Design, Synthesis, and Evaluation of Aza-Peptide Michael Acceptors as Human 20S Proteasome Inhibitors: Extension to the Prime Site

Current FDA-approved proteasome inhibitor (PI) drugs, such as bortezomib, carfilzomib, and ixazomib, have significantly improved the treatment of multiple myeloma (MM) over the past 20 years. However, due to their associated severe side effects and multidrug resistance developed by many MM patients, treatment typically falters within a year. Although other mechanisms within the ubiquitin-proteasome-dependent protein degradation machinery are targeted to overcome this drug resistance, inhibition of the proteasome itself remains an attractive target. To this end, we report aza-peptide Michael acceptors as a new class of PIs. Peptidyl analogues, such as bortezomib, carfilzomib, and ixazomib, employ chemical warheads at their C-termini, and therefore the peptide backbone engages with the nonprime sites of the substrate binding pocket. Here, we designed 11 aza-peptide Michael acceptors PIs to explore the binding specificities beyond the C-terminus (<i>i.e</i>. at the prime binding sites). Specifically, we utilized tripeptidyl, Cbz-Leu-Leu-ALeu, and tetrapeptidyl, Cbz-HPh-Leu-Phe-ALeu and Mp-HPh-Leu-Phe-ALeu backbone structures to measure specificity. These backbones also included C-terminal extensions incorporating ethyl ester, <i>N</i>-ethyl amide, benzyl ester, <i>N</i>-benzyl amide, or <i>N</i>,<i>N</i>-dibenzyl amide groups. Our top compound, Mp-HPh-Leu-Phe-ALeu-CHCH-CONHBn, is a submicromolar inhibitor of the catalytic ß5 subunit. The selectivity of these inhibitors over other classes of proteases makes them suitable for further development as candidate therapeutic agents to potentially treat multiple myeloma, neurodegenerative disorders, and infectious diseases.