Identification of noncovalent proteasome inhibitors with high selectivity for chymotrypsin-like activity by a multistep structure-based virtual screening

Noncovalent proteasome inhibitors introduce an alternative mechanism of inhibition to that of covalent inhibitors, e.g. carfilzomib, used in cancer therapy. A multistep hierarchical structure-based virtual screening (SBVS) of the 65,375 NCI lead-like compound library led to the identification of two compounds (9 and 28) which noncovalently inhibited the chymotrypsin-like (ChT-L) activity (K-i = 2.18 and 2.12 mu M, respectively) with little or no effects on the other two major proteasome proteolytic activities, trypsin-like (T-L) and post-glutamyl peptide hydrolase (PGPH) activities. A subsequent hierarchical similarity search over the full NCI database with the most active tripeptide-based inhibitor 9 resulted in the discovery of the beta 5/beta 6-specific tripeptide derivative 38 that noncovalently binds the ChT-L site (K-i = 0.42 mu M). The solution structure of 9 and 38 was solved by H-1 NMR spectroscopy and the binding mode of the inhibitors was elucidated by docking experiments using the yeast 20S proteasome. Compound 38 (IC50 = 26.7 mu M) is slightly more potent than 9 (IC50 = 34.3 mu M) at inhibiting survival of dexamethasone-resistant (MM.1R) human multiple myeloma cells. The identified ligand thus provides valuable insights for the future structure-based design of subtype-specific proteasome inhibitors.