Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries

摘要: A method is presented for the preparation and use of fluorogenic peptide substrates that allows configuration general substrate libraries to rapidly identify primary extended specificity proteases. The contain leaving group 7-amino-4-carbamoylmethylcoumarin (ACC). Substrates incorporating ACC show kinetic profiles comparable those with traditionally used 7-amino-4-methylcoumarin (AMC) group. bifunctional nature efficient production single by using 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase synthesis techniques. approximately 3-fold-increased quantum yield over AMC permits reduction in enzyme concentrations. As a consequence, greater number can be tolerated assay, thus enabling an increase diversity space library. Soluble positional protease 137,180 6,859 members, possessing amino acid at P4-P3-P2-P1 P4-P3-P2 positions, respectively, were constructed. Employing this screening method, we profiled specificities diverse array proteases, including serine proteases thrombin, plasmin, factor Xa, urokinase-type plasminogen activator, tissue granzyme B, trypsin, chymotrypsin, human neutrophil elastase, cysteine papain cruzain. resulting create pharmacophoric portrayal aid design selective potent inhibitors.