Peptidyl keto vinyl amides as improved inhibitors of cathepsin L

Cysteine protease cathepsin L (CatL) is linked to cancer, infection, and neurodegeneration, rendering it a compelling target for therapy. Expanding upon dipeptidyl enoates (keto vinyl esters, KVE), we introduce dipeptidyl keto vinyl amides (KVA) as a more stable, tunable Michael-acceptor scaffold. A first set of compounds (1-11), that utilized a Z-Leu-Ala backbone, was synthesized based on previous MD simulations and docking results to prioritize the variants for S'-subsite engagement. Fluorometric assays using recombinant CatL showed that all behaved as purely reversible inhibitors (non-time-dependent) with low-micromolar to mid-nanomolar K_i values from 1.47 μM to 116 nM, with KVA outperforming KVE. A second set of more hydrophobic compounds (42-43) was synthesized on Z-Phe-Leu and provided low nanomolar K_i values. Then, a series of long MD simulations was done to rationalize the kinetics. Computational results show that Cys25 is close to the warhead, while hydrogen-bonding to the oxyanion hole (Gln19) and contacts with Asp162 and Gly68, all stabilize binding. KVA 4 (Leu-Ala, 2,2-difluoroethyl) exhibited the most consistent specific interaction, in agreement with the lowest experimentally determined K_i values, providing a favorable starting point for further development. Overall, KVAs emerge as stable, potent CatL inhibitors and a promising platform for selectivity and in cellulo optimization.