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How lipophilic cannabinergic ligands reach their receptor sites
Prostaglandins & other Lipid Mediators 77 (2005) 210–218
Review
How lipophilic cannabinergic ligands reach their receptor sites Alexandros Makriyannis ∗ , Xiaoyu Tian, Jianxin Guo Center for Drug Discovery, Bouve College of Health Sciences, Northeastern University, 116 Mugar Life Sciences Building, 360 Huntington Avenue, Boston, MA 02115, USA Received 10 January 2004; accepted 11 January 2004
Abstract It is postulated that lipophilic ligands reach their sites of action on membrane-bound functional proteins through fast lateral diffusion across the membrane bilayer. We have shown using NMR experiments that such ligands when incorporated in a membrane system assume a preferred orientation and conformation. While occupying a specific location within the bilayer, these molecules undergo fast lateral diffusion which allows them to engage in productive interactions with their respective protein sites of action. The proposed model is discussed using a group of classical and non-classical cannabinoids as well as the endogenous cannabinoid ligand anandamide. © 2005 Elsevier Inc. All rights reserved. Keywords: Lipophilic ligands; Drug lateral diffusion; Drug membrane interaction; Drug orientation; NMR spectroscopy
Contents 1. 2. 3.
The role of membranes in drug action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ligand–membrane interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cannabinoids and cannabinergic ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abbreviations: NMR, Nuclear magnetic resonance; 8 -THC, (−)-8 -tetrahydrocannabinol; Me-8 -THC, (−)-O-methyl-8 -tetrahydrocannabinol ∗ Corresponding author. Tel.: +1 617 373 4200. E-mail address: [email protected] (A. Makriyannis). 1098-8823/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.prostaglandins.2004.01.010
A. Makriyannis et al. / Prostaglandins & other Lipid Mediators 77 (2005) 210–218
4. 5.
Non-classical cannabinoids (NCCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The endocannabinoid anandamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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It has been postulated that lipophilic ligands, in order to reach their catalytic site in a membrane-bound functional protein, first segregate within the bilayer leaflet of a cellular membrane [1–3]. Subsequently, they undergo fast lateral diffusion, and engage in a productive interaction with their target biological site, a membrane embedded functional protein (Fig. 1). Support for such a postulate has been provided by the argument that there is an entropic advantage for a ligand–protein interaction within the two-dimensional matrix of the membrane bilayer when compared to a situation in which the ligand approaches its protein binding site from the three-dimensional space of the extracellular aqueous medium. The argument was strengthened by extensive biophysical work on the structural and dynamic features of ligand–membrane interactions using small angle X-ray diffraction, solid state NMR and differential scanning calorimetry [4–7]. More recent work demonstrating the heterogeneity of biological membranes and the presence of lipid rafts within which protein–protein and ligand–protein interactions can occur are very congruent with a mechanism involving fast lateral diffusion within the membrane leaflet [8–10].
1. The role of membranes in drug action It can be argued that biological membranes feature a general set of biochemical properties that are primarily determined by the amphipathic nature of their phospholipid bilayer structure. Their structure includes the hydrophobic core of lipid chains from each of the two
Fig. 1. Diffusion of an amphipathic ligand to its receptor binding site.
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membrane leaflets sandwiched between two polar surfaces formed by the polar phospholipid head groups. This anisotopic molecular assembly characterizes some key membrane properties including their role as specialized cellular barriers capable of modulating the functions of membrane-bound proteins and of allowing for the selective diffusion of small ligands into and out of the cell. The role of cellular membranes is further diversified through variabilities in lipid composition and cholesterol content. Such variations can impart specialized functions to different cellular assemblies and can also lead to structural differentiation between inner and outer membrane leaflets as well as formation of transient microdomains within the same cell. An important example of such microdomains is the lipid rafts which appear to play a prominent role in membrane protein di- and oligo-merization [10]. In earlier work, we have explored the role of cell membrane in drug action. To this effect, we have studied the interactions of lipophilic ligands with model membranes and sought to identify the structural features within a drug molecule responsible modifying the dynamics of this molecular assembly (membrane perturbation) [11]. We have also aimed at identifying the structural features that play a role in determining the ligands’ orientation, location and conformation within the membrane bilayer [3,12–14].
