Lysosomotropism of basic cathepsin K inhibitors contributes to increased cellular potencies against off-target cathepsins and reduced functional selectivity

The lysosomal cysteine protease Cathepsin K is a target for osteoporosis therapy. The aryl-piperazine-containing Cathepsin K Inhibitor CRA-013783/L-006235 (1) displays greater than 4000-fold selectivity against the lysosomal/endosomal antitargets Cathepsin B, L, and S. However, 1 and other aryl-piperazine-containing analogues, including balicatib (10), are approximately 10-100-fold more potent in cell-based Enzyme occupancy assays than against each purified Enzyme. This phenomenon arises from their basic, lipophilic nature, which results in lysosomal trapping. Consistent with its lysosomotropic nature, 1 accumulates in cells and in rat tissues of high lysosome content. In contrast, nonbasic aryl-morpholino-containing analogues do not exhibit lysosomotropic properties. Increased off-target activities of basic Cathepsin K inhibitors were observed in a cell-based Cathepsin S antigen presentation assay. No potency increases of basic inhibitors in a functional Cathepsin K bone resorption whole cell assay were detected. Therefore, basic Cathepsin K inhibitors, such as 1, suffer from reduced functional selectivities compared to those predicted using purified Enzyme assays.