Stereochemistry-Guided Switch from Irreversible to Slow-Binding Cathepsin L Inhibition

Due to its involvement in a range of disease-related processes, such as cancer metastasis, viral entry, and immune dysregulation, cathepsin L (CatL), a lysosomal cysteine protease, has been proposed as an attractive therapeutic target. In this work, we present the design, synthesis, and mechanistic investigation of two epoxyketone-based dipeptidyl CatL inhibitors (D1 and D2) and two azido-based derivatives (AZA and AMK). From a combination of experimental and computational studies, we have demonstrated the importance of epoxide stereochemistry and azido functionality in determining the mode of inhibition and reactivity. Classical and hybrid QM/MM molecular dynamics (MD) simulations show that only (R)-epoxide (D1) occupies reactive conformations with respect to the catalytic triad (Cys25−His163−Asn187), leading to irreversible covalent inactivation via nucleophilic attack at the epoxide functionality. (S)-Epoxide (D2), on the other hand, occupies non-reactive conformations but can reach a less stable “flipped” conformation, suggesting a very slow SN2-type attack at the carbon adjacent to the azido moiety. The substitution of the epoxide with an azidomethyl ketone (AMK) preserved slow-binding covalent inhibition; however, exploration of the free energy landscape revealed a lower activation barrier, consistent with the retention of activity. Kinetic, mass spectrometry (MS), saturation transfer difference (STD) NMR, and thermal-shift studies support the computational predictions and show how slight modifications to the stereochemistry and the warhead of the inhibitor can tune CatL activity from fully irreversible to slow-binding reversible inhibition. These results provide a rational framework for designing covalent inhibitors with tunable residence times and dual reactivity toward CatL that are important in cancer progression and in viral processes.