Profiling of protease specificity using combinatorial fluorogenic substrate libraries

US6680178B2 · kind B2 · utility

Assignee

• The Regents of the University of California · US

Inventors

• Jennifer Harris · San Diego, US
• Bradley Backes · San Francisco, US
• Jonathan A. Ellman · Guilford, US
• Charles S. Craik · San Francisco, US

Key dates

• Filing date: May 25, 2001
• Grant date: Jan 20, 2004
• Expiry date: Nov 10, 2021

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C07K5/1024
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

A method is presented for the preparation and use of fluorogenic peptide substrates that allows for the configuration of general substrate libraries to rapidly identify the primary and extended specificity of enzymes, such as proteases. The substrates contain a fluorogenic-leaving group, such as 7-amino-4-carbamoylmethyl-coumarin (ACC). Substrates incorporating the ACC leaving group show comparable kinetic profiles as those with the traditionally used 7-amino-4-methyl-coumarin (AMC) leaving group. The bifunctional nature of ACC allows for the efficient production of single substrates and substrate libraries using solid-phase synthesis techniques. The approximately 3-fold increased quantum yield of ACC over AMC permits reduction in enzyme and substrate concentrations. As a consequence, a greater number of substrates can be tolerated in a single assay, thus enabling an increase in the diversity space of the library. Soluble positional protease substrate libraries of 137,180 and 6,859 members, possessing amino acid diversity at the P4-P3-P2-P1 and P4-P3-P2 positions, respectively, were constructed. Employing this screening method the substrate specificities of a diverse array of prote…