions identified at 16°C for 16 hours. Affinity chromatography using a Ni-NTA column facilitated the purification of PLpro, which demonstrated efficient cleavage activity on a synthetic peptide substrate derived from the viral polyprotein with a Km value of 13.7 µM and a Vmax value of 21.22 RFU/s. In vitro protease inhibitor screening identified two potential inhibitors, Lap1-1-0 and TC6, both exhibiting a competitive mode of inhibition with Ki values of 18.5 μM and 31.43 μM, respectively, suggesting TC6 has the highest binding affinity. Computational studies, including molecular docking and molecular dynamics simulations, were conducted to elucidate and compare the binding behavior of these inhibitors with SARS-CoV-2 PLpro. The results confirmed that both inhibitors bind to the BL2-Loop, with TC6 demonstrating the most potent binding efficiency, consistent with experimental findings. Crystallization of PLpro harboring the C111S mutation was also attempted. This study provides valuable insights into PLpro inhibition and serves as a foundation for the development of novel antiviral drugs targeting SARS-CoV-2 and related coronaviruses.