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 epoxy ketone-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 dyad (Cys25–His163), 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 the slow-binding covalent competition, but 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.

Reduced trajectories (5000 snapshots of each 0.5 µs MD data) of the non-covalent complex formed between the inhibitors (D1, D2, D2-flipped and AMK) and cathepsin L (CatL).