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Canna~Fangled Abstracts

2012 Division of Medicinal Chemistry Award Address: Trekking the Cannabinoid Road: A Personal Perspective.

By April 7, 2014No Comments
 2014 Apr 7. [Epub ahead of print]
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pm8Published in final edited form as:

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J Med Chem. Author manuscript; available in PMC Jun 20, 2014.
 

Published online May 1, 2014. doi:  10.1021/jm500220s

PMCID: PMC4064474
NIHMSID: NIHMS587463

2012 Division of Medicinal Chemistry Award Address: Trekking the Cannabinoid Road: A Personal Perspective

Abstract

My involvement with the field of cannabinoids spans close to three decades, and covers a major part of my scientific career. It also reflects the robust progress in this initially largely unexplored area of biology. During this period of time, I have witnessed the growth of modern cannabinoid biology, starting from the discovery of its two receptors and followed by the characterization of its endogenous ligands and the identification of the enzyme systems involved in their biosynthesis and biotransformation. I was fortunate enough to start at the beginning of this new era and participate in a number of the new discoveries. It has been a very exciting journey. By covering some key aspects of my work during this period of “modern cannabinoid research,” this perspective, in part historical, intends to give an account of how the field grew, the key discoveries, and the most promising directions for the future.

History

On October 30-31, 1986, the National Institute on Drug Abuse organized a “Technical Review” event in Building 1 of the National Institutes of Health (NIH) campus in Bethesda, MD, whose purpose was to bring together scientists involved in the field of cannabinoids to discuss the most current research efforts. I had just received my first cannabinoid grant and was invited by its organizer, Dr. Rao Rapaka, who had recently joined the extramural program of the National Institute on Drug Abuse, to help put together such an event. It was generally felt that scientific activity in this important aspect of drug abuse was at a low point and such an event may help energize the field.

By all accounts, this effort by the National Institute on Drug Abuse (NIDA) was very successful in achieving its goals. Publication of the conference proceedings217 was followed by a number of key discoveries, including the identification, cloning, characterization and imaging of the CB1 receptor; the discovery of the key endocannabinoids, anandamide and 2-arachidonylglycerol, and the design and synthesis of novel ligands that enabled the elucidation of the cannabinoid biochemical system and established its major role in mammalian physiology. It is now universally recognized that cannabinoids are a very active research area. Also, because of its pleiotropic nature and its drug-friendly targets, the endocannabinoid system has excellent prospects in serving as a basis for drug discovery.

The three decades preceding this conference had witnessed a great deal of exciting work aimed at accessing the therapeutic properties of cannabis and its ingredients and developing novel therapeutic medications. This involved the development of new chemistries for the synthesis of terpenoid analogs with structural similarities to cannabis’ endogenous constituents. The effort was led by Alexander Todd from University of Manchester35 and Roger Adams from the Noyes Chemical Laboratory at the University of Illinois, Urbana-Champaign,37 and produced new molecules with pronounced physiological effects when tested in different animal species. A major boost to the field was the isolation of the key bioactive constituent of cannabis and its subsequent synthesis by Raphael Mechoulam in Israel.3839 This gummy non-crystallizable compound, which was identified as a tricyclic terpene encompassing a middle pyran ring, a phenolic hydroxyl, and a linear 3-pentyl side chain attached to the aromatic ring, was named (-)-Δ1-tetrahydrocannabinol and later renamed (-)-Δ9-tetrahydrocannabinol (Δ9-THC). Its structure served as a prototype for additional synthetic efforts by a number of academic laboratories, including the Mechoulam, as well as the Razdan and Pars laboratories in Cambridge, MA.40

The above efforts were paralleled by significant programs within the pharmaceutical industry to develop cannabinoid-based medications principally as non-opioid effective analgesic agents. Notably, Lilly’s efforts had led to the synthesis and development of the drug Nabilone,41 which has been used by patients receiving cancer chemotherapy. Also, companies such as Abbott and Arthur D. Little Inc. were developing nitrogen containing analogs (eg. Nabitan) that were deemed to have more drug-like properties.40 One of the major programs was undertaken at Pfizer in Groton, Connecticut. The effort for the discovery of cannabinoid analgesics was led by two talented medicinal chemists, Larry Melvin and Ross Johnson, whose work led to their first clinical candidate, Levonantradol,42 a compound that was less lipophilic than the key phytocannabinoid Δ9-THC, and also was 10- to 100-fold more potent in analgesia tests. In their systematic SAR, they had developed a series of analogs lacking the middle ring of the tricyclic terpenoid structure which they named non-classical cannabinoids,17 the most prominent of which was CP-55940,17 a 3β-hydroxycyclohexane phenol in which the 3-pentyl chain of Δ9-THC was substituted with a 1’,1’-dimethylheptyl chain.

Melvin and Johnson were among the participants of this historic 1986 event, where they described the detailed SAR obtained from testing the non-classical cannabinoids for their analgesic effects.17 Their results underscored the remarkable correlation between analgesic potency with their respective absolute and relative stereochemistries,17 as well as subtle structural modifications. The results argued for the existence of a specific site of interaction through which the new cannabinoids were producing their effects. Coincidentally, an interesting presentation during the meeting was from a young investigator, Allyn Howlett,17who had been testing some of the phytocannabinoids for their effects on modulating the levels of cAMP. She showed that the most biologically-active of these compounds, including Δ9-THC, dose-dependently reduced the levels of cAMP, suggesting an inhibitory effect on the enzyme adenylyl cyclase. During that meeting, the Pfizer scientists offered to develop a tritiated analog from their non-classical cannabinoid series, which they provided to Howlett’s laboratory. She and her graduate student William Devane tested a variety of synthetic cannabinoid analogs, in a heterologous displacement assay, using the non-classical Pfizer analog CP55940 as the radioligand, and obtained high quality sigmoid curves that accurately reflected the in vivo potencies of the compounds.43

These successful results were immediately followed by the autoradiographic imaging of the putative receptor in rat brain by Miles Herkenham at the NIH, using the same radioligand,28and its cloning by Lisa Matsuda in Tom Bonner’s laboratory,44 which was next to that of Herkenham’s, thus allowing for a great deal of communication between the two laboratories.

