Title : Effect of cathepsin K inhibitor basicity on in vivo off - target activities

This article has not been copyedited and formatted. The final version may differ from this version. Abstract Cathepsin K is a lysosomal cysteine protease that is a pharmacological target for the treatment of osteoporosis. Previous studies showed that basic, lipophilic cathepsin K inhibitors are lysosomotropic and have greater activities in cell-based assays against cathepsin K, as well as the physiologically important lysosomal cysteine cathepsins B, L and S than expected on the basis of their potencies against these isolated enzymes. Chronic administration of the basic cathepsin K inhibitors L-006235 and balicatib to rats at a supratherapeutic dose of 500 mg/kg/d for four weeks resulted in increased tissue protein levels of cathepsin B and L, but no effect on cathepsin B and L message. This is attributed to the inhibitor engagement of these off-target enzymes and their stabilisation to proteolytic degradation. No such increase in these tissue cathepsins was detected at the same dose of L-873724, a potent non-basic cathepsin K inhibitor with a similar off-target profile, although all three inhibitors provided similar plasma exposures. Using an activity-based probe [ 125 I]-BIL-DMK, in vivo inhibition of cathepsins B, L and S was detected in tissues of mice given a single oral dose of L-006235 and balicatib, but not in mice given L-873724. In each case, similar tissue levels were achieved by all three compounds, thereby demonstrating the in vivo cathepsin selectivity of L-873724. In conclusion, basic cathepsin K inhibitors demonstrate increased off-target cysteine cathepsin activities than their non-basic analogues and potentially have a greater risk of adverse effects associated with inhibition of these cathepsins. This article has not been copyedited and formatted. The final version may differ from this version.