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

Abstract

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.

Figure 1

Structures, inhibition data against catS, and bioavailabilities (F%) of developed cathepsin S inhibitors and nirmatrelvir. Warheads are highlighted in blue.⁻

Figure 2

Aim and scope of the present study: (A) combinatorial SAR study through merging of two potent catS inhibitors and further substitutions, and (B) subsequent warhead replacement and reversible capping of the warhead. Created with BioRender.com.

Figure 3

(A) Dilution assay of DMSO control (red), compounds 51 (dark blue), 36 (reversible nitrile, blue), 49 (orange), 44 (purple), 50 (cyan), and irreversible control 52 (vinylsulfone, green). Inhibitors were incubated at 20-fold K_i or K_i^* app concentrations followed by 100-fold dilution, resulting in enzyme recovery in cases of DMSO and compounds 51, 36, 49 (linear progress curves), and 44 (time-dependent progress curve). Progress curves of (B) inhibitor 45, catS and (C) inhibitor 50, catS, showing the time-dependent inhibition by cpd 45 and non-time-dependent inhibition by cpd 50.

Figure 4

Noncovalent docking poses of 44 ((A) pink C atoms) and (50 (B), green C atoms) inside the active site of catS (pdb entry: 1MS6). Polar interactions between the inhibitors and active site amino acids are depicted as yellow dashed lines. The distance between the sulfur atom of Cys-25 and the electrophilic C atoms of 44 and 50 is depicted as a red dashed line and the distance is given in Å.

Figure 5

(A) Correlation of log P and permeability (P_e) for 19 compounds. (B) Correlation of TPSA and permeability (P_e), divided into two subsets (6 Acc nitriles and 13 others). “Cut-off” depicts the effective permeability value, above which compounds were considered relevantly permeable (P_e > 1 × 10^–6 cm/s).