My own involvement with cannabinoid chemistry and biochemistry had started in the early eighties, when the prevalent theory for cannabinoid activity was one invoking drug-induced specific membrane perturbations that modulated the properties of functional proteins within the cell membrane. Based on this approach, I was awarded a NIDA grant, which involved detailed structural and conformational studies aimed at developing correlations between ligand structure and function with their effects on model and cellular membrane systems. A turning point in my approach came when, in 1985, I heard a presentation by a Pfizer scientist describing their very extensive medicinal chemistry with their new cannabinoid-like ligands and the striking differences in the pharmacological properties based on subtle structural modifications. The results strongly argued for the presence of a yet to be identified functional protein, in all likelihood a G-protein coupled receptor.

With accumulating evidence for the existence of one or more cannabinoid receptors, I started a research program whose goals were to design and develop new biochemical and pharmacological tools that would allow us to characterize these new GPCRs and also decipher the circuitries of what was anticipated to be an intriguing and extensive biochemical system. An early effort in my laboratory to identify this putative receptor was through the design and synthesis of novel photoaffinity and electrophilic probes. This initial work by my student Avgui Charalambous led to a family of photoactivatable Δ8-tetrahydrocannabinol analogs designed to covalently attach to the receptor either through a carbene or an aliphatic nitrene mechanism.1 One of these, 5’-azido-Δ8-THC1, was subsequently radioiodinated and served as the first covalent probe for the CB1 receptor.2 This work was being carried out in collaboration with my colleague Sumner Burnstein, contemporaneously with the characterization of the CB1 receptor in the Howlett and Bonner laboratories. When rat brain membranes were allowed to photoreact with our 125I-labeled azido probe, the gel, when developed, showed two bands reflecting the presence of the corresponding proteins. This radiolabeling was prevented when the preparation was pretreated with Δ9-THC. The experiment provided the first chemical evidence for the presence of a ligand-cannabinoid receptor complex. Other information obtained from this early simple labeling experiment was the identification of a second, fainter band of lower molecular weight which allowed us to speculate on the presence of another cannabinoid receptor. This first photoaffinity labeling opened the door for future experiments in my laboratory to use covalent chemistry to characterize the structural, biochemical and biophysical properties of the cannabinoid receptors.

The story of the cannabinoid biochemical system was further expanded by the identification of the second cannabinoid receptor, designated as CB2, and initially described as peripheral because of its presumed absence in mammalian brain.45 More recently evidence was obtained for its CNS presence, albeit in low concentrations.4647 We also know now that CB2 levels can be very significantly enhanced in non-homeostatic conditions such as inflammation, neurodegeneration and cancer.

The AM Compounds

Through the use of target-based design and focused medicinal chemistry, over the past three decades, we have developed a wide variety of structurally-distinct analogs, the most interesting of which have found applications in pharmacology, biochemistry, and biophysics, as well as serving as early leads or advanced preclinical candidates for drug development. Under the AM acronym, many of these compounds are universally used by research laboratories. As pharmacological probes, AM compounds have played a substantial role in elucidating the physiological role of the endocannabinoid system. In this medicinal chemistry effort, we were assisted by an excellent collaboration with Marcus Tius at the University of Hawaii. Initial in vivo testing of key AM compounds were provided by a number of very productive collaborations with the laboratories of Toby Järbe (Northeastern University), John Salamone (University of Connecticut), Philip Malan (University of Arizona), Frank Porreca (University of Arizona), Andrea Hohmann (University of Indiana), Linda Parker (University of Guelph), and Keith Sharkey (University of Calgary).

Endocannabinoids

What are the endogenous molecules that engage and activate the two receptors? The first endocannabinoid anandamide (AEA) was isolated from pig brain in Mechoulam’s laboratory.48 This was followed by the characterization of 2-arachidonylglycerol (2AG) as the second endogenous cannabinoid.4950 We now know that the endocannabinoid system is modulated by a large family of structurally-related lipid mediators belonging to either the amide or ester families, all of which are involved in the activation of the cannabinoid receptors. Additionally, there is evidence that these same lipid molecules may also modulate other systems such as the vanilloid receptor 1 (VR1) and other transient receptor potential vanilloid (TRPV) channels, as well as the largely unexplored GPR55, GPR35, GPR18 and GPR119 receptors. This property of pleiotropic action by lipid modulators is an important feature of the cannabinoid system, and distinguishes it from other non-lipid neurotransmitters. Arguably, the levels of individual modulators, as an aggregate within a human or animal biological sample, may serve as a unique descriptor of the organism’s physiological state. This realization prompted us to develop an accurate LC/MS/MS-focused lipidomic assay for the measurement of the larger family of these endocannabinoid lipids, which we named the “endocannabinoid metabolome.”5154 This assay expands the family of endocannabinoid lipid modulators beyond its two major players AEA and 2AG, and affords us a more accurate description of the endocannabinoid system by providing quantitative information on its individual components. Such measurements can find utility in identifying endocannabinoid fluctuations that are drug-related or the result of cannabinoid-associated pathologies, such as depression and schizophrenia, where they could serve as important biomarkers.

