Discovery of novel covalent proteasome inhibitors through a combination of pharmacophore screening, covalent docking, and molecular dynamics simulations.

The ubiquitin-proteasome pathway plays a pivotal role in the regulation of cellular protein processing and degradation. Proteasome inhibitors (PIs) have enormous potential to treat multiple myeloma, solid tumors, parasites, inflammation, and immune diseases, which is spurring the development of new types of PIs with enhanced efficacy, fewer side effects, and reduced drug resistance. Nevertheless, virtual screening for covalent PIs has rarely been reported because calculating the covalent binding energy is a challenging task. The aim of this study was to discover new covalent inhibitors of the 20S proteasome. The structures of PIs were manually divided into two parts: a noncovalent binding part resulting from virtual screening, and an epoxyketone group that was pre-selected as a covalent binding part. The SPECS database was screened by noncovalent docking and a pharmacophore model built with the 20S proteasome. After validating the covalent conjugation, 88 hits with epoxyketone were covalently docked into the 20S proteasome to analyze the intermolecular interactions. Four compounds were selected after multiple filtration and validations. Molecular dynamics simulations were performed to check the stability of the noncovalent and covalent docked ligand-enzyme complexes and investigate the interaction patterns of the screened inhibitors. Finally, two compounds with novel aromatic backbones, reasonable interactions, and stable covalent binding modes were retained. These compounds can serve as potential hits for further biological evaluation.