Inhibition of cathepsin activity in a cell-based assay by a light-activated ruthenium compound

Light-activated inhibition of cathepsin activity was demonstrated in a cell-based assay. Inhibitors of cathepsin K, Cbz-Leu-NHCH2 CN (2) and Cbz-Leu-Ser(OBn)-CN (3), were caged within the complexes cis-[Ru(bpy)2 (2)2 ]Cl2 (4) and cis-[Ru(bpy)2 (3)2 ](BF4 )2 (5) (bpy=2,2'-bipyridine) as 1:1 mixtures of Δ and Λ stereoisomers. Complexes 4 and 5 were characterized by (1) H NMR, IR, and UV/Vis spectroscopies and electrospray mass spectrometry. Photochemical experiments confirm that 4 releases two molecules of 2 upon exposure to visible light for 15 min, whereas release of 3 by 5 requires longer irradiation times. IC50 determinations against purified cathepsin K under light and dark conditions with 4 and 5 confirm that inhibition is enhanced from 35- to 88-fold, respectively, upon irradiation with visible light. No apparent toxicity was observed for 4 in the absence or presence of irradiation in bone marrow macrophage (BMM) or PC3 cells, as determined by MTT assays, at concentrations up to 10 μM. Compound 5 is well tolerated at lower concentrations (<1 μM), but does show growth-inhibitory effects at higher concentrations. Confocal microscopy experiments show that 4 decreases intracellular cathepsin activity in osteoclasts with light activation. These results support the further development of caged nitrile-based inhibitors as chemical tools for investigating spatial aspects of proteolysis within living systems.