Structural Determinants for Recognition and Translocation by the Anandamide Transporter
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Proc. Natl. Acad. Sci. USA Vol. 96, pp. 5802–5807, May 1999 Pharmacology Structural determinants for recognition and translocation by the anandamide transporter D. PIOMELLI*†‡,M.BELTRAMO*, S. GLASNAPP*, S. Y. LIN§,A.GOUTOPOULOS§,XIANG-QUN XIE§, AND A. MAKRIYANNIS‡§ *The Neurosciences Institute, San Diego, CA 92121; †Department of Pharmacology, University of California, Irvine, CA 92697; and §Departments of Pharmaceutical Sciences and Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269 Communicated by L. L. Iversen, University of Oxford, Oxford, United Kingdom, March 16, 1999 (received for review December 4, 1998) ABSTRACT The biological actions of anandamide There is considerable experimental and medical interest in (arachidonylethanolamide), an endogenous cannabinoid understanding the mechanism of anandamide transport and in lipid, are terminated by a two-step inactivation process con- developing pharmacological agents that selectively interfere sisting of carrier-mediated uptake and intracellular hydroly- with it. Anandamide transport inhibitors may be used as sis. Anandamide uptake in neurons and astrocytes is medi- experimental tools to reveal the possible physiological func- ated by a high-affinity, Na1-independent transporter that is tions of this biologically active lipid. Many of these functions selectively inhibited by N-(4-hydroxyphenyl)-arachidonamide are still elusive despite a growing body of evidence suggesting (AM404). In the present study, we examined the structural that the endocannabinoid system is intrinsically active not only determinants governing recognition and translocation of sub- in brain and spinal cord (for review, see ref. 11), but also in strates by the anandamide transporter constitutively ex- peripheral tissues (12–14). Furthermore, anandamide trans- pressed in a human astrocytoma cell line. Competition ex- port inhibitors may offer a rational therapeutic approach to a periments with a select group of analogs suggest that sub- variety of disease states, including pain (13, 14), psychomotor strate recognition by the transporter is favored by a polar disorders (15), and multiple sclerosis (16–18), in which eleva- nonionizable head group of defined stereochemical configu- tion of native anandamide levels may bring about a more ration containing a hydroxyl moiety at its distal end. The favorable response and fewer side effects than direct activation of CB1 receptors by agonist drugs. secondary carboxamide group interacts favorably with the In our search for selective inhibitors of anandamide trans- transporter, but may be replaced with either a tertiary amide port, we found that the compound N-(4-hydroxyphenyl)- or an ester, suggesting that it may serve as hydrogen acceptor. arachidonamide (AM404) prevents [3H]anandamide uptake in Thus, 2-arachidonylglycerol, a putative endogenous cannabi- vitro and enhances various effects of anandamide administra- noid ester, also may serve as a substrate for the transporter. tion both in vitro and in vivo (4, 19). These results, together with Substrate recognition requires the presence of at least one cis data from initial experiments on the selectivity of [3H]anan- double bond situated at the middle of the fatty acid carbon damide uptake by rat brain astrocytes (4), suggest that the chain, indicating a preference for ligands whose hydrophobic interactions of anandamide with its putative transporter pro- tail can adopt a bent U-shaped conformation. On the other tein are governed by strict structural requirements. In the hand, uptake experiments with radioactively labeled sub- present study we have used a human astrocytoma cell line to strates show that no fewer than four cis nonconjugated double conduct a systematic investigation on the structure-activity bonds are required for optimal translocation across the cell relationship of substrates and inhibitors of anandamide trans- membrane, suggesting that substrates are transported in a port. Our results delineate the broad molecular requisites for folded hairpin conformation. These results outline the general this process, thus providing a basis for the design of more structural requisites for anandamide transport and may potent and selective inhibitors with potential applications to assist in the development of selective inhibitors with potential medicine. clinical applications. MATERIALS AND METHODS When administered as a drug, the endogenous cannabinoid anandamide (1, 2) elicits a spectrum of pharmacological Chemistry. All amides were synthesized by the reaction of responses that closely resemble those of D9-tetrahydrocannab- the fatty acid or fatty acid chloride with the appropriate amine inol, the main psychoactive constituent of marijuana (3). or aminoalcohol as described (20). 