Biochemical characterization and in vitro activity of AZ513, a noncovalent, reversible, and noncompetitive inhibitor of fatty acid amide hydrolase

Abstract Fatty acid amide hydrolase (FAAH) hydrolyzes several bioactive lipids including the endocannabinoid anandamide. Synthetic FAAH inhibitors are being generated to help define the biological role(s) of this enzyme, the lipids it degrades in vivo , and the disease states that might benefit from its pharmacological modulation. AZ513 inhibits human FAAH (IC 50  = 551 nM), is 20-fold more potent against rat FAAH (IC 50  = 27 nM), and is inactive at 10 μM against the serine hydrolases acetylcholinesterase, thrombin, and trypsin. In contrast to most other potent FAAH inhibitors, AZ513 showed no evidence of covalently modifying the enzyme and displayed reversible inhibition. In an enzyme cross-competition assay, AZ513 did not compete with OL-135, an inhibitor that binds to the catalytic site in FAAH, which indicates that AZ513 does not bind to the catalytic site and is therefore noncompetitive with respect to substrate. AZ513 has good cell penetration as demonstrated by inhibition of anandamide hydrolysis in human FAAH-transfected HEK293 cells (IC 50  = 360 nM). AZ513 was tested in a rat spinal cord slice preparation where CB 1 activation reduces excitatory post-synaptic currents (EPSCs). In this native tissue assay of synaptic activity, AZ513 reduced EPSCs, which is consistent with inhibiting endogenous FAAH and augmenting endocannabinoid tone. AZ513 has a unique biochemical profile compared with other published FAAH inhibitors and will be a useful tool compound to further explore the role of FAAH in various biological processes.