Optimal Subsite Occupancy and Design of a Selective Inhibitor of Urokinase

Song-Hua Ke,Gary S Coombs,Kathy Tachias,David R Corey,Edwin L Madison

doi:10.1074/jbc.272.33.20456

Abstract

Human urokinase type plasminogen activator (u-PA) is a member of the chymotrypsin family of serine proteases that can play important roles in both health and disease. We have used substrate phage display techniques to characterize the specificity of this enzyme in detail and to identify 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 target sequence, the peptide substrates selected in this study were cleaved as much as 120 times more efficiently by u-PA than by tissue type plasminogen activator (t-PA), an intimately related enzyme. Analysis of the selected peptide substrates strongly suggested that the primary sequence SGRSA, from position P3 to P2', represents optimal subsite occupancy 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 inhibited u-PA approximately 70 times more rapidly than it inhibited t-PA. These observations provide a solid foundation for the design of highly 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.

Highlights

Human urokinase type plasminogen activator (u-PA) is a member of the chymotrypsin family of serine proteases that can play important roles in both health and disease
Unlike peptides containing the physiological target sequence, the peptide substrates selected in this study were cleaved as much as 120 times more efficiently by u-PA than by tissue type plasminogen activator (t-PA), an intimately related enzyme
K91 (Fϩ) and MC1061 (FϪ) strains of E. coli were provided by Steve Cwirla (Affymax). mAb 3-E7 was purchased from Gramsch Laboratories (Schwabhausen, FRG). u-PA was obtained from Jack Henkin (Abbott Laboratories)

Summary

Introduction

Human urokinase type plasminogen activator (u-PA) is a member of the chymotrypsin family of serine proteases that can play important roles in both health and disease. Analysis of the selected peptide substrates strongly suggested that the primary sequence SGRSA, from position P3 to P2؅, represents optimal subsite occupancy 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 inhibited u-PA approximately 70 times more rapidly than it inhibited t-PA. U-PA and t-PA are very closely related members of the chymotrypsin gene family These two proteases possess extremely high structural similarity [7, 8], share the same primary physiological substrate (plasminogen) and inhibitor (plasminogen activator inhibitor, type 1) [3], and, unlike plasmin, exhibit remarkably stringent substrate specificity (9 –11). Mice lacking t-PA, for example, are resistant to specific excitotoxins that cause extensive neurodegeneration in wild type mice [13], and mice lacking u-PA exhibit defects in the proliferation and/or migration of smooth muscle cells in a model of restenosis following vascular injury [5, 6]

Methods

Results

Conclusion