| dc.description.abstract | In the era of high-throughput drug discovery against enzyme targets, the utilization of rational drug design principles has become a path less traveled. This rational approach can provide researchers more intuitive methods to address challenging drug targets by elucidating the mechanistic details of an enzyme and designing molecules to exploit key interactions. Herein, we detail our application of synthetic chemistry, kinetic analysis, and rational drug discovery against a variety of pathogenic targets.
To guide our efforts in developing inhibitors of the cysteine protease cruzain, from Trypanosoma cruzi, we synthesized and kinetically characterized fluorogenic dipeptide substrates. Upon appending a fixed electrophilic warhead to our varied peptide scaffolds, we identified a linear correlation between the Km of our substrates and Ki* of inhibitors, suggesting the binding affinity of the peptide scaffold plays a dominate role in inhibitor potency.
Beyond cruzain, we developed covalent inactivators to target the enzyme isocitrate lyase (ICL1) from Mycobacterium tuberculosis. Through screening succinate-based substrate analogs, we uncovered that cis-epoxy succinate is an uncompetitive inactivator of ICL1 that forms a covalent bond with the catalytic cysteine 191 (Cys₁₉₁) of the enzyme. In a different vein, we identified 5-descarboxy-5-nitro-D-isocitric acid (5-NIC) as a highly efficient mechanism-based inactivator of ICL1. Retro-aldol cleavage of 5-NIC by the enzyme forms glyoxylate and 3-nitropropionate (3-NP) in the ICL1 active site, which allows facile formation of a thiohydroxamate adduct with Cys₁₉₁ via reaction with the nitronate form of 3-NP.
Shifting away from ICL1, we synthesized and kinetically characterized peptide substrates containing varied lysine post-translational modifications against the sole NAD+-dependent lysine deacylase from Mycobacterium tuberculosis (Rv1151c or Mt-Sirt). This revealed that Mt-Sirt has a strong specificity for long chain fatty acyl and succinyllysine substrates, thus allowing us to leverage this specificity to develop potent mechanism-based inhibitors that mimic myristoyllysine.
Finally, we identified a vinyl-sulfone-containing cysteine protease inhibitor, K777, as a potent inhibitor of SARS-CoV-2 in mammalian cells. Using activity-based protein profiling, we identified that the alkyne analog of K777 targeted human cathepsin B and L, but that only cathepsin L was able to process the SARS-CoV-2 spike protein in vitro. | |
