(35) Research Capstone Experiences: Design, Synthesis, In Silico Analysis, and Assay Development Towards Prokaryotic Proteasome Antagonists

Objective : Herein, we report the development of antagonists of the Mycobacterium tuberculosis prokaryotic proteasome system (PPS) to address drug-resistant tuberculosis infections.

Methods: As part of their capstone research experience, PharmD students in our laboratory undertook an integrated, multi-disciplinary approaches towards the stated objective. We used computational docking to predict the binding sites of non-competitive inhibitors that target protein binding sites with the PPS, to screen selected FDA-approved drugs for potential PPS binding and drug repurposing, and to inform the synthesis of mechanism-based substrate analogues of the PPS. Next, using standard medicinal chemistry and characterization techniques, we synthesized tripeptide substrate mimics containing stable phosphodiester isosteres. Lastly, we developed and optimized a thermal assay to evaluate binding between the PPS, known ligands and inhibitors, computational hits, and our prepared substrate mimics.

Results: We performed computational in silico docking between PPS enzymes and known cofactors, uncompetitive inhibitors, select FDA-approved drugs, and our tripeptide substrate mimics. Known cofactors and ligands bound their anticipated binding sites with modest affinity (-7.6–-7.9 kcal/mol). In contrast, uncompetitive inhibitors bound allosteric sites (-6.6–-9.9 kcal/mol), the FDA-approved drugs mostly bound the active site (-6.0–-9.9 kcal/mol), and our tripeptide substrate mimics bound with weaker binding affinities (-4.1–-5.0 kcal/mol). Moreover, we completed the synthesis, purification, and characterization of seven tripeptide analogues in 3–5 linear steps. Lastly, using our thermal assay, we determined the melting temperature of a PPS enzyme (Tm=42–44 °C) and evaluated whether cofactors or select drugs effect enzyme stability.

Conclusions: We developed a comprehensive approach towards PPS inhibitor development and further optimization of our initial designed substrate mimics will be reported in subsequent studies. Drugs that selectively inhibit the M. tuberculosis PPS hold tremendous potential as novel antimycobacterials.