As mentioned above, the endocannabinoid family comprises a variety of ethanolamides and glycerol long chain fatty acid esters. However, among those, AEA,, also known as anandamide, and 2AG are the most potent and best studied. AEA and 2AG are substrates for the enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL), respectively, as well as other hydrolytic enzymes, and consequently have relatively short in vivo half lives, a property that limits their usefulness as probes for the in vivopharmacological characterization of the endocannabinoid system. An early goal in my cannabinoid journey was to develop more stable analogs, encompassing the key structural features of the endogenous ligands. Design of the metabolically-stable anandamides was based on introducing substituents in the vicinity of the sessile amide bond to impede access of the ligand to the enzyme’s catalytic site. Alternatively, AEA can be modified in a manner which maintains all features required for the recognition of CB1 and CB2 receptors while impeding its fit within the enzyme’s catalytic domain. These approaches led to the design of ligands with significantly enhanced stability toward the key deactivating enzyme FAAH. The first of these, AM356, an anandamide analog substituted at the headgroup with a chiral methyl group, and also known as R-methanandamide, was a successful probe with significantly higher metabolic stability and substantially improved CB1 potency.5557AM356 has gained popularity among pharmacologists and is universally used as a surrogate for the endogenous AEA. A second generation of such probes was inspired by our early observation that the inverse amides or retroamide analogs of anandamide were significantly more resistant to enzymatic hydrolysis.57 Experimentation around that concept resulted in the development of AM1346,5859 a very potent and metabolically-stable anandamide, which we named “mahanandamide” or superanandamide, a designation contributed by Dr. Rapaka. Ongoing efforts in our laboratory toward the development of hydrolytically-stable 2AG have led to the dimethylated headgroup analog AM10336 (K. Vadivel and A. Makriyannis, unpublished).

The Role of Cell Membrane in Cannabinoid Activity

Prior to the discovery of the cannabinoid receptors, the dominant postulate was that these compounds act by perturbing the cell membrane, thus, indirectly affecting the function of key membrane proteins. This concept coincided with my first entry into the cannabinoid field, during which time we adapted or developed a variety of biophysical methods to study the effects of cannabinoids on model and natural membranes. The methods included solution60,61 and deuterium solid state NMR,2962 as well as X-ray316263 and neutron small angle diffraction.6465 These approaches, which were initially used to study the molecular mechanism of cannabinoid action, were subsequently directed to a more general program aimed at understanding the interaction of lipophilic and amphipathic ligands, including cannabinoids and endocannabinoids with cell membranes.

Because of their lipophilic properties, cannabinergic compounds have very limited aqueous solubility, raising the question of how they can access their sites of action within the respective membrane-bound proteins? Our studies provided evidence that cannabinoids and amphipathic ligands in general, assume a preferred orientation within the membrane bilayer.9,336667 In early works, we introduced the concept that amphipathic cannabinoids favorably partition into the membrane and, through fast lateral diffusion, interact with the receptor.966,67 We postulated that the ligand aligns itself within the bilayer, in a favorable orientation and conformation which optimizes its ability for a productive interaction with the respective functional protein.967 To provide experimental evidence for our postulate, we used high-resolution NMR to study the conformational properties of select cannabinergic ligands.6061,68 We also developed 2H-solid state NMR,336970 small angle x-ray31627172 and neutron diffraction approaches65 described in a series of publications that allowed us to identify the manner by which the ligand aligns itself within the membrane.637275 Our studies demonstrated that because of their amphipathic properties, cannabinergic ligands align themselves in an orientation in which the polar components of the molecule interact with the respective membrane headgroups. Such an orientation enhances their ability to engage in fast lateral diffusion within the bilayer and allows for a productive collision at the receptor site.933 We, thus, demonstrated that Δ9-THC aligns itself in an “awkward” orientation, in which its phenolic hydroxyl interacts with the polar headgroup within the bilayer, while its hydrophobic tricyclic component orients with its access orthogonal to those of phospholipid chains.293172 Cannabinoids with two or more hydroxyl groups such as the Pfizer analog CP55940 align in a manner in which all three OHs interact with the polar membrane components.9 We also showed that this “amphipathic reciprocal alignment” of amphipathic ligands within a membrane bilayer has general applicability with endogenous and exogenous lipid-friendly ligands such as steroids, including neurosteroids637476 and estrogens. Conversely, hydrophobic ligands with no polar groups capable of hydrogen bonding (e.g. OH, NH2), such as O-methyl-Δ9-THC, align with their long axis parallel to the membrane bilayer chains.717274 The endocannabinoid anandamide aligns with its extended arachidonyl chain parallel with the membrane chains and its polar ethanolamide group interacting with the phospholipid headgroups.33

In the above series of biophysical experiments, we provided substantial experimental evidence supporting our postulate that hydrophobic and amphipathic ligands access their sites of action by fast lateral diffusion after preferentially partitioning in the cellular membrane bilayer.933 This “through membrane” access of ligands to their respective GPCR binding domains is now generally well-accepted within the GPCR scientific community.

Endocannabinoid Transport Across the Cell Membrane

Endocannabinoids have been shown to access their intracellular targets through a yet to be fully identified transport mechanism.1077 The search for an endocannabinoid transport mechanism was spearheaded by the discovery of AM404,10 and subsequently AM1172,1977both lipophilic ligands that were shown to inhibit the transport of endocannabinoids across the cell membrane. This area of research, which we pursued through a close collaboration with Daniele Piomelli, provided excellent insights on the physiological effects resulting from modulating the intracellular levels of endocannabinoids and the potentially very promising opportunities for utilizing this approach for therapeutic gain.717478 To date, the endocannabinoid transport mechanism has remained quite elusive, with different postulates being proposed.79 Notwithstanding this uncertainty, the value of developing transport inhibitors as pharmacological probes, and potentially as useful medications, remains high.