2. Ligand–membrane interactions Much of our work on the structural and dynamic features of the ligand:membrane bilayer complexes employed model systems consisting of lipophilic cannabinergic agents and multilamellar bilayers generally prepared from hydrated phosphadidylcholines. Subsequently, the general nature of our findings were confirmed by parallel experiments utilizing membrane models of different composition as well as native membranes [15]. Cannabinergic compounds are generally amphipathic in nature and carry one or more polar groups (OH or NH) attached to larger hydrophobic cyclic and/or chain-like moietes. Data from our laboratory has demonstrated that amphipathic ligands align themselves within a membrane bilayer in a preferred orientation and location as determined by the electronic and stereochemical features of the compound [4,16]. While in this location, the drug molecule undergoes fast lateral diffusion within a fixed plane parallel to the bilayer surface. We have also shown that the relative regio-and stereo-chemical arrangements of the polar groups within a molecule can play a major role in determining its conformation [3], orientation and location in the bilayer and can also significantly modulate its biological properties.
3. Cannabinoids and cannabinergic ligands (−)-9 -Tetrahydrocannabinol (9 -THC), the most recognized biologically active ingredient of cannabis is a terpenoid tricyclic compound with a phenolic hydroxyl at the 1-position and a five carbon-long side chain at the 3-position of phenyl ring A. The 3-alkyl substitution which characterizes all cannabinoids appears to be a key pharmacophoric feature within this class of analogs and variations in the nature of these substitutes can lead to large differences in the potencies of individual analogs.
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Fig. 2. Classical cannabinoids align with their polar groups facing the polar side of the membrane bilayer.
Using a combination of stationary 2 H-solid state NMR and small angle X-ray diffraction, we studied the interactions of 9 -THC with membrane preparations [4]. We showed that, within the amphipathic environment of the bilayer, 9 -THC preferentially orients with the long axis of its tricyclic ring structure perpendicular to the membrane surface and the phenolic hydroxyl near the bilayer interface. This awkward orientation allows the amphipathic ligand to align its polar phenolic group towards the respective polar side of the membrane in order to undergo a hydrogen bonding interaction at the bilayer interface. This “counter intuitive” orientation of 9 -THC was shown to hold true within a wide range of membrane preparations including native synaptosomal membranes. The thermodynamic incentive for such an orientation is obtained from the reciprocal interactions between the polar and hydrophobic components of the ligand and the bilayer. On the other hand the ligand’s n-pently side chain aligns parallel with the membrane phospholipids chain. Such an orientation is accomplished when the first methylene segment of the chain assumes a cisconformation with the remaining segments existing in an extended all-trans conformation
We have further demonstrated that cannabinoids with two polar hydroxyl groups within the tri cyclic cannabinoid structure, such as the two diastereomeric (−)-11hydroxyhexahydrocannabinols align within the membrane in a manner that allows both hydroxyl groups to face the polar membrane interface [17] (Fig. 2).
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Fig. 3. Non-classical cannabinoids prefer a conformation in which all of their polar hydroxyl groups face the polar side of the bilayer and engage in hydrogen bonding interactions at the phospholipid bilayer interface.
4. Non-classical cannabinoids (NCCs) In this later generation of cannabinergic ligands developed by Pfizer, the middle tetrahydropyran ring of the tricyclic ring structure of classical cannabinoids is removed allowing for greater flexibility within the molecule. Also, the most potent members of this class of compounds carry an additional hydroxyl group attached to the C-ring through a carbon chain or a carbocyclic ring. This novel pharmacophore generally designated as southern aliphatic hydroxyl (SAH) exhibits well defined sterochemical requirements for cannabinergic activity. To develop an understanding on the pharmacophoric requirements in this class of compounds, we studied the conformational properties of three representative analogs (Fig. 3) of progressive structural complexity and correspondingly enhanced potency using NMR and molecular modelling [3]. CP-47497 is the NCC prototype with the most simplified structure while CP-55940 carries an additional beta hydroxypropyl group (SAH) on the C-ring. CP-55244 is the most structurally elaborate analog in the class and has the hydroxypropyl group of CP55940 is conformationally restricted by means of a third cycloxexyl ring.
