Method: Km and kcat values were determined for the degradation of type-I collagen by the cathepsin K/C4-S complex. Michalis-Menten kinetics and a Lineweaver-Burk analysis were used to evaluate the data. Structurally-related putative cathepsin K/C4-S complex formation inhibitors were evaluated for their ability to prevent cathepsin K/C4-S-mediated degradation of collagen in a non-active site inhibitory mechanism. An IC50was determined for each effective compound, and the accessibility of the active site of cathepsin K was determined using a synthetic fluorogenic peptide substrate, Z-FR-MCA.
Result: The kinetic values for cathepsin K/C4-S-mediated degradation of collagen are as follows: Km = 1.6 µM, kcat = 6 h-1 and kcat/Km = 1.062 M-1s-1. Six out of the 8 putative cathepsin K inhibitors were able to prevent the degradation of collagen: Aurintricarboxylic Acid (IC50 = 9.6 μM); Ellipticine (IC50 = ~100 μM); Epigallocatechin gallate (IC50 = 52 μM); Raloxifene (IC50 = 85 μM); Suramin (IC50 = 6.7 μM); and Tamoxifen (IC50= ~150 μM). All but Suramin appeared to be non-active site inhibitors. Interestingly, Tamoxifen and Raloxifene have been clinically demonstrated to inhibit bone resorption via an estrogen related pathway.
Conclusion: Degradation of collagen by the Cathepsin K:C4-S complex can be inhibited by a variety of compounds in a non-active site manner.