Abstract 1021: The development of non-covalent non-peptide inhibitors of the proteasome: exploring a series of proteasome-inhibiting pyrazoles

The purpose of this study was to develop reversible, non-peptide, non-covalent inhibitors of the 20S proteasome. The FDA approval of the peptide-based proteasome inhibitors bortezomib and carfilzomib in 2003 and 2012, respectively, has validated the proteasome as an important target in the treatment of multiple myeloma. Additionally, these inhibitors have demonstrated in vitro as well moderate in vivo efficacy in preclinical models of solid tumors. Despite this, clinical trials utilizing these drugs for the treatment of solid tumors have shown disappointing results. Although the reasons for these failures remain unclear, one possibility is that existing proteasome inhibitors such as bortezomib and carfilzomib lack the pharmacokinetic and pharmacodynamic properties needed to sufficiently inhibit proteasomes in solid tumors . With this in mind, it has been postulated that reversible, non-peptide proteasome inhibitors could be developed with improved properties and that these inhibitors could serve both as novel tools to advance our understanding of the proteasome and as potential therapeutic options for the treatment of solid tumors. In order to identify novel proteasome inhibitors with non-peptide scaffolds, we conducted an in silico screen of more than 340,000 structures followed by in vitro assays using purified proteasomes. This screening effort lead to several novel non-peptide hits. One hit, a substituted pyrazole derivative, was used as the lead compound to synthesize a small library of analogs. These compounds were evaluated for their ability to inhibit proteasome catalytic activity using specific fluorogenic substrates and a structure-activity relationship was developed. In vitro cell viability assays were conducted against three non-small cell lung cancer (NSCLC) cell lines. The in vivo antitumor activity of the most promising analog was also determined using a NSCLC xenograph model. Our lead compound was found to have reversible inhibitory activity against the chymotrypsin-like subunits of the 20S proteasome and immunoproteasome with single-micromolar IC50 values. We found that our lead compound was equipotent against three NSCLC cell lines despite varied sensitivity to bortezomib and carfilzomib. IC50 values against these cell lines were less than 10 μM. Using an analog with improved potency and promising drug-like properties, inhibition of in vivo tumor growth was also observed. Studies designed to validate the proteasome as the target of this s