Our work has demonstrated that all the three analogs share some common preferred sterochemical features in hydrophobic and amphipathic media where the A-ring is approximately perpendicular to the C-ring and the phenolic proton points away from the C-ring.
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In a membrane environment, the dimethylheptyl side chain adopts a conformation almost perpendicular to the plane of the aromatic ring and parallel to the bilayer chains while the beta 9-hydroxyl group (NAH) and the phenolic hydroxyl orient towards the polar surface of the bilayer. Our studies also demonstrated that in a membrane environment both CP-55940 and CP55244 prefer a conformation in which the southern aliphatic hydroxyl also orients towards the polar side of the bilayer (Makriyannis and Xie, unpublished data). Thus, all three analogs when placed in the amphipathic environment align themselves in a manner which allows for an optimal interaction of their polar groups with the membrane polar surface while all hydrophobic components are embedded within the membrane’s hydrophobic core (Fig. 3). Notably, CP-97587 the epimer of CP-55244, the SAH pharmacophore is conformationally prohibited from optimizing its interaction with the polar membrane surface, a property which is reflected in its lower potency. The conformational properties of the three potent NCC analogs described above, support a postulate according to which these compounds incorporate into biological membranes with all their polar hydroxyl facing the polar surface. Within the bilayer, the NCCs engage in a fast lateral diffusion an approach the cannabinoid receptor in an orientation which is highly
Fig. 4. The potent non-classical cannabinoid CP-55244 orients with all its hydroxyl groups facing the membrane interface and undergoes in fast lateral diffusion in order to reach its binding site on the receptor.
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favorable for receptor recognition and a productive collision with their respective binding sites (Fig. 4).
5. The endocannabinoid anandamide N-arachidonoylethanolamine (anandamide) was initially isolated from mammalian brain and shown to be an endogenous ligand for the cannabinoid receptors [18]. This lipophilic molecule is not stored in a cellular compartment but is produced upon demand from cell membrane phospholipid components through the consecutive actions of membrane-bound enzymes. Anandamide produces its effects by interacting with the cannabinoid receptors (CB1, CB2) and is, subsequently, deactivated through an intracellular transport [19] process followed by enzymatic degradation [20,21]. Anandamide can also be released following the activation of a number of post-synaptic receptors [19]. Upon its release, this endocannabinoid modulator is believed to be carried by specialized carrier proteins and
Fig. 5. The endocannabinoid anandamide acquires an extended conformation in the bilayer with its polar group at the same level as the polar phospholipid head groups. It reaches the CB1 receptor by fast lateral diffusion and interacts with a hydrophobic groove in helix 6.
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allowed to interact with the cannabinoid receptors that are found in presynaptic nerve terminals.
It can, thus, be argued that anandamide engages the cannabinoid receptors through a fast lateral diffusion within the cellular membrane leaflet. For this reason, information on its conformational and dynamic properties within the membrane can provide useful insights on the nature of anandamide–cannabinoid receptor interactions. Earlier computational work has shown that anandamide can adopt any of three general conformations designated as hairpin (U-shaped). J-shaped and extended [22]. To obtain experimental information on the preferred conformation of anandamide in a membrane environment, we carried out solid-state NMR experiments (Makriyannis and Tian, unpublished results). Using rotational echo double resonance (REDOR) we were able to accurately measure intra- or intermolecular distances between specific 13 C, 2 H and 15 N labels that were strategically introduced in both the anandamide ligand and the membrane phospholipids. The results indicated that in membrane systems anandamide preferentially adopts an extended conformation with its headgroup near the lipid–water interface and the end of its fatty acid tail near the bilayer center. (Fig. 5). The findings support our initial postulate according to which anandamide predominantly resides within the membrane bilayers where it undergoes fast lateral diffusion. This hypothesis is congruent with data from our laboratory suggesting that the terminal five-carbon chain of anandamide interacts with the CB1 cannabinoid receptor through a hydrophobic groove involving hydrophobic residues V6.43 and I6.46 of helix 6 and situated at the level of the bilayer center (Makriyannis and Picone, unpublished results). Arguably such an extended anandamide conformation may facilitate its interaction with the CB1 receptor.