Cannabinoid Diffusion across the Cell Membrane

Xenobiotic cannabinoids have been shown to diffuse passively across the cell membrane. Using well-targeted biophysical experiments with representative ligands and a number of different membrane preparations, we were able to obtain evidence on how cannabinergic compounds engage in this diffusion process and, thus, provide a basis for understanding the structural features within a ligand required for brain penetration and oral bioavaibility. The above series of experiments involved studies on the conformation, location, and orientation of a carefully-selected group of cannabinergic ligands in model and cell membranes. We showed that hydrophobic molecules with no hydrogen bearing heteroatoms, such as O-methyl-THC, initially partition within the outer membrane leaflet and reside below the polar surface. When this partitioning becomes thermodynamically unstable, a fraction of the drug partitions in the middle of the bilayer, while remaining in equilibrium with the ligand population near the polar headgroup. In an interesting study using solid state 2H NMR, we demonstrated the presence of these two drug populations within the membrane and, thus, provided a basis for a mechanism by which hydrophobic compounds cross the cell membrane. Conversely, amphipathic compounds with hydrogen-bearing heteroatoms (e.g. Δ8-THC), first partition in the outer membrane leaflet from where they interact through H-bonding with polar membrane components and subsequently engage in a flip-flop action that allows them to translocate to the inner membrane leaflet where they can access the cytosolic cellular sites. Our work also demonstrated that this flip-flop mechanism, involving amphipathic ligands, is substantially faster than the diffusion of hydrophobic molecules.23

Cannabinergic Carrier Proteins

To address the question of how cannabinoids and endocannabinoids can cross the “aqueous” intercellular space between two adjacent cells (e.g. neural synapses), we invoked the action of specialized proteins such as albumin. In a series of biophysical experiments, we provided experimental evidence of how ligands can be “picked up” and carried by albumin from a cell membrane surface and subsequently deposited on the adjacent cell surface. In an experiment, initially carried out using 2H-NMR,27 and later continued using 19F-NMR, we were able to identify the binding domains of our ligand on albumin.218081 More recently, my laboratory has been involved in studying the role of another class of carrier proteins, known as fatty acid binding proteins (FABPs). These intracellular proteins are believed to have a role in transporting endocannabinoid ligands from the inner membrane leaflet to other intracellular functional proteins including FAAH, the endocannabinoid deactivating enzyme. We recently cloned one of these proteins and used 15N-labelling to obtain high resolution NMR spectra, with which we can study in significant detail the interactions of this protein with endocannabinoids and xenocannabinoids.4

CB2 Selective Ligands

The initial concept that cannabinoid-induced analgesia was an exclusively CB1 associated response was challenged in early work published by Porreca and Makriyannis.82Subsequently, the analgesic effects of CB2 agonists were established with the synthesis of CB2 selective ligands including HU308 from the Mechoulam laboratory83 and our own more potent and in vivo more efficacious AM1241.8486 In a series of detailed publications involving collaborative work with Phil Malan at the University of Arizona and Andrea Hohmann at the University of Indiana, we documented the role of CB2 activation in peripheral and chronic analgesia, as well as inflammation.8794 More recently, in collaboration with Andrea Hohmann, using three different CB2 selective chemotypes, including AM1241, AM1710, and AM1714, we demonstrated that CB2 activation effectively relieves chemotherapy-induced neuropathy.9598 We also showed that the protective and therapeutic effects of these compounds were superior to those of gabapentin (Neurontin), the most commonly used medication for the different neuropathies. Our early success demonstrating the analgesic and anti-inflammatory effects obtained through activation of the CB2 receptor motivated a number of pharmaceutical companies to establish active research programs for the development of therapeutically-useful CB2 agonists. Unfortunately, the limited data obtained from testing CB2-selective agonists in humans did not fully validate the animal-based results. It can be argued that the limited clinical trials in which CB2 agonists were tested for their analgesic properties involved suboptimal therapeutic indications. I propose that more successful clinical results may be expected by choosing better targeted therapies.

Moreover, the development of novel CB2-selective agonists within academic and industrial laboratories opened the door for understanding the role of CB2 activation as a potential target for other therapeutic indications, such as bone disorders.99 It has also become clear that, contrary to earlier findings, CB2 is expressed in brain.46 Although brain expression under homeostatic conditions is limited, it becomes much more pronounced in pathological conditions, such as inflammation, neurodegeneration and cancer, where the CB2 is highly overexpressed both in brain and the periphery, suggesting that CB2 is a suitable target for such conditions as amyotrophic lateral sclerosis100 and colitis.101

Very recent evidence has identified a role for CB2 activation in addiction,102 which, arguably, can be considered a drug-induced inflammatory syndrome, and offers opportunities for additional therapeutic intervention.

Ligand Assisted Protein Structure

Following their discovery, the structure and function of GPCRs has been a major goal of biologists and pharmacologists. Although these proteins were cloned and expressed over three decades ago, their structural and functional characterization remained elusive and relied mostly on indirect biophysical approaches. Based on crystal structure determination, first of bacterial rhodopsin, and later of rhodopsin,103 computational methods were developed to obtain homology models of individual GPCRs, and their respective ligand receptor complexes based on the rhodopsin structure. Only recently were the crystal structures of pharmacologically-interesting GPCRs achieved. First among these was the beta adrenergic receptor, an important accomplishment through collaborative efforts between the Brian Kobilka and Ray Stevens laboratories, which required significant protein engineering of the GPCR protein.104 In parallel, Chris Tate and his collaborators at MRC, UK,105 used extensive receptor mutations to obtain crystallizable GPCR samples.

In my laboratory over the past ten years, we have been developing a novel approach aimed at obtaining experimental information on the binding motifs and functional characteristics of cannabinergic ligands with their respective receptors. This approach, which we named “Ligand Assisted Protein Structure” (LAPS), relies on the availability of covalent CB1 and CB2 probes. To identify the binding motif of individual ligands within CB1 and CB2, we use a dual methodology which includes the development of targeted CB1 and CB2 mutants and a focused proteomic characterization of the complex using LC/MS/MS.

CB1/CB2 Targeted Mutants

Our initial targets for CB1 and CB2 labeling were the cysteine residues within the seven transmembrane helical receptor domain, and developed comprehensive libraries of single or multiple cysteine mutants for each receptor. In early work, we showed that the isothiocyanate group was the optimal electrophile that reacted exclusively with cysteine residues near or at the ligand’s binding domain under the experimental conditions we used.34106108 In parallel, for a photoaffinity-based approach, we chose aliphatic azide groups as suitable moieties capable of covalently labeling the receptor upon irradiation.6109 By placing the reactive groups in different positions within a ligand, we were able to identify the respective reactive amino acid residues involved in its binding. Such experimentally-derived information was subsequently used to model the ligand-receptor complex. Conversely, our data were used to refine further existing CB1 and CB2 computational models.

