The Consequences of Lysosomotropism on the Design of Selective Cathepsin K Inhibitors

Abstract

Many drug candidates contain a basic functional group that results in lysosomotropism—the accumulation of drug in the acidic lysosomes of a cell. When evaluating inhibitors of lysosomal enzymes, such as the cathepsins, this physical property can have a dramatic impact on the functional selectivity of the test compounds. A basic P3 substituent in cathepsin K inhibitors provides a means of achieving potent and selective enzyme inhibition. To evaluate the whole‐cell selectivity of the basic cathepsin K inhibitor L‐006235, we identified the irreversible pan‐selective cathepsin probe BIL‐DMK and used it to design whole‐cell enzyme‐occupancy assays. These cell‐based assays showed a dramatic reduction in selectivity against cathepsins B, L, and S relative to the selectivities observed in enzyme assays. Two‐photon confocal fluorescence microscopy showed punctated subcellular localization of L‐006235, which colocalized with BODIPY‐labelled Lysotracker, consistent with compound lysosomotropism. To address this potential problem, a series of potent cathepsin K inhibitors was developed by replacing the P2P3 amide bond with a metabolically stable trifluoroethylamine moiety. X‐ray crystallography has identified the binding of this functional group to active‐site residues in cathepsin K. This modification resulted in increased potency and selectivity that allowed the removal of the basic P3 substituent. The resulting nonbasic inhibitor L‐873724 is a 0.2 nM inhibitor of cathepsin K with cathepsin B, L, and S potencies that were not shifted between purified enzyme and whole‐cell assays; thus indicating that this compound is not lysosomotropic. L‐873724 exhibits excellent pharmacokinetics and is orally active in a monkey model of osteoporosis at 3 mg kg⁻¹ q.d.