Development of in vitro assays for fatty acid amide hydrolase inhibitors

Sara Pritchard, Julian Reeves

Glaxo SmithKline, Harlow Essex, UK

Fatty Acid Amide Hydrolase (FAAH) inhibitors exhibit analgesic, anxiolytic and neuroprotective properties in pre-clinical animal models and therefore they represent developable agents for multiple conditions. Various in vitro assays for assessment of their inhibitory activity have been described, including fluorescent substrate assays compatible with high throughput screening (Ramarao et al., 2005) and radioactive substrate conversion assays compatible with cell and tissue homogenates (Boldrup et al, 2004). However little information is available on the suitability of the radioactive substrate conversion assay for determining activity in whole cells. In this study we describe an adapted in vitro radioactive substrate assay for measurement of FAAH activity in whole cell and native whole cell formats.

Various cell lines and primary human cells were examined for their endogenous expression of FAAH by Taqman. HEK293, COS-1 and human isolated granulocyte cell membranes, positive for FAAH expression, PC-3 cells negative for FAAH expression, and rat brain membranes, were examined in a 3H anandamide conversion assay (Boldrup et al., 2004). FAAH activity was confirmed in cell membranes positive for FAAH expression and in rat brain membranes. Initial assays using whole cells showed a low dynamic range, and adaptions were made to address this, including the introduction of radioactive signal detection by Lumaplates, which increased signals ∼10-fold to an acceptable level.

Using this adapted technique, COS-1 and HEK293 whole cells were able to demonstrate conversion of 3H anandamide to 3H ethanolamine. The pIC50 for a standard tool compound URB597 to inhibit 3H anandamide conversion in COS-1 and HEK293 whole cells was 7.68 + 0.12 and 8.60 + 0.10, respectively. Human isolated granulocytes and diluted whole blood also demonstrated URB597-inhibitable activity in this assay (pIC50s 8.75 +0.04, 8.04 + 0.04 respectively). Data are the mean+ S.E.M of 4 experiments.

This study has identified a suitable method for measuring FAAH inhibitory activity in both a whole cell and native cell assay format

Ramarao MK, et al. (2005) Analytical Biochemistry. 343(1):143-51

Boldrup L, et al. (2004) Journal of Biochemical & Biophysical Methods. 60(2):171-7