Cathepsin K is a papain-like human cysteine peptidase with a catalytic diad consisting of a Cys−His+ ion pair. The enzyme is predominantly expressed in osteoclasts and is a crucial peptidase involved in bone remodelling due to its capability to cleave collagen types I and II besides other proteins in the extracellular matrix. Regulation of proteolytic activity is important for normal functioning of an organism. Excessive activity of cathepsin K has been associated with osteoporosis, osteoarthritis and rheumatoid arthritis. Thus, inhibitors which would reduce the proteolytic activity to its optimum are potential candidates for therapeutic use.
Biguanides form stable complexes with metal ions as N,N-bidenate ligands. Phenformin (N-phenylethylbiguanide), a formerly used drug for diabetes type II, is especially interesting in the context of cysteine cathepsins inhibition. Structural comparison of benzyl-L-arginine amide (BAA), a synthetic protease substrate, and phenformin-metal ion complex suggests that the phenyl group binds to S2 substrate biding site, a major determinant of substrate preference for cysteine cathepsins, and metal ion interacts with the catalytic diad.
In this work we studied the effects of zinc(II) and lead(II) ions in combination with biguanide phenformin on the activity of cathepsin K. We determined the EC50 value for phenformin and how the addition of phenformin influences the EC50 values for zinc(II) and lead(II) ions by measuring initial velocity of hydrolysis of fluorogenic substrate Z-FR↓AMC. Our results show that phenformin is a hyperbolic inhibitor of cathepsin K and it increases the inhibitory potency of lead(II) ions. The same effect was not observed with zinc(II) ions. We also analysed the binding of phenformin, Zn2+, Pb2+ and their complexes on cathepsin K by using GaudiMM programme. Docking results predict biding of the phenyl moiety into the S2 biding site and interaction of metal ions with Cys25 from the catalytic diad.