OPTIMAL SUBSITE OCCUPANCY AND DESIGN OF A SELECTIVE INHIBITOR OF UROKINASE

Human urokinase type plasminogen activator (u-PA) is a member of the c hymotrypsin family of serine proteases that can play important roles i n both health and disease, We have used substrate phage display techni ques to characterize the specificity of this enzyme in detail and to i dentify peptides that are cleaved 840-5300 times more efficiently by u -PA than peptides containing the physiological target sequence of the enzyme, In addition, unlike peptides containing the physiological targ et sequence, the peptide substrates selected in this study were cleave d as much as 120 times more efficiently by u-PA than by tissue type pl asminogen activator (t-PA), an intimately related enzyme. Analysis of the selected peptide substrates strongly suggested that the primary se quence SGRSA, from position P3 to P2', represents optimal subsite occu pancy for substrates of u-PA. Insights gained in these investigations were used to design a variant of plasminogen activator inhibitor type 1, the primary physiological inhibitor of both u-PA and t-PA, that inh ibited u-PA approximately 70 times more rapidly than it inhibited t-PA . These observations provide a solid foundation for the design of high ly selective, high affinity inhibitors of u-PA and, consequently, may facilitate the development of novel therapeutic agents to inhibit the initiation and/or progression of selected human tumors.