Our first success story was with AM841, a classical cannabinoid analog carrying an isothiocyanate group at the end carbon of its dimethylheptyl chain.34 By sequentially mutating the individual cysteines within the seven transmembrane helical domain of the CB1 receptor to other isosteric residues that were unreactive with the NCS group, for example serine or alanine, we demonstrated that the reactive cysteine was the one within the conserved CWXP motif of helix 6.5 AM841 was also shown to attach to the same cysteine within the Hx6 of CB2, although through an overall different binding motif.20 For both receptors, the functional potencies of this ligand exceeded by 20-50 fold that of its non-covalent counterparts, an observation we recently confirmed in in vivo tests. For this reason, AM841 was designated as a “megagonist.”20

The above approach, targeting individual cysteines within the intramembrane sites in CB1 and CB2, was used to characterize the binding motifs of cannabinergic ligands from a variety of distinct chemotype classes, and supplemented with ligands carrying other reactive electrophiles, such as the carbamate and nitrate groups.52036

CB1/CB2 Proteomic Characterization

To complement our targeted mutant approach, we use LC/MS/MS methodology to identify more directly the site(s) of covalent ligand attachment within the GPCR structure. Such approaches have been used very effectively in my laboratory to characterize the structures and functions of different cannabinergic enzymes, including rat and human FAAH and MGL,110111 and more recently human N-acylethanolamine-hydrolyzing acid amidase (NAAA).30112 However, its application to GPCRs proved to be more laborious and experimentally demanding.

Our proteomic studies with CB1 and CB2 required developing efficient methods for receptor expression, purification under non-denaturing conditions, and further proteomic analysis of the targeted proteins. For these studies, we expressed the receptors in baculovirus and developed methods for obtaining purified samples of functionally-competent receptors capable of selectively and effectively interacting with our covalent ligands. The preparations were subsequently subjected to selective enzymatic digestion and the resulting peptides analyzed using LC/MS/MS methods.113116 We have been successful in perfecting this approach, and have used it to characterize the sites of attachment for a number of electrophilic ligands.16

Currently, we are expanding the LAPS approach to include a wider variety of covalent probes aimed at multiple amino acid residues within the orthosertic or allosteric sites of functional proteins. We are also exploring the use of newly designed homo- and heterobifunctional probes. These interesting covalent ligands target two points of attachment within the GPCR by incorporating two reactive groups that are either identical (eg. bisNCS; homobifunctional) or different ones (eg. NCS/N3; heterobifunctional) within the same molecule and are intended to identify more accurately the ligands’ binding motifs.

CB1/CB2 Ligand Motifs and Functional Selectivity

To date, the LAPS approach has been extended to a number of ligand chemotypes,36 and has targeted both CB1 and CB2. Our early data provide robust evidence that the CB1 and CB2 covalent ligands we have tested clearly have distinctive receptor biding motifs, as well as differentiated signaling profiles. It is tempting to propose that the CB1 and CB2 functional selectivity observed with our covalent ligands can be correlated with the different motifs of the ligand-receptor complexes. According to this postulate, individual classes of ligands induce distinct, active receptor conformations that are represented by different signaling profiles. This opens the door for developing functionally selective CB1 and CB2 compounds by using the individual ligand-receptor complexes as pharmacophoric templates for our design. Such an effort could lead to novel medications possessing either reduced undesirable side effects or more selective pharmacological profiles.

CB1 Receptor Antagonists

The development of rimonabant (SR141716A, Acomplia), a selective CB1 antagonist, by Sanofi as a potential antiobesity medication was initially greeted enthusiastically by cannabinoid researchers. When tested in humans, the drug was shown to reduce body weight and to ameliorate dyslipidemias, diabetes and metabolic syndrome. However, this enthusiasm was dampened by later observations of undesirable side effects, which included nausea, anxiety, depression, and in some cases, suicidal tendencies, which led to its withdrawal from the market in October 2008.117118

In my laboratory, we have been developing CB1 receptor antagonists since the late nineties,119123 among which, AM251 and AM281 received special attention as useful pharmacological probes with inverse agonist pharmacological profiles.119120122 AM281 was later used to demonstrate the first in vivo imaging of the receptor in non-human primates using SPECT technology.119121 In tandem, and anticipating potential CNS side effects resulting from CB1 antagonism, we also made serious efforts to develop novel compounds with no or reduced side effect profiles. We followed two distinct approaches. The first was to develop effective CB1 antagonists with no inverse agonist or weak inverse agonist properties. This approach led to a family of compounds with in vitro neutral antagonist profiles, i.e. no or minimal effect on cAMP levels. The best known of these, AM4113 and AM6527,124135when tested in rodents, reproduced all of the therapeutic effects associated with rimonabant and other inverse CB1 antagonists being developed by the pharmaceutical industry, including reduction in food consumption, weight loss, and ability to antagonize the effects of stimulant and nicotine addiction. When tested in animals, these compounds did not exhibit any of the undesirable side effects of inverse agonists in a number of animal models. The testing for side effect profiles included nausea, using emesis experiments in ferrets127 and rats,126128gastrointestinal motility in mice, as well as anxiety,129130 depression and anhedonia132 in rats. The above results are very encouraging, but require further validation to determine how the data can be translated into improved and novel CB1antagonists with favorable therapeutic index profiles.

