Structural Modifications of Covalent Cathepsin S Inhibitors: Impact on Affinity, Selectivity, and Permeability.

Cathepsin S (catS) is a member of the cysteine protease family with limited tissue distribution, which is predominantly found in antigen-presenting cells. Due to overexpression and overactivity of catS in numerous cancers, inhibition of catS is supposed to improve the antitumor response. Here, we explore the potential of small-molecule catS inhibitors emphasizing their in vitro pharmacodynamics and pharmacokinetics. Membrane permeability of selected inhibitors was measured with a Parallel Artificial Membrane Permeation Assay and correlated to calculated physicochemical parameters and inhibition data. The binding kinetics and inhibition types of potent and selective new inhibitors with unexplored warheads were investigated. Our unique approach involves reversible masking of these potent warheads, allowing for further customization without compromising affinity or selectivity. The most promising inhibitors in this study include covalent aldehyde and ketone derivatives reversibly masked as hydrazones as potential candidates for therapeutic interventions targeting catalytic enzymes and modulating the immune response in cancer.