Synthetic substrate for high specificity enzymatic assays

• US 7,291,698 B2
• Filed: 09/03/2002
• Issued: 11/06/2007
• Est. Priority Date: 09/04/2001
• Status: Expired due to Fees

• Current Assignee: Stephen Eliot Zweig
• Original Assignee: Stephen Eliot Zweig
• Inventors: Zweig, Stephen Eliot
• Primary Examiner(s): Lankford, Jr.; Leon B.
• Assistant Examiner(s): HANLEY, SUSAN MARIE
• Application Number:
  US10/233,908
• Publication Number:
  US 20030113768A1
• Time in Patent Office: 1,890 Days
• Field of Search: 435/23, 435/7.72, 435/174, 435/28, 435212-219, 530/300, 530/800, 530324-332
• US Class Current: 530/300
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C12Q 1/42 involving phosphatase
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: G01N 2500/04 Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Abstract

Novel synthetic enzyme substrates, enhanced to have improved enzymatic specificity, are disclosed. These synthetic enzyme substrates consist of a substrate peptide that has had its specificity further improved by additional synthetic moieties, selected by combinatorial chemistry techniques, that act to sterically block non-target enzymes. These “steric restrictor” moieties may be labeled to produce a detectable signal upon enzymatic reaction. These novel substrates are particularly useful for improved enzyme substrate microarrays. Specific applications for improved protease substrate microarrays are discussed. A variety of applications for these improved protease substrate microarrays are also disclosed, including proteomics research, protease discovery, protease binding site characterization, diagnosis of the protease composition of biological samples, monitoring the angiogenic status of a tumor, monitoring the status of arthritis and other inflammatory diseases, and the discovery and optimization of novel drugs that modify or inhibit protease activity.

Claims

1. A synthetic enzyme substrate, comprising:

a peptide substrate moiety capable of independently serving as a substrate for a target protease that cleaves a bond between two amino acids of said peptide substrate moiety;

a first and second streric restrictor group, wherein the N-terminus and C-terminus of said peptide substrate moiety are each covalently coupled to one of said steric restrictor groups, wherein said steric restrictor groups are polymers comprised of two or more non-identical D-amino acid monomers, wherein the sequences of said D-amino acid monomers of each of said steric restrictor groups are selected based upon the ability of said restrictor groups to protect said peptide substrate from serving as a substrate for non-target proteases, while preserving the ability of said peptide substrate to serve as a substrate for said target protease;

wherein a first label moiety incorporated into said first steric restrictor group produces a detectable signal that is quenched by a second label moiety incorporated into said second steric restrictor group, and in which enzymatic cleavage of the peptide substrate moiety by said target protease produces a change in the detectable signal.

2. The synthetic substrate of claim 1, in which the first label moiety is a fluorophore, and the second label moiety note to quench the fluorescence of the first label moiety.

3. The synthetic substrate of claim 1, wherein said substrate is bound to a solid support, and used as an assay device for protease activity.