References [1] Rhodes DG, Sarmiento JG, Herbette LG. Kinetics of binding of membrane-active drugs to receptor sites. Diffusion-limited rates for a membrane bilayer approach of 1,4-dihydropyridine calcium channel antagonists to their active site. Mol Pharmacol 1985;27:612–23. [2] Makriyannis A, Banijamali A, Van der Schyf C, Jarrell H. Interactions of cannabinoids with membranes. The role of cannabinoid stereochemistry and absolute configuration and the orientation of -9-THC in the membrane bilayer. NIDA Res Monogr 1987;79:123–33. [3] Xie XQ, Melvin LS, Makriyannis A. The conformational properties of the highly selective cannabinoid receptor ligand CP-55,940. J Biol Chem 1996;271:10640–7. [4] Makriyannis A, Banijamali A, Jarrell HC, Yang DP. The orientation of (−)- 9-tetrahydrocannabinol in DPPC bilayers as determined from solid-state 2 H NMR. Biochim Biophys Acta 1989;986:141–5. [5] Makriyannis A, Yang DP, Griffin RG, Das Gupta SK. The perturbation of model membranes by (−)- 9-tetrahydrocannabinol. Studies using solid-state 2 H and 13 C NMR. Biochim Biophys Acta 1990;1028: 31–42. [6] Mavromoustakos T, Yang DP, Charalambous A, Herbette LG, Makriyannis A. Study of the topography of cannabinoids in model membranes using X-ray diffraction. Biochim Biophys Acta 1990;1024:336–44.
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[7] Mavromoustakos T, Yang DP, Makriyannis A. Topography and thermotropic properties of cannabinoids in brain sphingomyelin bilayers. Life Sci 1996;59:1969–79. [8] Lai EC. Lipid rafts make for slippery platforms. J Cell Biol 2003;162:365–70. [9] Edidin M. The state of lipid rafts: from model membranes to cells. Annu Rev Biophys Biomol Struct 2003;32:257–83. [10] Wollscheid B, von Haller PD, Yi E, et al. Lipid raft proteins and their identification in T lymphocytes. Subcell Biochem 2004;37:121–52. [11] Makriyannis A, Yang DP, Mavromoustakos T. Combined use of solid-state nuclear magnetic resonance spectroscopy, small-angle X-ray diffraction, and differential scanning calorimetry in studies of cannabinoid: membrane interactions. NIDA Res Monogr 1991;112:106–28. [12] Yang DP, Mavromoustakos T, Makriyannis A. Small angle X-ray diffraction studies of (−)- 8tetrahydrocannabinol and its O-methyl analog in membranes. Life Sci 1993;53:PL117–22. [13] Mavromoustakos T, Yang DP, Makriyannis A. Small angle X-ray diffraction and differential scanning calorimetric studies on O-methyl-(−)- 8-tetrahydrocannabinol and its 5 iodinated derivative in membrane bilayers. Biochim Biophys Acta 1995;1237:183–8. [14] Guo J, Pavlopoulos S, Tian X, et al. Conformational study of lipophilic ligands in phospholipid model membrane systems by solution NMR. J Med Chem 2003;46:4838–46. [15] Mavromoustakos T, Yang DP, Broderick W, Fournier D, Makriyannis A. Small angle X-ray diffraction studies on the topography of cannabinoids in synaptic plasma membranes. Pharmacol Biochem Behav 1991;40:547–52. [16] Xie XQ, Pavlopoulos S, DiMeglio CM, Makriyannis A. Conformational studies on a diastereoisomeric pair of tricyclic nonclassical cannabinoids by NMR spectroscopy and computer molecular modeling. J Med Chem 1998;41:167–74. [17] Yang DP, Banijamali A, Charalambous A, Marciniak G, Makriyannis A. Solid state 2 H NMR as a method for determining the orientation of cannabinoid analogs in membranes. Pharmacol Biochem Behav 1991;40:553–7. [18] Devane WA, Hanus L, Breuer A, et al. Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 1992;258:1946–9. [19] Beltramo M, Stella N, Calignano A, Lin SY, Makriyannis A, Piomelli D. Functional role of high-affinity anandamide transport, as revealed by selective inhibition. Science 1997;277:1094–7. [20] Deutsch DG, Ueda N, Yamamoto S. The fatty acid amide hydrolase (FAAH). Prostaglandins Leukot Essent Fatty Acids 2002;66:201–10. [21] Piomelli D, Beltramo M, Glasnapp S, et al. Structural determinants for recognition and translocation by the anandamide transporter. Proc Natl Acad Sci USA 1999;96:5802–7. [22] Barnett-Norris J, Guarnieri F, Hurst DP, Reggio PH. Exploration of biologically relevant conformations of anandamide, 2-arachidonylglycerol, and their analogues using conformational memories. J Med Chem 1998;41:4861–72.
