Inhibition of Urokinase by 4-substituted Benzo[ft]thiophene-2-carboxamidines: An Important New Class of Selective Synthetic Urokinase Inhibitor

I'rokinase-type plasminogen activator (uPA) is an important mediator of cellular invasiveness. Specifically, cell surface receptor-bound uPA ac tivates plasminoceli to the potent general protease plasmin, which then degrades extracellular matrix or basement membrane either directly or via proteolytic activation of latent collagenases. Thus, cell surface uPA initiates an extracellular proteolytic cascade with which invasive cells eliminate barriers to movement. Since cellular invasiveness plays impor tant roles in several disease states, including cancer metastasis and inva sion, arthritis and inflammation, and diabetic retinal neovascularization, the development of synthetic uPA inhibitors is an attractive therapeutic goal. Here we show that 4-substituted henzo[A]thiophene-2-carhoxamidines represent an important new class of potent and selective synthetic uPA inhibitor. Two compounds in this class, B428 and B623. inhibit human uPA in plasminogen-linked assays with median inhibition concentration de '.„i values of 0.32 and 0.07 UM. respectively. This level of inhibition represents 20and 100-fold increases in potency, respectively, relative to the 6—7UMpotencies reported for amiloride and 4-chlorophenylguanidine, the two most potent selective synthetic uPA inhibitors previously de scribed. Importantly, both compounds show -300-fold selectivity for uPA relative to tissue-type pla.sminogen activator and > 1000-fold selectivity relative to plasmin. Lineweaver-Burk analyses show uPA inhibition by B428 and B623 to be competitive in nature with inhibition constants i A, i of 0.53 and 0.16 UM,respectively. Since it is cell surface uPA and not free or secreted uPA that is primarily responsible for cellular invasiveness, biologically effective uPA inhibitors must be capable of inhibiting cell surface uPA. B428 and B623 meet this criterion by inhibiting cell surface uPA on HT1080 human fibrosarcoma cells with l( .,, values of 0.54 and 0.20 u\i, respectively. Moreover, degradation of [-'H]fibronectin by HT 1080 cells via cell surface uPA-mediated, plasminogen-dependent mechanisms is inhibited by B428 and B623, with K Ü,values of 1.5 and 0.39 UM,respectively. In summary, 4-substituted benzo[¿>]thiophene-2-carboxamidines such as B428 and B623 represent the most potent class of competitive synthetic uPA inhibitors currently known. Their ability to selectively inhibit both free and cell surface uPA as well as cell surface uPA-mediated cellular degradative functions suggests that this class of compounds may hold significant promise for further development as antiinvasiveness drugs.