Our second approach for obtaining CB1 antagonists with reduced undesirable side effect profiles was the development of peripherally-active compounds. This effort was reinforced by existing clinical data from the rimonabant trials, suggesting that some of the drug’s key therapeutic effects were peripherally mediated through a mechanism involving modulation in lipid metabolism and energy balance. The most prominent result of our effort was AM6545, a peripherally-acting neutral antagonist. When tested in rodents, this compound was found to produce weight loss, as well as improved lipid profile and insulin sensitivity. Currently, AM6545 is in advanced preclinical testing and being considered for development for the treatment of non-alcoholic fatty liver disease (NAFLD), as well as liver fibrosis.136139Current SAR work on the 2-pyrrolidinone scaffold has led to AM10009 or molecule with distinct chiral features (J. Garcia et al, unpublished).

Cannabinoid Agonists with Controlled Deactivation

A program we recently initiated based on the “soft drug” approach aims at developing safer and more effective cannabinergic compounds with controllable deactivation, improved druggability and an overall safer pharmacological profile.

To this end, we incorporate a metabolically vulnerable ester group within the structure of a successful cannabinergic ligand.140141 Hydrolysis at the esteratic site leads to products devoid of pharmacological activity and low or no toxicity. In our controlled deactivation/detoxification design, the compound’s systemic half-life is determined by two factors. The first is the extent to which the ligand is sequestered within the body before it is released for systemic circulation (depot effect). This process is dependent on the compound’s physicochemical properties and can be modulated by adjusting log P and PSA. The second parameter is the rate of enzymatic hydrolysis by blood esterases. This can be calibrated by incorporating suitable stereochemical features in the vicinity of the hydrolyzable group (enzymatic effect). In recent publications involving analogs with an ester group at the side chain or as a lactone structure within the cannabinoid C-ring of the cannabinoid structure, we demonstrated our ability to modulate the rates of hydrolysis, as well as the drug’s depot effects. Key compounds synthesized in our laboratory show great promise as pharmacological probes and potential drug leads. Currently, we are pursuing this approach for the design of peripherally-acting analgesic agents, as well as potential medications in THC substitution therapy.

Endocannabinoid Deactivating Enzymes

Fatty Acid Amide Hydrolase

The first enzyme to be identified was a specific amidase involved in the hydrolysis of anandamide and other long-chain fatty acid amides and ethanolamides. The role of this intriguing enzyme was first identified by Dale Deutsch, with whom we collaborated and developed the first inhibitors for this enzyme, the most prominent of which was the sulfonyl fluoride AM374, a relatively simple compound with surprisingly high selectivity for FAAH and capable of crossing the BBB.142143 To enhance our ability to test FAAH inhibitors, we developed two assays.144145 We also were first to purify this enzyme from rat brain membranes using an affinity column designed by my student Andreas Goutopoulos.146 The enzyme which we had initially named anandamide amidase was later given the name fatty acid amide hydrolase (FAAH).

Our efforts to characterize this important enzyme were subsequently superseded by the work of Ben Cravatt, who cloned and purified it, and subsequently, through a collaboration with Ray Stevens, produced informative crystal structures of the inhibitor enzyme complex.147The development of transgenic mice in which the enzyme was deleted was a big step in understanding the enzyme’s pharmacology. This very productive effort by the Scripps investigators opened a new chapter in cannabinoid research.

A major effort by the academic and industrial research communities led to a plethora of novel FAAH inhibitors, which allowed for the exploration of the therapeutic value of indirect activation of the cannabinoid receptors. A prominent early inhibitor, URB597 by Daniele Piomelli,148 was used to demonstrate the pharmacological effects associated with FAAH inhibition and enhancement of endocannabinoid tone and the potential for using such agents in depression. Our own AM3506 and AM5206 were shown to have potent neuroprotective properties using a model involving glutamatergic excitotoxicity in collaboration with my colleague Ben Bahr.1218149152 Subsequently, a productive programe by the Pfizer group led to a high quality series of FAAH inhibitors encompassing electrophilic ureas, of which PF-04457845 was the first to advance to clinical trials.153 Unfortunately, when this compound was tested in humans for osteoarthritis, it failed to show substantial efficacy. Notwithstanding the unsatisfactory clinical results, I am convinced that there is a promising future for the development of FAAH inhibitor medications by targeting more relevant therapeutic indications.

This key endocannabinoid presynaptically-localized enzyme is an esterase involved in the hydrolytic deactivation of the endocannabinoid ester 2AG, which also is its optimal endogenous substrate. Chemical inactivation of this enzyme leads to an increase in 2AG levels with the concomitant enhancement of cannabinoid activity. The human enzyme was cloned by a number of laboratories and expressed in E. coli. Additionally, a number of crystal structures have been produced for the enzyme-ligand complexes.154155 Our involvement with MGL stemmed from an interest in obtaining detailed information on its structure and function beyond what was available in the literature, and in using it to design and synthesize novel inhibitors covering a broad spectrum of potency, reversibility, and selectivity profiles. Additionally, we were interested in exploring MGL species differences with the intent of developing novel successful inhibitors that were equally potent in human and rodent enzymes to ensure that if successful, these compounds could be advanced beyond the preclinical stage.

The work involved cloning and expression of the enzyme in E. coli and purification using affinity chromatography.26110111156 We subsequently used MGL as a lead project to develop biophysical methods which we plan to apply to other endocannabinoid targets. Structural experiments were based principally on the combined use of mass spectrometry and high resolution NMR,14156 accompanied by computer modeling. For example, we identified, at a distance of 22Å from the reactive serine, a tryptophan residue, which, when substituted with other residues, rendered the enzyme practically inactive. The NMR experiments provided detailed information on the H-bonding network required to enhance the nucleophilic properties of the catalytic serine,14 while maintaining the active enzyme conformation. The work opened the door for designing allosteric enzyme inhibitors by targeting key enzyme residues away from the catalytic site, but distantly involved in the catalytic process. The work also identified the two conformations (active and inactive) observed with other esterases, where the enzyme lid is respectively in the open or closed conformations. Thus, we were able to develop specific conditions under which each of the two enzyme conformational states could be isolated and structurally characterized in solution.156

Although a cytosolic enzyme, MGL’s activity is enhanced by its association with a membrane environment. To study these MGL membrane interactions, we used nanodiscs in combination with Hydrogen/Deuterium Exchange Mass Spectrometry (HXMS) technology.26 In parallel, we have produced triply-labeled (2H,13C,15N) enzyme preparations for the purpose of identifying residue resonances through a 3D-analysis. This work, which will serve as a basis for using SAR by NMR methods to study inhibitor-enzyme interactions and design optimized inhibitors, is currently underway.

