The endocannabinoid hydrolase FAAH is an allosteric enzyme

Abstract

Fatty acid amide hydrolase (FAAH) is a membrane-bound homodimeric enzyme that in vivo controls content and biological activity of N-arachidonoylethanolamine (AEA) and other relevant bioactive lipids termed endocannabinoids. Parallel orientation of FAAH monomers likely allows both subunits to simultaneously recruit and cleave substrates. Here, we show full inhibition of human and rat FAAH by means of enzyme inhibitors used at a homodimer:inhibitor stoichiometric ratio of 1:1, implying that occupation of only one of the two active sites of FAAH is enough to fully block catalysis. Single W445Y substitution in rat FAAH displayed the same activity as the wild-type, but failed to show full inhibition at the homodimer:inhibitor 1:1 ratio. Instead, F432A mutant exhibited reduced specific activity but was fully inhibited at the homodimer:inhibitor 1:1 ratio. Kinetic analysis of AEA hydrolysis by rat FAAH and its F432A mutant demonstrated a Hill coefficient of ~1.6, that instead was ~1.0 in the W445Y mutant. Of note, also human FAAH catalysed an allosteric hydrolysis of AEA, showing a Hill coefficient of ~1.9. Taken together, this study demonstrates an unprecedented allosterism of FAAH, and represents a case of communication between two enzyme subunits seemingly controlled by a single amino acid (W445) at the dimer interface. In the light of extensive attempts and subsequent failures over the last decade to develop effective drugs for human therapy, these findings pave the way to the rationale design of new molecules that, by acting as positive or negative heterotropic effectors of FAAH, may control more efficiently its activity. Endocannabinoids form a relevant class of lipids that are widespread in tissues, where they exert diverse biological actions 1. Endocannabinoids have been demonstrated to reduce pain and inflammation, modulate energetic homeostasis and appetite, and have anticancer, anxiolytic, and neuroprotective effects through type-1 and/or type-2 cannabinoid receptors (CB 1 and CB 2), as well as via stimulation of transient receptor potential vanilloid channels, peroxisome proliferator-activated receptors, and additional targets 2-4. Their degradation is due to the activity of several metabolic enzymes, among which FAAH has a prominent role also in vivo 5. In addition, biotransformation of endocannabinoids is catalysed by lipoxygenases (LOXs) or cyclooxygenase-2 (COX-2) 6-8. FAAH is a membrane-bound homodimer that is able to hydrolyse AEA and, to a lesser extent, 2-arachidonoylglycerol (2-AG), N-oleoylethanolamine 9,10 , N-palmitoylethanolamine 11 , and N-oleoyltaurine 5,12,13. The biological activity of AEA, 2-AG and related compounds within the central nervous system 2,3 and at the periphery has been recently reviewed 6. The 3D structures of a truncated form of rat FAAH lacking the N-terminal region 14 (hereafter referred to as rFAAH), and of its humanized form containing residues of the human active site 15 , have been resolved at high resolution, and some details of the catalytic mechanism of the enzyme ha...

A Guide to Targeting the Endocannabinoid System in Drug Design

“…For MAGL, it was shown that its irreversible inhibitors, through prolonged inactivation of this enzyme, may cause physical dependence, CB1 desensitization, and impaired eCB-dependent synaptic plasticity [68]. Moreover, MAGL and FAAH form homodimers [66,69]. Recently, FAAH dimers have been proven to be dependent on the signaling between their subunits and its allosteric regulation.…”

“…Recently, FAAH dimers have been proven to be dependent on the signaling between their subunits and its allosteric regulation. Thus, it may be possible to design allosteric modulators of FAAH [69]. TRPV1 is a non-selective channel.…”

UPLC-MS/MS Method for Analysis of Endocannabinoid and Related Lipid Metabolism in Mouse Mucosal Tissue

“…These methods are optimized for quantitating the rate of (i) MAG biosynthesis in intestinal epithelium and lung tissue via DGL, (ii) MAG degradation in intestinal epithelium and lung tissue via MGL, (iii) MAG degradation via ABHD6 in lung tissue, and (iv) FAE degradation in intestinal epithelium and lung tissue via FAAH. Notably, we provide novel methods that do not require radioactive substrates to assess activity of FAAH in mouse mucosal tissues as described elsewhere (Fu et al, 2007;Dainese et al, 2020), and expand and optimize to lung tissue application of our previously-reported UPLC-MS/MS-based assays of DGL and MGL activity in intestinal epithelium (Batugedara et al, 2018;Argueta et al, 2019). Furthermore, the UPLC-MS/MS methods described here are ideal for detecting discrete changes in the activity of enzymes that metabolize eCBs and related lipids given that these enzymatic reactions involve hydrolysis of substrates leading to detectable changes in substrate mass.…”

“…These methods are optimized for quantitation of the rate of metabolism of eCBs by DGL, MGL, ABHD6, and FAAH. Moreover, novel methods are described for measuring FAAH activity that does not require use of radioactive compounds as substrates (Fu et al, 2007;Dainese et al, 2020).…”

Targeting Endocannabinoid Metabolism: an Arrow with Multiple Tips Against Multiple Sclerosis

“…Very recently, Maccarrone and co‐workers provided evidence for the existence of an allosteric regulation of FAAH, involving a functional cross‐talk between its monomers. This evidence offers the potential opportunity to elegantly fine‐tune FAAH activity by targeting regulatory sites [137] …”