Structural basis for selectivity of a small molecule, S1-binding, submicromolar inhibitor of urokinase-type plasminogen activator

Introduction: Urokinase-type plasminogen activator (uPA) is a protease asso ciated with tumor metastasis and invasion. Inhibitors of uPA may have poten tial as drugs for prostate, breast and other cancers. Therapeutically usefu l inhibitors must be selective for uPA and not appreciably inhibit the rela ted, and structurally and functionally similar enzyme, tissue-type plasmino gen activator (tPA), involved in the vital blood-clotting cascade, Results: We produced mutagenically deglycosylated low molecular weight uPA and determined the crystal structure of its complex with 4-iodobenzo[b]thio phene-2-carboxamidine (K-i = 0.21 +/- 0.02 mu M), To probe the structural d eterminants of the affinity and selectivity of this inhibitor for uPA we al so determined the structures of its trypsin acid thrombin complexes, of apo -trypsin, apo-thrombin and ape-factor Xa, and of uPA, trypsin and thrombin bound by compounds that are less effective uPA inhibitors, benzo [b]thiophe ne-2-carboxamidine, thieno[2,3-b]pyridine-2-carboxamidine and benzamidine, The K-i values of each inhibitor toward UPA, tPA, trypsin, tryptase, thromb in and factor Xa were determined and compared. One selectivity determinant of the benzo[b]thiophene-2-carboxamidines for uPA involves a hydrogen bond at the S1 site to O gamma(Ser190) that is absent in the Ala190 proteases, t PA, thrombin and factor Xa. Other subtle differences in the architecture of the S1 site also influence inhibitor affinity and enzyme-bound structure. Conclusions: Subtle structural differences in the S1 site of uPA compared w ith that of related proteases, which result in part from the presence of a serine residue at position 190, account for the selectivity of small thioph ene-2-carboxamidines for uPA, and afford a framework for structure-based de sign of small, potent, selective uPA inhibitors.