Chemical probes to potently and selectively inhibit PNAS PLUS endocannabinoid cellular reuptake Andrea Chiccaa,1, Simon Nicolussia,1, Ruben Bartholomäusb, Martina Blunderc,d, Alejandro Aparisi Reye, Vanessa Petruccia, Ines del Carmen Reynoso-Morenoa,f, Juan Manuel Viveros-Paredesf, Marianela Dalghi Gensa, Beat Lutze, Helgi B. Schiöthc, Michael Soeberdtg, Christoph Abelsg, Roch-Philippe Charlesa, Karl-Heinz Altmannb, and Jürg Gertscha,2 aInstitute of Biochemistry and Molecular Medicine, National Centre of Competence in Research NCCR TransCure, University of Bern, 3012 Bern, Switzerland; bDepartment of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zurich, 8093 Zurich, Switzerland; cDepartment of Neuroscience, Biomedical Center, Uppsala University, 751 24 Uppsala, Sweden; dBrain Institute, Universidade Federal do Rio Grande do Norte, Natal 59056- 450, Brazil; eInstitute of Physiological Chemistry, University Medical Center of the Johannes Gutenberg University Mainz, D-55099 Mainz, Germany; fCentro Universitario de Ciencias Exactas e Ingenierías, University of Guadalajara, 44430 Guadalajara, Mexico; and gDr. August Wolff GmbH & Co. KG Arzneimittel, 33611 Bielefeld, Germany Edited by Benjamin F. Cravatt, The Scripps Research Institute, La Jolla, CA, and approved May 10, 2017 (received for review March 14, 2017) The extracellular effects of the endocannabinoids anandamide and ECs over arachidonate and other N-acylethanolamines (NAEs) 2-arachidonoyl glycerol are terminated by enzymatic hydrolysis after (15–19). However, although suitable inhibitors are available for crossing cellular membranes by facilitated diffusion. The lack of potent most targets within the ECS (20), the existing AEA uptake in- and selective inhibitors for endocannabinoid transport has prevented hibitors lack potency and show poor selectivity over the other the molecular characterization of this process, thus hindering its components of the ECS, in particular FAAH (21, 22). biochemical investigation and pharmacological exploitation. Here, Given the lack of appropriate inhibitors, it is not unexpected we report the design, chemical synthesis, and biological profiling of that the process of EC cellular uptake has remained largely natural product-derived N-substituted 2,4-dodecadienamides as a selec- uncharacterized at the biochemical level and, therefore, is also = tive endocannabinoid uptake inhibitor. The highly potent (IC50 10 nM) controversially discussed (21). Here, building on previous work on inhibitor N-(3,4-dimethoxyphenyl)ethyl amide (WOBE437) exerted pro- N-alkyl-2,4-dodecadienamides from Echinacea purpurea (L.) PHARMACOLOGY nounced cannabinoid receptor-dependent anxiolytic, antiinflammatory, Moench, which have been shown to interact with the ECS (22, 23), and analgesic effects in mice by increasing endocannabinoid levels. a series of derivatives and analogs of these natural unsaturated A tailored WOBE437-derived diazirine-containing photoaffinity fatty acid amides were synthesized, and their effects on EC probe (RX-055) irreversibly blocked membrane transport of transport were investigated. This work has resulted in the both endocannabinoids, providing mechanistic insights into this com- identification of (2E,4E)-N-[2-(3,4-dimethoxyphenyl)ethyl] plex process. Moreover, RX-055 exerted site-specific anxiolytic effects dodeca-2,4-dienamide (WOBE437; 1) as a highly potent and on in situ photoactivation in the brain. This study describes suitable in- selective EC uptake inhibitor, which was extensively profiled. In hibitors to target endocannabinoid membrane trafficking and uncovers addition, we have designed and synthesized the WOBE437- an alternative endocannabinoid pharmacology. derived photoaffinity probe RX-055 (2) as a potent and irre- endocannabinoid reuptake | 2-AG | inhibitor | endocannabinoid system | versible EC uptake inhibitor, which has enabled unambiguous lipid transport insights into the uptake process. Significance he endocannabinoid system (ECS) is a pan-organ lipid sig- Tnaling network that modulates numerous biological pro- cesses, including neurotransmission and immune function (1, 2). Suitable chemical tools have been instrumental in the discov- The major endogenous agonists [i.e., endocannabinoids (ECs)] ery and characterization of the endocannabinoid system. However, the lack of potent and selective inhibitors for endo- for cannabinoid receptors CB1 and CB2 are the arachidonic acid (AA)-derived lipids 2-arachidonoyl glycerol (2-AG) and cannabinoid transport has prevented the molecular character- N-arachidonoylethanolamine [anandamide (AEA)]. Altered EC ization of this process. Current uptake inhibitors are poorly signaling in the brain has been implicated in nociception (3), bioavailable