Structure-directed discovery of potent non-peptidic inhibitors of human urokinase that access a novel binding subsite

Background: Human urokinase-type plasminogen activator has been implicated in the regulation and control of basement membrane and interstitial protein degradation. Because of its role in tissue remodeling, urokinase is a cent ral player in the disease progression of cancer, making it an attractive ta rget for design of an anticancer clinical agent. Few urokinase inhibitors h ave been described, which suggests that discovery of such a compound is in the early stages. Towards integrating structural data into this process, a new human urokinase crystal form amenable to structure-based drug design ha s been used to discover potent urokinase inhibitors. Results: On the basis of crystallographic data, 2-naphthamidine was chosen as the lead scaffold for structure-directed optimization. This cc-crystal s tructure shows the compound binding at the primary specificity pocket of th e trypsin-like protease and at a novel binding subsite that is accessible f rom the 8-position of 2-napthamidine. This novel subsite was characterized and used to design two compounds with very different 8-substituents that in hibit urokinase with K-i values of 30-40 nM. Conclusions: Utilization of a novel subsite yielded two potent urokinase in hibitors even though this site has not been widely used in inhibitor optimi zation with other trypsin-like proteases, such as those reported for thromb in or factor Xa. The extensive binding pockets present at the substrate-bin ding groove of these other proteins are blocked by unique insertion loops i n urokinase, thus necessitating the utilization of additional binding subsi tes. Successful implementation of this strategy and characterization of the novel site provides a significant step towards the discovery of an antican cer clinical agent.