Early evidence from the Cravatt laboratory and ours has suggested a level of synergy between the two key endocannabinoid deactivating enzymes FAAH and MGL.15157158 To explore these findings, we designed and synthesized a family of dual MGL/FAAH inhibitors. To date, our program on novel FAAH, MGL and MGL/FAAH inhibitors (eg. AM6701) has identified promising leads, which are being pursed as potential medications for neurodegenerative diseases and addiction.

Paracannabinoid Targets

Because of the pleiotropic nature of its endogenous ligands, cannabinoid biology is connected with other biochemical systems involving lipid modulators, such as the VR1 ion channel, a number of recently deorphanized GRPCs, including GPR55, as well as the arachidonic acid metabolizing enzyme cyclooxygenase-2 and the more recently identified NAAA. Because of their functional proximity with the endocannabinoid system, some of these targets are being explored in my laboratory. Recently, we have cloned, expressed, and purified GPR55, and are prepared to subject it to our LAPS approach in order to gain more detailed structural and functional information.

Also, we have cloned, expressed, and purified hNAAA, a lysosomal amidase which has shown promise as a therapeutic target for inflammation. Our mammalian expression system has allowed us to produce pure enzyme in milligram quantities.112 We used proteomic approaches to gain information on the catalytic mechanism of this unique cysteine amidase, where the apoenzyme is activated through an intramolecular scission, leading to two chain components, which form the active enzyme heterodimer.30112 Although NAAA hydrolyzes anandamide, its optimal native substrate is palmitylethanolamide, a lipid modulator generally associated with the nuclear receptor PPAR-alpha.

Recently, we have obtained good quality 1H NMR spectra of the purified enzyme, which allows us to follow the enzymatic catalysis in real NMR time, and to design novel inhibitors. The most successful of these, AM9053, is a chemically- and metabolically-stable compound with substantial in vitro potency (Ki = 30 nM). Very recently, its in vivo efficacy was demonstrated in a colon inflammation model.

Now and the future

Almost three decades after its discovery, the endocannabinoid system offers rich opportunities for the chemist. It has been identified as a major biochemical system, playing major roles in brain function, autonomic physiology, the immune system, and as a key player in maintaining homeostasis. Much of this biology needs to be further elaborated and new, related pathways remain to be discovered. The medicinal chemist should have a major role in developing or participating in the development of molecular tools in this exciting endeavor. These include selective pharmacological probes, in vivo imaging agents for PET and SPECT, as well as in vitro fluorescent and radiolabelling reagents. There continues to be a need for new ligands for structural biology work involving NMR, x-ray, and mass spectrometry that will assist in understanding the function of some key endocannabinoid proteins, including the two GPCRs and the enzymes and proteins involved in endocannabinoid biosynthesis, metabolism, and transport. The pleiotropic nature of the endocannabinoids encourages the exploration of noncannabinoid functional proteins associated with the endocannabinoid system, and identified in this perspective as paracannabinoid targets, as potential therapeutic targets. These may include other GPCRs and channels that are modulated by lipid ligands, as well as enzymes involved in their biotransformations.

As a therapeutic target, the endocannabinoid system has found to date only modest success, and the failure of Sanofi’s CB1 antagonist Acomplia was a major setback in this endeavor. However, a number of new findings, coupled with current efforts for cannabinoid-based drug development, point to a more successful future. CB1 antagonist approaches, including the peripherally-acting compounds, show promise as medications for metabolic disorders, liver damage and fat metabolism. In this regard, early candidates, such as AM6545, with combined neutral antagonist/peripheral profiles, may reduce the peripheral side effect profile in this class of compounds. As for the brain penetrant neutral CB1 antagonists, these may find usefulness in addiction disorders, including those from nicotine, opioids, and alcohol.

Notwithstanding the initial unsatisfactory results from the limited in vivo trials, CB2 agonists continue to show promise as potential medications for inflammatory and neuropathic pain, as well as in neurodegenerative conditions, including ALS and multiple sclerosis. Arguably, the development of CB1 agonists with reduced side effects, either through peripheralization or the controlled deactivation approaches I have outlined earlier, still holds great promise as analgesic medications for pain management, lacking some of the side effects associated with opioid anaglesia.

Therapeutic approaches involving indirectly-acting cannabinoid agonists, such as FAAH inhibition, are attractive because of their more favorable side effect profiles, notably the absence of THC-like effects in the CNS. However, initial in vivo trials with a FAAH inhibitor for osteoarthritis have not proven successful. Notwithstanding this failure, such compounds still offer promise as anti-inflammatory and neuroprotective medications, if properly targeted. MGL inhibitors, acting, at least in part, through a non-cannabinoid mechanism, are being developed as potential anticancer medications. Also, the observed synergy accompanying the simultaneous inhibition of both deactivating enzymes FAAH and MGL invites the development of optimized dual inhibitors. Indirectly-acting cannabinoid agonists also hold promise in drug abuse as potential substitution therapies. Early results identify NAAA inhibitors as potential anti-inflammatory agents in conditions such as irritable bowel syndrome. Recent data with our selective NAAA inhibitor AM9053 are very exciting. Such compounds may also prove useful as peripherally-acting analgesics.

Finally, CB1 positive or negative allosteric modulators offer interesting prospects for developing medications lacking CB1-related undesirable side effects. Although substantial research has identified several scaffolds that exhibit in vitro allosteric profiles, in vivoexperiments have shown only modest efficacy results. This is an area which would benefit from novel or improved scaffolds.

