title: Computer-assisted mutagenesis of ecotin to engineer its secondary binding site for urokinase inhibition.
abstract: Inhibitors of urokinase-type plasminogen activator (uPA) were selected in vitro from two ecotin phage-display libraries to study the effect on binding of amino acid substitutions at critical positions 108, 110, 112, and 113 within the 100s loop (RNKL, respectively, in wild type ecotin). The first, a focused library, was the result of a computation-assisted approach using the three-dimensional structure of the ecotin-trypsin complex to guide the modeling of amino acid substitutions predicted to increase affinity for uPA. The second, a complete library, allowed for all substitutions at the above identified positions. The consensus sequences selected from the focused, and complete libraries were RRWS and R(R/N)QL, respectively. Inhibition constant determinations showed ecotin variants containing these sequences to be similarly potent (K(i) = 1-2 nm). These substitutions were combined with previously identified substitutions in another critical region of ecotin. One of these combinations (D70R/M84R/RRQL) is the tightest (K(i) = 50 pm) ecotin variant inhibitor of uPA. The blending of combinatorial methods and computer algorithms designed to predict stronger binders has allowed us to obtain protein derivatives that exhibit greatly increased affinity for a predetermined target. This technology can be applied to select for enhanced binding interactions at protein-protein interfaces and accelerate the process of protease inhibitor development.
authors.affiliation: Departamento de Ciências Farmacêuticas, Faculdade de Ciências da Saúde, Universidade de Brasilia, Campus Universitário Darcy Ribeiro, 70910-900 Brasilia, DF Brasil.
authors.forename: Martha C A
authors.initials: MC
authors.lastname: Laboissière
authors.forename: Malin M
authors.initials: MM
authors.lastname: Young
authors.forename: Rilva G
authors.initials: RG
authors.lastname: Pinho
authors.forename: Stephen
authors.initials: S
authors.lastname: Todd
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Fletterick
authors.forename: Irwin
authors.initials: I
authors.lastname: Kuntz
authors.forename: Charles S
authors.initials: CS
authors.lastname: Craik
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics*; D001665:Binding Sites; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005818:Genetic Engineering; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D019151:Peptide Library; D033903:Periplasmic Proteins; D011485:Protein Binding / Q000235:genetics; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors*
chemical_list: D001426:Bacterial Proteins; C039076:Eco protein, E coli; D029968:Escherichia coli Proteins; D019151:Peptide Library; D033903:Periplasmic Proteins; D014568:Urokinase-Type Plasminogen Activator