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.

Investigation of the Compatibility between Warheads and Peptidomimetic Sequences of Protease Inhibitors-A Comprehensive Reactivity and Selectivity Study.

Covalent peptidomimetic protease inhibitors have gained a lot of attention in drug development in recent years. They are designed to covalently bind the catalytically active amino acids through electrophilic groups called warheads. Covalent inhibition has an advantage in terms of pharmacodynamic properties but can also bear toxicity risks due to non-selective off-target protein binding. Therefore, the right combination of a reactive warhead with a well-suited peptidomimetic sequence is of great importance. Herein, the selectivities of well-known warheads combined with peptidomimetic sequences suited for five different proteases were investigated, highlighting the impact of both structure parts (warhead and peptidomimetic sequence) for affinity and selectivity. Molecular docking gave insights into the predicted binding modes of the inhibitors inside the binding pockets of the different enzymes. Moreover, the warheads were investigated by NMR and LC-MS reactivity assays against serine/threonine and cysteine nucleophile models, as well as by quantum mechanics simulations.