to the central nervous system (CNS) and weakly learning and memory (4), anxiety (5), and depression (6). The selective because they also inhibit fatty acid amide hydro- indirect modulation of EC levels may lead to fewer side effects lase (FAAH), the major anandamide-degrading enzyme. Few studies have addressed the uptake inhibition of 2-arachidonoyl than the direct activation of CB1 receptors in terms of neuro- transmission, metabolism, and immunomodulation (7). glycerol (2-AG), which is the major endocannabinoid. Here, we report a highly potent and selective endocannabinoid reuptake CB1 receptor agonists are intrinsically associated with strong central side effects that are far less pronounced for increasing inhibitor. Our data indicate that endocannabinoid transport EC levels upon blockage of the main EC hydrolytic enzymes fatty across the membrane can be targeted, leading to general acid amide hydrolase (FAAH) and monoacylglycerol lipase antiinflammatory and anxiolytic effects in mice. (MAGL). In addition to general antiinflammatory and analgesic Author contributions: A.C., S.N., M.B., B.L., K.-H.A., and J.G. designed research; A.C., S.N., effects, the modulation of EC tissue concentrations is a prom- R.B., M.B., A.A.R., V.P., I.d.C.R.-M., J.M.V.-P., and M.D.G. performed research; M.S., C.A., ising therapeutic approach to treat diseases related to the central and R.-P.C. contributed new reagents/analytic tools; A.C., S.N., R.B., M.B., A.A.R., V.P., nervous system (CNS) (8, 9). Pharmacological strategies to treat I.d.C.R.-M., J.M.V.-P., B.L., H.B.S., M.S., and J.G. analyzed data; and A.C., S.N., M.S., neuropsychiatric disorders currently focus on the inhibition of K.-H.A., and J.G. wrote the paper. EC degradation (10). FAAH and MAGL inhibitors such as The authors declare no conflict of interest. URB597 (11) and JZL184 (12), respectively, have been in- This article is a PNAS Direct Submission. strumental to elucidate the role of AEA and 2-AG in rodent 1A.C. and S.N. contributed equally to this work. models of anxiety and depression (6, 12–14). Although AEA and 2To whom correspondence should be addressed. Email: [email protected]. 2-AG have different intracellular fates, they may share a com- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mon mechanism of membrane trafficking that is selective for 1073/pnas.1704065114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1704065114 PNAS Early Edition | 1of10 Downloaded by guest on September 30, 2021 Results 2,691; and 1,242 nM, respectively (Fig. 1A and SI Appendix,Table Discovery of WOBE437 as a Highly Potent AEA Uptake Inhibitor. Based on S1). The most potent compounds were those with an N-phenethyl the natural product (2E,4E)-N-isobutylamidedodeca-2,4-dienamide head group, which showed IC50 values in the low nanomolar range SI Appendix (3)(SI Appendix,Fig.S1andTableS1) from the medicinal plant ( ,TableS1). Compared with the unsubstituted parent 8 = E. purpurea as a starting point, we synthesized a library of 634 ana- compound (IC50 1,142 nM), the presence of a single methoxy group at different positions of the aryl system (20, 21,and22) was logs and derivatives with varying alkyl chain lengths and structure of = SI Appendix SI Experimental associated with approximately fourfold increased potency (IC50 the head group moiety ( ,Fig.S1and – A B SI Appendix Procedures). All new derivatives were tested for AEA uptake in- 198 271 nM) (Fig. 1 and and ,TableS1). Potency SI Appendix SI could be increased dramatically by 3,4-dimethoxylation, providing the hibition in U937 cells by using a screening assay ( , 1 Experimental Procedures highly potent and selective inhibitor (WOBE437)withanIC50 value ). From a total of 348 analogs, we identified ± 2E,4E N of 10 8 nM for AEA uptake inhibition (using 100 nM total AEA) the dodeca- -dienoyl -alkylamide scaffold as the most prom- and an outstanding 1,000-fold selectivity over FAAH (Fig. 1 A and B ising framework for the development of EC transport inhibitors SI Appendix > A and ,TableS1). The structurally related benzodioxole being highly selective over FAAH ( 100-fold selectivity; Fig. 1 and (24) and dihydrobenzodioxine (25)analogswere∼13timeslesspo- B N N ). For instance, -(pyridin-3-yl)ethyl, -(2-methoxyphenyl)ethyl, tent (Fig. 1 A and B and SI Appendix,Fig.S2). Modifications in the N E E and -(2,3-dihydro-1,4-benzodioxin-6-yl)ethyl dodeca-2 ,4 - acyl part of WOBE437 (chain length and degree of unsaturation) led 14 20 25 dienamide ( , ,and , respectively) exhibited IC50 values in the to less potent analogs (SI Appendix,Fig.S3). The similarity between nanomolar range for AEA uptake and 60- to 105-fold selectivity over the head groups in WOBE437 and the minor EC N-arachidonoyl AEA hydrolysis