Assay methods

• US 20040014131A1
• Filed: 07/09/2002
• Published: 01/22/2004
• Est. Priority Date: 07/09/2002
• Status: Abandoned Application

• Current Assignee: Pfizer Inc.
• Original Assignee: Pfizer Inc.
• Inventors: Boyd, Helen Frances, Benson, Neil, Stacey, Peter, Singleton, David Harlan, Contillo, Leonard Gabriel
• Application Number:
  US10/192,407
• Publication Number:
  US 20040014131A1
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: G01N 2500/02 Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
• 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)
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching

Abstract

The invention relates to methods for identifying agents, which bind to and/or modulate exopeptidases or endopeptidases, preferably human soluble secreted endopeptidase (SEP), oxytocinase, neutral endopeptidase (NEP), or non-human SEP, preferably comprising contacting said agents with a polypeptide comprising: SEQ ID NO: 2, an amino acid sequence encoded by the polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 5; or a polypeptide encoded by the cDNA of NCIMB 41110, and determining whether binding and/or modulation occurs. Preferably, said method is carried out in the presence of a substrate peptide labelled with at least one fluorescent donor dye and at least one fluorescence acceptor dye, with the method used to detect said agents being a competitive binding Fluorescence Resonance Energy Transfer (FRET) assay.

Claims

1. A method for identifying an agent which binds to and/or modulates a SEP polypeptide comprising contacting said agent with a polypeptide comprising:

SEQ ID NO;

2, an amino acid sequence encoded by the polynucleotide sequence of SEQ ID NO;

1 or SEQ ID NO;

5;

or a polypeptide encoded by the cDNA of NCIMB 41110, and determining whether binding and/or modulation occurs.

2. The method of claim 1, comprising contacting said SEP polypeptide with a SEP substrate peptide in the presence of said agent, wherein said substrate peptide is capable of providing a detectable signal in response to cleavage by said SEP polypeptide, wherein said agent is identified as a modulator of said SEP polypeptide if there is a difference in the detectable signal in the presence and in the absence of said agent.

3. The method of claim 2, wherein said method identifies an agent that decreases said detectable signal and is a SEP polypeptide inhibitor.

4. The method of claim 2 or claim 3, wherein said substrate peptide is labelled with at least one fluorescent donor dye and said signal is detected by Fluorescence Resonance Energy Transfer (FRET) assay.

5. The method of claim 4, wherein said labelled substrate peptide is Rhodamine green-Gly-Gly-dPhe-Leu-Arg-Arg-Val-CyS(QSY™

7)-β

Ala-NH₂, 5-(and

     6) tetramethyl rhodamine Gly-Gly-dPhe-Leu-Arg-Arg-Val-CyS(QSY™

7)-β

Ala-NH₂ or 5-carboxyfluorescein-Gly-Gly-dPhe-Leu-Arg-Arg-Val-Cys(5-(and

     6)tetramethylrhodamine)-β

Ala-NH₂.

6. The method of any one of claims 1 to 5, wherein binding between said SEP polypeptide and said agent is detected.

7. The method of claim 6, wherein said method is a competitive binding assay.

11. An agent identified by the method of any one of claims 1 to 10;

wherein said agent is not phosphoramidon, thiorphan, fasidotrilat, omapatrilat or FR901533.

12. An agent identified by the method of any one of claims 1 to 7;

wherein said agent has a greater than 30-fold selectivity for SEP over neutral endopeptidase NEP EC 3.4.24.11 and angiotensin converting enzyme (ACE).

13. An agent according to claim 12;

wherein said agent has a greater than 50-fold selectivity for SEP over neutral endopeptidase NEP EC 3.4.24.11 and angiotensin converting enzyme (ACE).

14. An agent identified by the method of any one of claims 1 to 7;

wherein said agent has a greater than 100-fold selectivity for SEP over endothelin converting enzyme (ECE).

8. A method for identifying an agent which inhibits or selectively inhibits a peptidase comprising contacting said peptidase and a peptidase substrate peptide selected from the group consisting of Rhodamine green-Gly-Gly-dPhe-Leu-Arg-Arg-Val-Cys(QSY™

7)-β

Ala-NH₂, 5-(and

     6) tetramethyl rhodamine Gly-Gly-dPhe-Leu-Arg-Arg-Val-CyS(QSY™

7)-β

Ala-NH₂ and 5-carboxyfluorescein-Gly-Gly-dPhe-Leu-Arg-Arg-Val-Cys (5- (and

     6) tetramethylrhodamine)-β

Ala-NH₂, in the presence of said agent, wherein said substrate peptide is capable of providing a detectable signal in response to cleavage by said peptidase as detected by Fluorescence Resonance Energy Transfer (FRET) assay, wherein said agent is identified as an inhibitor of said peptidase if there is a decrease in the detectable signal in the presence of said agent as compared to in the absence of said agent.

9. The method of claim 8, wherein said peptidase is an exopeptidase or an endopeptidase.

10. The method of claim 8, wherein said peptidase is oxytocinase, neutral endopeptidase (NEP), or non-human SEP.