Assay for inhibitors of fatty-acid amide hydrolase

• US 6,699,682 B2
• Filed: 06/28/2001
• Issued: 03/02/2004
• Est. Priority Date: 06/12/1995
• Status: Expired due to Term

• Current Assignee: The Scripps Research Institute
• Original Assignee: The Scripps Research Institute
• Inventors: Cravatt, Benjamin F., Lerner, Richrd A., Gilula, Norton B.
• Primary Examiner(s): Marx, Irene
• Application Number:
  US09/894,790
• Publication Number:
  US 20020187542A1
• Time in Patent Office: 978 Days
• Field of Search: 435/228, 435/134, 435/227, 435/129, 435/18, 532/62, 436/34, 436/37
• US Class Current: 435/18
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/20 Hypnotics; Sedatives
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07C 233/09 with carbon atoms of carboxamide groups bound to carbon atoms of an acyclic unsaturated carbon skeleton
• CPC Class Codes: C07C 327/22 having carbon atoms of esterified thiocarboxyl groups bound to hydrogen atoms or to acyclic carbon atoms
• CPC Class Codes: C07C 45/45 by condensation
• CPC Class Codes: C07C 49/227 containing halogen
• CPC Class Codes: C07C 57/03 Monocarboxylic acids
• CPC Class Codes: C07F 9/5407 Acyclic saturated phosphonium compounds
• CPC Class Codes: C12N 9/80 acting on amide bonds in linear amides (3.5.1)
• CPC Class Codes: C12N 9/99 Enzyme inactivation by chemical treatment
• CPC Class Codes: C12P 7/6418 by hydrolysis of fatty acid esters

Abstract

The soporific activity of cis-9,10-octadecenoamide and other soporific fatty acid primary amides is neutralized by hydrolysis in the presence of fatty-acid amide hydrolase (FAAH). Hydrolysis of cis-9,10-octadecenoamide by FAAH leads to the formation of oleic acid, a compound without soporific activity. FAAH has be isolated and the gene encoding FAAH has been cloned, sequenced, and used to express recombinant FAAH. Inhibitors of FAAH are disclosed to block the hydrolase activity.

Claims

1. A method for ascertaining the inhibitory activity of a candidate inhibitor of fatty-acid amide hydrolase (FAAH), the method comprising the following steps:

Step A;

forming mixture “

A”

by combining a FAAH having an amino acid residue sequence selected from the group consisting of SEQ ID NO;

36, SEQ ID NO;

40, and SEQ ID NO;

43 and a fatty-acid primary amide substrate under reaction conditions;

Step B;

forming mixture “

B”

by combining the mixture “

A”

of said Step A with the candidate inhibitor under the reaction conditions of Step A;

Step C;

measuring the conversion of said fatty-acid primary amide substrate to a hydrolysis product within mixture “

A”

;

Step D;

measuring the conversion of said fatty-acid primary amide substrate to said hydrolysis product within mixture “

B”

; and

then

Step E;

comparing the conversion of substrate to hydrolysis product in Step C to the conversion of substrate to hydrolysis product in Step D, wherein a finding of decreased conversion in Step D as compared to Step C indicates the inhibitory activity of the candidate inhibitor.

2. The method of claim 1 wherein said fatty-acid primary amide substrate is selected from the group consisting of cis-9,10-octadecenoamide, anandamide, myristic amide, palmitic amide and stearic amide.