Integration of computational and experimental techniques for the discovery of PL^pro covalent inhibitors. When large...

Papain-like protease (PL^pro) and 3-chymotrypsin-like protease (3CL^pro or Mpro) are viral enzymes essential for the replication of SARS-CoV-2, the virus causing coronavirus disease of 2019 (COVID-19) infection. While 3CL^pro has been the main target of many potential antivirals including nirmatrelvir (active ingredient of Paxlovid), PL^pro has proven to be more challenging to target and only a handful of inhibitors have been disclosed. However, PL^pro inhibitors would enrich the therapeutic arsenal against COVID-19 resistant strains to 3CL^pro inhibitors and in future coronavirus pandemics. Combining our experience with 3CL^pro covalent inhibitors with our expertise in structure-based covalent drug discovery, we rationally designed PL^pro inhibitors achieving a maximum potency (IC₅₀) of 13 μM through fusion of GRL0617 and VIR-251 PL^pro inhibitors. In parallel, we launched an integrated large scale virtual screening/experimental approach, identifying four novel chemical series active at micromolar concentrations against PL^pro. We report herein our investigations including rational design, virtual screening, synthesis of selected structures and in vitro assays leading to novel PL^pro inhibitors. Surprisingly, these two very distinct approaches led to structures with similar features.