(4-Aminomethyl)phenylguanidine derivatives as nonpeptidic highly selectiveinhibitors of human urokinase

Increased expression of the serine protease urokinase-type plasminogen acti vator (uPA) in tumor tissues is highly correlated with tumor cell migration , invasion, proliferation, progression, and metastasis. Thus inhibition of uPA activity represents a promising target for antimetastatic therapy. So f ar. only the x-ray crystal structure of uPA inactivated by H-Glu-Gly-Arg-ch loromethylketone has been reported, thus limited data are available for a r ational structure-based design of uPA inhibitors. Taking into account the t rypsin-like arginine specificity of uPA, (4-aminomethyl)phenylguanidine was selected as a potential P1 residue and iterative derivatization of its ami no group with various hydrophobic residues, and structure-activity relation ship-based optimization of the spacer in terms of hydrogen bond acceptor/do nor properties led to N-(1-adamantyl)-N'-(4-guanidinobenzyl)urea as a highl y selective nonpeptidic uPA inhibitor. The x-ray crystal structure of the u PA B-chain complexed with this inhibitor revealed a surprising binding mode consisting of the expected insertion of the phenylguanidine moiety into th e S1 pocket, but with the adamantyl residue protruding toward the hydrophob ic S1' enzyme subsite, thus exposing the ureido group to hydrogen-bonding i nteractions. Although in this enzyme-bound state the inhibitor is crossing the active site, interactions with the catalytic residues Ser-195 and His-5 7 are not observed, but their side chains are spatially displaced for steri c: reasons. Compared with other trypsin-like serine proteases, the S2 and S 3/S4 pockets of uPA are reduced in size because of the 99-insertion loop. T herefore, the peculiar binding mode of the new type of uPA inhibitors offer s the possibility of exploiting optimized interactions at the S1'/S2' subsi tes to further enhance selectivity and potency. Because crystals of the uPA /benzamidine complex allow inhibitor exchange by soaking procedures, the st ructure-based design of new generations of uPA inhibitors can rely on the a ssistance of x-ray analysis.