Dual blockade of FAAH and MAGL identifies behavioral processes regulated by endocannabinoid crosstalk in vivo Jonathan Z. Longa, Daniel K. Nomuraa, Robert E. Vannb, D. Matthew Walentinyb, Lamont Bookerb, Xin Jina, James J. Burstonb, Laura J. Sim-Selleyb, Aron H. Lichtmanb, Jenny L. Wileyb, and Benjamin F. Cravatta,1 aThe Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA 92037; and bDepartment of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA 23298-0613 Edited by Michael A. Marletta, University of California, Berkeley, CA, and approved October 6, 2009 (received for review August 19, 2009) ⌬9-Tetrahydrocannabinol (THC), the psychoactive component of mar- serve as key points of control over specific endocannabinoid ijuana, and other direct cannabinoid receptor (CB1) agonists produce signaling events in vivo (13). Fatty acid amide hydrolase (FAAH) a number of neurobehavioral effects in mammals that range from the is the major degradative enzyme for AEA (14–16). In contrast, beneficial (analgesia) to the untoward (abuse potential). Why, how- although several enzymes can hydrolyze 2-AG, this reaction ap- ever, this full spectrum of activities is not observed upon pharmaco- pears to be primarily catalyzed by monoacylglycerol lipase (MAGL) logical inhibition or genetic deletion of either fatty acid amide in the nervous system (17). The designation of FAAH and MAGL hydrolase (FAAH) or monoacylglycerol lipase (MAGL), enzymes that as principal AEA and 2-AG degradative enzymes, respectively, is regulate the two major endocannabinoids anandamide (AEA) and supported by a combination of genetic and pharmacological stud- 2-arachidonoylglycerol (2-AG), respectively, has remained unclear. ies. For instance, FAAH(Ϫ/Ϫ) mice or animals treated with Here, we describe a selective and efficacious dual FAAH/MAGL in- selective FAAH inhibitors possess Ͼ10-fold elevations in brain hibitor, JZL195, and show that this agent exhibits broad activity in the levels of AEA and other N-acyl ethanolamines (18–20) but no tetrad test for CB1 agonism, causing analgesia, hypomotilty, and alterations in 2-AG (20–22). Conversely, mice treated with the catalepsy. Comparison of JZL195 to specific FAAH and MAGL inhibi- selective MAGL inhibitor JZL184 show 8- to 10-fold increases in tors identified behavioral processes that were regulated by a single brain 2-AG levels without changes in AEA content (23, 24). endocannabinoid pathway (e.g., hypomotility by the 2-AG/MAGL Pharmacological studies have revealed that selective FAAH and pathway) and, interestingly, those where disruption of both FAAH MAGL inhibitors produce an intriguing subset of the behavioral and MAGL produced additive effects that were reversed by a CB1 effects observed with direct CB1 agonists, including analgesia in antagonist. Falling into this latter category was drug discrimination multiple acute and chronic pain models (23, 25–27). Inhibition of behavior, where dual FAAH/MAGL blockade, but not disruption of MAGL, but not FAAH, also causes CB1-dependent hypomotility either FAAH or MAGL alone, produced THC-like responses that were (23). However, neither FAAH nor MAGL inhibitors induce the reversed by a CB1 antagonist. These data indicate that AEA and 2-AG cataleptic behavioral responses observed with direct CB1 agonists. signaling pathways interact to regulate specific behavioral processes Moreover, FAAH inhibitors have proved inactive in models of drug in vivo, including those relevant to drug abuse, thus providing a abuse, including drug discrimination assays (28). Collectively, these potential mechanistic basis for the distinct pharmacological profiles of findings indicate that selective blockade of FAAH or MAGL can direct CB1 agonists and inhibitors of individual endocannabinoid disassociate some of the beneficial and undesirable effects of CB1 degradative enzymes. activation. Although the different pharmacological profiles of CB1 agonists hydrolase ͉ inhibitor ͉ metabolism and endocannabinoid hydrolase inhibitors are potentially exciting from a therapeutic perspective, they also present a frustrating -arachidonoyl ethanolamine (anandamide or AEA) (1) and mechanistic conundrum. Simply put, why do selective FAAH and N2-arachidonoylglycerol (2-AG) (2, 3) are lipid transmitters MAGL inhibitors differ in their actions, and why do they produce that serve as endogenous ligands for the cannabinoid G-protein- only a subset of the behavioral effects observed with direct CB1 coupled receptors CB1 and CB2. These lipids and receptors, agonists? One possibility is that AEA and 2-AG signaling pathways along with the enzymes that biosynthesize and degrade AEA and interact in the nervous system to affect behavioral processes beyond 2-AG, form the endogenous