1- and 2-Arachidonylglyc- Unlike D9-tetrahydrocannabinol, however, anandamide has a erols were prepared by a modification of the procedure established by Serdarevich and Carroll (21) for the synthesis of very short duration of action because of a rapid inactivation fatty acid monoglycerides. Briefly, 1,3-O-benzylidine-sn- process consisting of carrier-mediated transport into cells glycerol, prepared by the reaction of glycerol with benzalde- followed by intracellular hydrolysis. Indeed, rat brain neurons hyde in the presence of p-toluenesulfonic acid, was allowed to and astrocytes in primary culture avidly take up radioactively react with arachidonyl chloride in the presence of pyridine. labeled anandamide through a mechanism that meets four key Subsequent treatment with boric acid in triethylborate to criteria of a carrier-mediated transport: temperature depen- remove the benzylidine moiety gave 2-arachidonylglycerol. For dence, high affinity, substrate selectivity, and saturation (2, 4, the preparation of 1(3)-arachidonylglycerol, commercially 5). Within cells, anandamide is hydrolyzed to arachidonic acid available 1,2-O-isopropylidene-sn-glycerol was esterified with and ethanolamine by a membrane-bound amidohydrolase that arachidonic acid in a similar manner, followed by removal of also reacts with other fatty acid amides and esters (6–10). the isopropylidene group by treatment with bromodimethyl- borane (22). The synthesis of compounds 32–34 will be de- The publication costs of this article were defrayed in part by page charge scribed in detail elsewhere. Radioactively labeled fatty acid payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. ‡To whom reprint requests should be addressed. e-mail: PNAS is available online at www.pnas.org. [email protected] or [email protected]. 5802 Downloaded by guest on September 24, 2021 Pharmacology: Piomelli et al. Proc. Natl. Acad. Sci. USA 96 (1999) 5803 ethanolamides were prepared by the reaction of acid chlorides from the Sybyl package. Charges were calculated for all (Nu-Check Prep, Elysian, MN) with [3H]ethanolamine (10–30 molecules by the semiempirical method (MOPAyAM1), and Ciymmol; American Radiolabeled Chemicals, St. Louis) as energies were minimized by using the Tripos force field in two described (6). All compounds were purified by HPLC or flash stages. First, the steepest descent method was applied for the column chromatography, and their identities were established first 200 steps, followed by the conjugate gradient method until by NMR andyor gas chromatography-mass spectrometry. Ad- the maximum derivative was less than 0.001 kcalymole per Å. ditional compounds were purchased from Avanti Polar Lipids, The conformation of anandamide was refined by using the Cayman Chemicals (Ann Arbor, MI), Nu-Check Prep, Re- Tripos random search module on the six rotatable single bonds search Biochemicals, or Sigma. within the group of four nonconjugated cis double bonds. [3H]Anandamide Competition Assay. Human CCF-STTG1 Conformer A depicted in Fig. 4 has a hairpin conformation astrocytoma cells (American Type Culture Collection) were similar to that described by others for anandamide (23) and grown in RPMI 1640 culture medium containing 10% FBS and arachidonic acid (24). The preferred conformers of all anan- 1 mM glutamine. For standard competition assays, confluent damide analogs were generated from A by appropriate struc- cells grown in 24-well plates were rinsed and preincubated for tural modifications followed by minimization. 10 min at 37°C in Tris-Krebs’ buffer (136 mM NaCly5mM y y y KCl 1.2 mM MgCl2-6H20 2.5 mM CaCl2-2H20 10 mM glu- cosey20 mM Trizma base) containing 0.1–0.3% DMSO or RESULTS 0.1–0.3% DMSO plus test compounds at their final concen- [3H]Anandamide Transport in Astrocytoma Cells. As ex- trations (0.1–100 mM). After having discarded the preincuba- pected of a carrier-mediated process, [3H]anandamide accu- 5 tion media, the cells were incubated for 4 min in 0.4 ml of mulation in human astrocytoma cells is rapid (t1/2 3 min), Tris-Krebs’ buffer containing 30 nM [3H]anandamide (220 temperature dependent, and saturable, displaying an apparent y 6 m Ci mmol, New England Nuclear) and 0.1–0.3% DMSO, or Michaelis constant (Km)of0.6 0.1 M and a maximal 6 y 0.1–0.3% DMSO plus test compounds. Reactions were accumulation rate (Vmax) of 14.7 01.5 pmol min per mg of stopped by removing the incubation media and rinsing the cells protein (n 5 5). The accumulation is not affected by replace- three times with 0.4 ml of ice-cold Tris-Krebs’ buffer contain- ment of Na1 with choline, suggesting that it is mediated by a ing 0.1% fatty acid-free BSA (Sigma). Radioactive material in