Design, synthesis and X-ray crystal structure of a new class of selective uPA-inhibitors

Increased expression of the serine protease urokinase-type plasminogen activator (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 anti-metastatic therapy. By structure-based design of diversomers of different arginine mimetics, a new class of low molecular mass and highly selective uPA inhibitors was identified, which consists of hydrophobic derivatives of 4-aminomethyl-phenylguanidine. SAR-based optimization of these inhibitors in terms of hydrogen bond acceptor/donor properties led to JV(l-adamantly)-JV-(4-guanidinobenzyl)urea (WX-293T). The X-ray crystal structure of this inhibitor was solved, using soakable crystals of the uPA B-chain. It revealed an unconventional binding mode, involving both the SI and SI' pocket. Despite the crossing of the active site, interactions with the catalytic residues Ser-195 and His-57 are not observed, although the side chains of these residues are spatially displaced for steric reasons. Compared to other trypsin-like serine proteases, the S2 and S3/S4 pockets of uPA are reduced in size due to the 99-insertion-loop. Therefore, the peculiar binding mode of the new type of uPA inhibitors offers the possibility to exploit optimized interactions at the S1VS2' subsites to further enhance selectivity and potency.