Review
How lipophilic cannabinergic ligands reach their receptor sites Alexandros Makriyannis ∗ , Xiaoyu Tian, Jianxin Guo Center for Drug Discovery, Bouve College of Health Sciences, Northeastern University, 116 Mugar Life Sciences Building, 360 Huntington Avenue, Boston, MA 02115, USA Received 10 January 2004; accepted 11 January 2004
Abstract It is postulated that lipophilic ligands reach their sites of action on membrane-bound functional proteins through fast lateral diffusion across the membrane bilayer. We have shown using NMR experiments that such ligands when incorporated in a membrane system assume a preferred orientation and conformation. While occupying a specific location within the bilayer, these molecules undergo fast lateral diffusion which allows them to engage in productive interactions with their respective protein sites of action. The proposed model is discussed using a group of classical and non-classical cannabinoids as well as the endogenous cannabinoid ligand anandamide. © 2005 Elsevier Inc. All rights reserved. Keywords: Lipophilic ligands; Drug lateral diffusion; Drug membrane interaction; Drug orientation; NMR spectroscopy
Contents 1. 2. 3.
The role of membranes in drug action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ligand–membrane interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cannabinoids and cannabinergic ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
211 212 212
Abbreviations: NMR, Nuclear magnetic resonance; 8 -THC, (−)-8 -tetrahydrocannabinol; Me-8 -THC, (−)-O-methyl-8 -tetrahydrocannabinol ∗ Corresponding author. Tel.: +1 617 373 4200. E-mail address: [email protected] (A. Makriyannis). 1098-8823/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.prostaglandins.2004.01.010
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4. 5.
Non-classical cannabinoids (NCCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The endocannabinoid anandamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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It has been postulated that lipophilic ligands, in order to reach their catalytic site in a membrane-bound functional protein, first segregate within the bilayer leaflet of a cellular membrane [1–3]. Subsequently, they undergo fast lateral diffusion, and engage in a productive interaction with their target biological site, a membrane embedded functional protein (Fig. 1). Support for such a postulate has been provided by the argument that there is an entropic advantage for a ligand–protein interaction within the two-dimensional matrix of the membrane bilayer when compared to a situation in which the ligand approaches its protein binding site from the three-dimensional space of the extracellular aqueous medium. The argument was strengthened by extensive biophysical work on the structural and dynamic features of ligand–membrane interactions using small angle X-ray diffraction, solid state NMR and differential scanning calorimetry [4–7]. More recent work demonstrating the heterogeneity of biological membranes and the presence of lipid rafts within which protein–protein and ligand–protein interactions can occur are very congruent with a mechanism involving fast lateral diffusion within the membrane leaflet [8–10].
1. The role of membranes in drug action It can be argued that biological membranes feature a general set of biochemical properties that are primarily determined by the amphipathic nature of their phospholipid bilayer structure. Their structure includes the hydrophobic core of lipid chains from each of the two
Fig. 1. Diffusion of an amphipathic ligand to its receptor binding site.
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membrane leaflets sandwiched between two polar surfaces formed by the polar phospholipid head groups. This anisotopic molecular assembly characterizes some key membrane properties including their role as specialized cellular barriers capable of modulating the functions of membrane-bound proteins and of allowing for the selective diffusion of small ligands into and out of the cell. The role of cellular membranes is further diversified through variabilities in lipid composition and cholesterol content. Such variations can impart specialized functions to different cellular assemblies and can also lead to structural differentiation between inner and outer membrane leaflets as well as formation of transient microdomains within the same cell. An important example of such microdomains is the lipid rafts which appear to play a prominent role in membrane protein di- and oligo-merization [10]. In earlier work, we have explored the role of cell membrane in drug action. To this effect, we have studied the interactions of lipophilic ligands with model membranes and sought to identify the structural features within a drug molecule responsible modifying the dynamics of this molecular assembly (membrane perturbation) [11]. We have also aimed at identifying the structural features that play a role in determining the ligands’ orientation, location and conformation within the membrane bilayer [3,12–14].