cannabinoid (endocannabinoid) those regulated by either endocannabinoid alone. To test this idea, system, which regulates a diverse number of physiological pro- we describe herein an inhibitor, JZL195, that inactivates both cesses in mammals, including pain, cognition, emotionality, FAAH and MAGL with high efficacy and selectivity in vivo. Using neurodegeneration, feeding, and inflammation (4). JZL195, we discover behavioral processes in which elevations in CB1 and CB2 receptors are also activated by ⌬9-tetrahydrocan- AEA and 2-AG combine to give additive effects. These behaviors nabinol (THC), the psychoactive component of marijuana (4). include catalepsy and THC-like drug discrimination responses, Most of the neurobehavioral effects of THC and other direct indicating that some of the untoward effects of direct CB1 agonists cannabinoid receptor agonists are mediated by the CB1 receptor (5, constitute points of crosstalk between endogenous AEA and 2-AG 6), likely reflecting its widespread and abundant expression in the signaling pathways. nervous system (7, 8). CB1 agonism produces medicinally useful activities, such as analgesia, but also a number of undesirable side Author contributions: J.Z.L., D.K.N., A.H.L., J.L.W., and B.F.C. designed research; J.Z.L., effects, including locomotor and cognitive impairments, as well as D.K.N., R.E.V., D.M.W., L.B., X.J., J.J.B., and L.J.S.-S. performed research; J.Z.L. contributed abuse liability. To date, it has proved difficult to uncouple these new reagents/analytic tools; J.Z.L., D.K.N., A.H.L., J.L.W., and B.F.C. analyzed data; and beneficial and untoward properties, thus limiting the therapeutic J.Z.L., A.H.L., J.L.W., and B.F.C. wrote the paper. utility of direct CB1 agonists. The authors declare no conflict of interest. Inhibitors of AEA and 2-AG degradation offer a potentially This article is a PNAS Direct Submission. attractive alternative strategy to stimulate the endocannabinoid 1To whom correspondence should be addressed. E-mail: [email protected]. system (9–12). Indeed, despite the structural similarity shared by This article contains supporting information online at www.pnas.org/cgi/content/full/ AEA and 2-AG, distinct enzymes inactivate these lipids and thus 0909411106/DCSupplemental. 20270–20275 ͉ PNAS ͉ December 1, 2009 ͉ vol. 106 ͉ no. 48 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0909411106 Downloaded by guest on September 28, 2021 Fig. 1. Development of a dual FAAH and MAGL inhibitor, JZL195. (A) Structures of the FAAH-selective inhibitors PF-622 and PF-3845, the MAGL-selective inhibitor JZL184, and dual FAAH-MAGL inhibitors. (B) Serine hydrolase activity profiles of brain membranes after incubation with dual FAAH-MAGL inhibitors (1 ␮M) as determined by competitive ABPP using the serine hydrolase-directed probe fluorophosphonate-rhodamine (FP-Rh). (C) Concentration-dependent BIOCHEMISTRY effects of JZL195 on mouse brain membrane serine hydrolases. (D and E) Blockade of AEA (D) and 2-AG (E) hydrolysis activity for MAGL and FAAH, respectively, recombinantly expressed in COS7 cells (black traces), or from mouse brain membranes (blue traces). For B–E, samples were treated with inhibitor for 30 min at 37 °C before addition of FP-Rh (1 ␮M) (B and C), 100 ␮M AEA (D), or 100 ␮M 2-AG (E). For D and E, data are presented as means Ϯ SEM of three independent experiments. Results effort culminated in the generation of JZL195, an N-(3- Development of JZL195, a Selective Dual Inhibitor of AEA and 2-AG phenoxybenzyl) piperazine carbamate (Fig. 1A and Fig. S1), that Hydrolysis. Previous attempts to characterize dual inhibition of produced near-complete blockade of FP-Rh labeling of both mouse FAAH and MAGL have used fluorophosphonate reagents, such as brain FAAH and MAGL at concentrations as low as 100 nM (IC50 isopropyldodecylfluorophosphonate (IDFP) (29). However, inter- values of 13 and 19 nM, respectively; Fig. 1C and Fig. S2), while preting the pharmacology of IDFP is difficult for several reasons. showing only modest and incomplete inhibitory activity against Ͼ ␮ Ϸ First, IDFP inhibits many serine hydrolases, including not only NTE (IC50 5 M, 50% maximal inhibition; Fig. 1C, Fig. S2). FAAH and MAGL but also the alternative 2-AG hydrolase At higher concentrations, JZL195 inhibited ABHD6 (Fig. 1C and ABHD6, hormone sensitive lipase (HSL), neuropathy target es- Fig. S2), but not any of the other brain serine hydrolases detected in our competitive ABPP assays (Fig. 1C). We furthermore con- terase (NTE), and the ether–lipid metabolic enzyme KIAA1363 ␮ (29). Second, IDFP displaces CB1 receptor agonist binding with an firmed that JZL195 (100 M) does not inhibit mouse brain acetylcholinesterase activity (Fig. S3). Substrate hydrolysis