Characterization of fatty acid amide hydrolase activity by a fluorescence-based assay

Florian M Dato; Andreas Maaßen; Bernd Goldfuß; Markus Pietsch

doi:10.1016/j.ab.2018.01.026

Characterization of fatty acid amide hydrolase activity by a fluorescence-based assay

Anal Biochem. 2018 Apr 1:546:50-57. doi: 10.1016/j.ab.2018.01.026. Epub 2018 Jan 31.

Authors

Florian M Dato¹, Andreas Maaßen¹, Bernd Goldfuß², Markus Pietsch³

Affiliations

¹ Institute II of Pharmacology, Center of Pharmacology, Medical Faculty, University of Cologne, Gleueler Str. 24, D-50931 Cologne, Germany; Institute of Organic Chemistry, Department of Chemistry, University of Cologne, Greinstr. 4, D-50939 Cologne, Germany.
² Institute of Organic Chemistry, Department of Chemistry, University of Cologne, Greinstr. 4, D-50939 Cologne, Germany.
³ Institute II of Pharmacology, Center of Pharmacology, Medical Faculty, University of Cologne, Gleueler Str. 24, D-50931 Cologne, Germany. Electronic address: markus.pietsch@uk-koeln.de.

PMID: 29408178
DOI: 10.1016/j.ab.2018.01.026

Abstract

Fatty acid amide hydrolase (FAAH) is involved in many human diseases, particularly cancer, pain and inflammation as well as neurological, metabolic and cardiovascular disorders. Therefore, FAAH is an attractive target for the development of low-molecular-weight inhibitors as therapeutics, which requires robust assays that can be used for high-throughput screening (HTS) of compound libraries. Here, we report the development of a fluorometric assay based on FAAH's ability to effectively hydrolyze medium-chain fatty acid amides, introducing N-decanoyl-substituted 5-amino-2-methoxypyridine (D-MAP) as new amide substrate. D-MAP is cleaved by FAAH with an 8-fold larger specificity constant than the previously reported octanoyl-analog Oc-MAP (V_max/K_m of 1.09 and 0.134 mL min^-1 mg^-1, respectively), with both MAP derivatives possessing superior substrate properties and much increased aqueous solubility compared to the respective p-nitroaniline compounds D-pNA and Oc-pNA. The new assay with D-MAP as substrate is highly sensitive using a lower enzyme concentration (1 μg mL^-1) than literature-reported fluorimetric FAAH assays. In addition, D-MAP was validated in comparison to the substrate Oc-MAP for the characterization of FAAH inhibitors by means of the reference compounds URB597 and TC-F2 and was shown to be highly suitable for HTS in both kinetic and endpoint assays (Z' factors of 0.81 and 0.78, respectively).

Keywords: 5-Amino-2-methoxypyridine; Enzyme kinetics; Fatty acid amide hydrolase; Fluorescent assay; Substrates.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amides / chemical synthesis
Amides / chemistry
Amides / metabolism
Amidohydrolases / antagonists & inhibitors
Amidohydrolases / metabolism*
Colorimetry
Enzyme Inhibitors / chemistry
Enzyme Inhibitors / pharmacology
Fluorescence*
Fluorescent Dyes / chemical synthesis
Fluorescent Dyes / chemistry
High-Throughput Screening Assays
Humans
Hydrolysis
Kinetics
Molecular Structure
Pyridines / chemistry
Pyridines / metabolism

Substances

Amides
Enzyme Inhibitors
Fluorescent Dyes
Pyridines
Amidohydrolases
fatty-acid amide hydrolase