Universität zu Köln

Dato, Florian M., Maassen, Andreas ORCID: 0000-0002-1446-0994, Goldfuss, Bernd ORCID: 0000-0002-1814-8818 and Pietsch, Markus (2018). Characterization of fatty acid amide hydrolase activity by a fluorescence-based assay. Anal. Biochem., 546. S. 50 - 58. SAN DIEGO: ACADEMIC PRESS INC ELSEVIER SCIENCE. ISSN 1096-0309

Full text not available from this repository.

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-n, 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 mu g 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).

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Dato, Florian M. UNSPECIFIED UNSPECIFIED UNSPECIFIED Maassen, Andreas UNSPECIFIED orcid.org/0000-0002-1446-0994 UNSPECIFIED Goldfuss, Bernd UNSPECIFIED orcid.org/0000-0002-1814-8818 UNSPECIFIED Pietsch, Markus UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-189823
DOI:	10.1016/j.ab.2018.01.026
Journal or Publication Title:	Anal. Biochem.
Volume:	546
Page Range:	S. 50 - 58
Date:	2018
Publisher:	ACADEMIC PRESS INC ELSEVIER SCIENCE
Place of Publication:	SAN DIEGO
ISSN:	1096-0309
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Chemistry > Institute of Organic Chemistry
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language ENDOCANNABINOID SYSTEM; CLINICAL-TRIAL; MONOACYLGLYCEROL LIPASE; INHIBITOR URB597; DISCOVERY; PAIN; FAAH; PF-04457845; K(CAT)/K-M; EXPRESSION Multiple languages Biochemical Research Methods; Biochemistry & Molecular Biology; Chemistry, Analytical Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/18982