A novel scintillation proximity assay for fatty acid amide hydrolase compatible with inhibitor screening.
Anal Biochem
; 354(1): 35-42, 2006 Jul 01.
Article
en En
| MEDLINE
| ID: mdl-16707086
ABSTRACT
A binding assay for human fatty acid amide hydrolase (FAAH) using the scintillation proximity assay (SPA) technology is described. This SPA uses the specific interactions of [3H]R(+)-methanandamide (MAEA) and FAAH expressing microsomes to evaluate the displacement activity of FAAH inhibitors. We observed that a competitive nonhydrolyzed FAAH inhibitor, [3H]MAEA, bound specifically to the FAAH microsomes. Coincubation with an FAAH inhibitor, URB-597, competitively displaced the [3H]MAEA on the FAAH microsomes. The released radiolabel was then detected through an interaction with the SPA beads. The assay is specific for FAAH given that microsomes prepared from cells expressing the inactive FAAH-S241A mutant or vector alone had no significant ability to bind [3H]MAEA. Furthermore, the binding of [3H]MAEA to FAAH microsomes was abolished by selective FAAH inhibitors in a dose-dependent manner, with IC50 values comparable to those seen in a functional assay. This novel SPA has been validated and demonstrated to be simple, sensitive, and amenable to high-throughput screening.
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Banco de datos:
MEDLINE
Asunto principal:
Conteo por Cintilación
/
Inhibidores Enzimáticos
/
Amidohidrolasas
Tipo de estudio:
Diagnostic_studies
/
Screening_studies
Límite:
Animals
/
Humans
Idioma:
En
Año:
2006
Tipo del documento:
Article