2. Ligand–membrane interactions Much of our work on the structural and dynamic features of the ligand:membrane bilayer complexes employed model systems consisting of lipophilic cannabinergic agents and multilamellar bilayers generally prepared from hydrated phosphadidylcholines. Subsequently, the general nature of our findings were confirmed by parallel experiments utilizing membrane models of different composition as well as native membranes [15]. Cannabinergic compounds are generally amphipathic in nature and carry one or more polar groups (OH or NH) attached to larger hydrophobic cyclic and/or chain-like moietes. Data from our laboratory has demonstrated that amphipathic ligands align themselves within a membrane bilayer in a preferred orientation and location as determined by the electronic and stereochemical features of the compound [4,16]. While in this location, the drug molecule undergoes fast lateral diffusion within a fixed plane parallel to the bilayer surface. We have also shown that the relative regio-and stereo-chemical arrangements of the polar groups within a molecule can play a major role in determining its conformation [3], orientation and location in the bilayer and can also significantly modulate its biological properties.
3. Cannabinoids and cannabinergic ligands (−)-9 -Tetrahydrocannabinol (9 -THC), the most recognized biologically active ingredient of cannabis is a terpenoid tricyclic compound with a phenolic hydroxyl at the 1-position and a five carbon-long side chain at the 3-position of phenyl ring A. The 3-alkyl substitution which characterizes all cannabinoids appears to be a key pharmacophoric feature within this class of analogs and variations in the nature of these substitutes can lead to large differences in the potencies of individual analogs.
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Fig. 2. Classical cannabinoids align with their polar groups facing the polar side of the membrane bilayer.
Using a combination of stationary 2 H-solid state NMR and small angle X-ray diffraction, we studied the interactions of 9 -THC with membrane preparations [4]. We showed that, within the amphipathic environment of the bilayer, 9 -THC preferentially orients with the long axis of its tricyclic ring structure perpendicular to the membrane surface and the phenolic hydroxyl near the bilayer interface. This awkward orientation allows the amphipathic ligand to align its polar phenolic group towards the respective polar side of the membrane in order to undergo a hydrogen bonding interaction at the bilayer interface. This “counter intuitive” orientation of 9 -THC was shown to hold true within a wide range of membrane preparations including native synaptosomal membranes. The thermodynamic incentive for such an orientation is obtained from the reciprocal interactions between the polar and hydrophobic components of the ligand and the bilayer. On the other hand the ligand’s n-pently side chain aligns parallel with the membrane phospholipids chain. Such an orientation is accomplished when the first methylene segment of the chain assumes a cisconformation with the remaining segments existing in an extended all-trans conformation
We have further demonstrated that cannabinoids with two polar hydroxyl groups within the tri cyclic cannabinoid structure, such as the two diastereomeric (−)-11hydroxyhexahydrocannabinols align within the membrane in a manner that allows both hydroxyl groups to face the polar membrane interface [17] (Fig. 2).
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Fig. 3. Non-classical cannabinoids prefer a conformation in which all of their polar hydroxyl groups face the polar side of the bilayer and engage in hydrogen bonding interactions at the phospholipid bilayer interface.
4. Non-classical cannabinoids (NCCs) In this later generation of cannabinergic ligands developed by Pfizer, the middle tetrahydropyran ring of the tricyclic ring structure of classical cannabinoids is removed allowing for greater flexibility within the molecule. Also, the most potent members of this class of compounds carry an additional hydroxyl group attached to the C-ring through a carbon chain or a carbocyclic ring. This novel pharmacophore generally designated as southern aliphatic hydroxyl (SAH) exhibits well defined sterochemical requirements for cannabinergic activity. To develop an understanding on the pharmacophoric requirements in this class of compounds, we studied the conformational properties of three representative analogs (Fig. 3) of progressive structural complexity and correspondingly enhanced potency using NMR and molecular modelling [3]. CP-47497 is the NCC prototype with the most simplified structure while CP-55940 carries an additional beta hydroxypropyl group (SAH) on the C-ring. CP-55244 is the most structurally elaborate analog in the class and has the hydroxypropyl group of CP55940 is conformationally restricted by means of a third cycloxexyl ring.
