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.