Species specificity of amidine-based urokinase inhibitors

Inhibition of the proteolytic activity of urokinase has been shown to inhib it the progression of tumors in rodent models and is being investigated for use in human disease. Understanding the rodent/ human species-specificity of urokinase inhibitors is therefore critical for interpretation of rodent cancer progression models that use these inhibitors. We report here studies with a panel of 11 diverse urokinase inhibitors in both human and mouse en zymatic assays. Inhibitors such as amiloride, B428, and naphthamidine, that occupy only the S1 subsite pocket were found to be nearly equipotent betwe en the human and the murine enzymes. Inhibitors that access additional, mor e distal, pockets were significantly more potent against the human enzyme b ut there was no corresponding potency increase against the murine enzyme. X -ray crystallographic structures of these compounds bound to the serine pro tease domain of human urokinase were solved and examined in order to explai n the human/mouse potency differences. The differences in inhibitor potency could be attributed to four amino acid residues that differ between murine and human urokinases: 60, 99, 146, and 192. These residues are Asp, His, S er, and Gln in human and Gln, Tyr, Glu, and Lys in mouse, respectively. Com pounds bearing a cationic group that interacts with residue 60 will prefere ntially bind to the human enzyme because of favorable electrostatic interac tions. The hydrogen bonding to residue 192 and steric considerations with r esidues 99 and 146 also contribute to the species specificity. The nonparal lel human/mouse enzyme inhibition observations were extended to a cell-cult ure assay of urokinase-activated plasminogen-mediated fibronectin degradati on with analogous results. These studies will aid the interpretation of in vivo evaluation of urokinase inhibitors.