Our work has demonstrated that all the three analogs share some common preferred sterochemical features in hydrophobic and amphipathic media where the A-ring is approximately perpendicular to the C-ring and the phenolic proton points away from the C-ring.
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In a membrane environment, the dimethylheptyl side chain adopts a conformation almost perpendicular to the plane of the aromatic ring and parallel to the bilayer chains while the beta 9-hydroxyl group (NAH) and the phenolic hydroxyl orient towards the polar surface of the bilayer. Our studies also demonstrated that in a membrane environment both CP-55940 and CP55244 prefer a conformation in which the southern aliphatic hydroxyl also orients towards the polar side of the bilayer (Makriyannis and Xie, unpublished data). Thus, all three analogs when placed in the amphipathic environment align themselves in a manner which allows for an optimal interaction of their polar groups with the membrane polar surface while all hydrophobic components are embedded within the membrane’s hydrophobic core (Fig. 3). Notably, CP-97587 the epimer of CP-55244, the SAH pharmacophore is conformationally prohibited from optimizing its interaction with the polar membrane surface, a property which is reflected in its lower potency. The conformational properties of the three potent NCC analogs described above, support a postulate according to which these compounds incorporate into biological membranes with all their polar hydroxyl facing the polar surface. Within the bilayer, the NCCs engage in a fast lateral diffusion an approach the cannabinoid receptor in an orientation which is highly
Fig. 4. The potent non-classical cannabinoid CP-55244 orients with all its hydroxyl groups facing the membrane interface and undergoes in fast lateral diffusion in order to reach its binding site on the receptor.
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favorable for receptor recognition and a productive collision with their respective binding sites (Fig. 4).
5. The endocannabinoid anandamide N-arachidonoylethanolamine (anandamide) was initially isolated from mammalian brain and shown to be an endogenous ligand for the cannabinoid receptors [18]. This lipophilic molecule is not stored in a cellular compartment but is produced upon demand from cell membrane phospholipid components through the consecutive actions of membrane-bound enzymes. Anandamide produces its effects by interacting with the cannabinoid receptors (CB1, CB2) and is, subsequently, deactivated through an intracellular transport [19] process followed by enzymatic degradation [20,21]. Anandamide can also be released following the activation of a number of post-synaptic receptors [19]. Upon its release, this endocannabinoid modulator is believed to be carried by specialized carrier proteins and
Fig. 5. The endocannabinoid anandamide acquires an extended conformation in the bilayer with its polar group at the same level as the polar phospholipid head groups. It reaches the CB1 receptor by fast lateral diffusion and interacts with a hydrophobic groove in helix 6.
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allowed to interact with the cannabinoid receptors that are found in presynaptic nerve terminals.
It can, thus, be argued that anandamide engages the cannabinoid receptors through a fast lateral diffusion within the cellular membrane leaflet. For this reason, information on its conformational and dynamic properties within the membrane can provide useful insights on the nature of anandamide–cannabinoid receptor interactions. Earlier computational work has shown that anandamide can adopt any of three general conformations designated as hairpin (U-shaped). J-shaped and extended [22]. To obtain experimental information on the preferred conformation of anandamide in a membrane environment, we carried out solid-state NMR experiments (Makriyannis and Tian, unpublished results). Using rotational echo double resonance (REDOR) we were able to accurately measure intra- or intermolecular distances between specific 13 C, 2 H and 15 N labels that were strategically introduced in both the anandamide ligand and the membrane phospholipids. The results indicated that in membrane systems anandamide preferentially adopts an extended conformation with its headgroup near the lipid–water interface and the end of its fatty acid tail near the bilayer center. (Fig. 5). The findings support our initial postulate according to which anandamide predominantly resides within the membrane bilayers where it undergoes fast lateral diffusion. This hypothesis is congruent with data from our laboratory suggesting that the terminal five-carbon chain of anandamide interacts with the CB1 cannabinoid receptor through a hydrophobic groove involving hydrophobic residues V6.43 and I6.46 of helix 6 and situated at the level of the bilayer center (Makriyannis and Picone, unpublished results). Arguably such an extended anandamide conformation may facilitate its interaction with the CB1 receptor.
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