The ubiquitous presence of the endocannabinoid system presents difficulties in targeting it for therapeutic gain. For this reason, it is important that indications for cannabinergic drug development be explored very thoughtfully. In this regard, a better understanding of cannabinoid receptor-related functional selectivity should assist in the development of drugs with safer pharmacological profiles, and also identify additional therapeutic opportunities. Thus, compounds belonging to the same pharmacological class could be optimized for different indications, i.e. developing drugs from the same family with many flavors to address specific needs. Notwithstanding some of the earlier failures in developing cannabinoid-related medications, there is ample accumulated knowledge to predict that the endocannabinoid system offers great promise as a potential source of future therapies.

Figure 1

Technical Review Proceedings Cover Page17
Figure 2

Phyto- and Xeno-Cannabinoids1,2
Figure 3

Autoradiography of 10 nM [3H]CP55,940 binding in a sagittal section of rat brain. Br St, brainstem; Cer, cerebellum; Col, collculi; CP, caudate-putamen; Cx, cerebral cortex; Ep, entopeduncular nucleus (homolog of GPi); GP, globus pallidus (e, external; 
Figure 4

Select AM Compounds. AM630 is the first CB2 antagonist.3,4 AM411 is the first adamantyl cannabinoid,8while AM4054 is a later generation analog.11 Both are early CB1 receptor agonists. AM2389 is a very potent long-acting CB1 agonist;13 AM404 is the first 
Figure 5

Endocannabinoid Analogs
Figure 6

Drug and endogenous ligands partition within the cellular membrane where they can: (a) diffuse passively across the bilayer to enter the intracellular space; (b) are tranlocated intracellularly through a yet to be fully identified transport mechanism; 
Figure 7

(I) Representative solid state 2H-NMR spectra from dipalmitoylphosphatidyl choline (DPPC) model membranes (42°C) containing five cannabinoids each having two deuterium labels at 2 and 4-positions: 2,4-d2-Me-Δ8-THC(A), 2,4-d2-Δ- 
Figure 8

Electron density profile differences inside the bilayer: Curve B-A is the difference between profiles of dimyristoylphosphatidyl choline (DMPC) + Δ8-THC and DMPC, curve C-A is the difference between those of DMPC + 5’-I-Δ8-THC 
Figure 9

A ligand-membrane-receptor model representing the trans-membrane diffusion of CP55940 en route to interacting with the cannabinoid receptor. According to our hypothesis, the ligand preferentially partitions in the membrane bilayer where it assumes a proper 
Figure 10

2H-solid state NMR experiments identifying the endocannabinoid anandamide assuming an extended conformation in the bilayer with its polar group at the same level as the polar phospholipid head groups. It diffuses laterally to the binding site of the CB1 
Figure 11

On the left, inhibition of [3H]anandamide accumulation by astrocytes by AM404. On the right, effects of AM404 on anandamide-induced inhibition of adenylyl cyclase activity in cortical neurons. AM403 is a control inactive ligand. In all experiments, cells 
Figure 12

Lipophilic ligands are transported intracellularly by means of extracellular carrier proteins (eg. albumin) and deposited initially at the outer membrane leaflet. Hydrophobic ligands (A, e.g. Me-Δ9-THC) initially occupy a location in the outer 
Figure 13

Human Fatty Acid Binding Protein (FABP7)
Figure 14

CB2 Agonists
Figure 15

Covalent Ligands. AM841 is a CB1/CB2 covalent megagonist.5 AM3677 is an anandamide covalent CB1 receptor agonist;6 AM9017 is an anandamide covalent CB2 agonist (K. Vadivel and A. Makriyannis, unpublished); AM967 is a photoactivatable CB2 covalent probe; 
Figure 16

Illustration of the CB2 R*/AM841 Binding Site from Modeling Studies
Figure 17

(a) Chemical structure of AM841. (b) Saturation-binding assay of [3H] CP-55940 radioligand of FLAG-hCB2R-6His in membranes from Sf-21 cells overexpressing this receptor. Membrane incubation with AM841 prior to [3H] CP-55940 binding (▲) reduced 
Figure 18

CB1 Antagonists
Figure 19

Example of controlled-deactivation cannabinergic ligand
Figure 20

Diagrammatic representation of the major hydrolytic inactivation and oxidative biotransformation pathways implicated in anandamide and 2-arachidonyl glycerol catabolism. (٠) Endocannabinoid proteins being studied by AM.
Figure 21

AM3506 was covalently docked to the catalytic Ser241 of rFAAH in the acyl chain binding channel. There is significant hydrogen bonding with the oxyanion hole (formed by the backbone of Ile238, Gly239, Gly240, and Ser241), and also with the backbone carbonyl 
Figure 22

AM5206 affords neuronal protection in the hippocampus after KA-induced excitotoxicity in vitro. Organotypic hippocampal slice cultures were used, and a low-power photomicrograph shows their characteristic maintenance of native cellular organization. 
Figure 23

AM5206 affords seizure and neuronal protection after KA induced excitotoxicity in vivo. Seizures were induced by i.p. injection of 9.5 mg/kg KA (n=12 rats), and following the KA administration animals were immediately injected with either vehicle or 8 
Figure 24

FAAH and MGL Inhibitors
Figure 25

Hydrogen bonding network in the active site of hMGL
Figure 26

MD simulation of hMGL interaction with membrane phospholipid bilayer. (a)Structure of AM6580. Snapshots of hMGL based on HXMS experimental derived data with a phospholipid bilayer membrane of the same composition as in experimental nanodiscs (POPC: POPG, 
Figure 27

3D-cube from the HNCA spectrum of hMGL (900 MHz spectrometer with cryo-probe, 2 days experiment. Triple resonance HNCA and HN(CO)CA spectra were recorded at 310 K on a Bruker Avance 900 MHz NMR spectrometer for the assignment of backbone resonances. All 
Figure 28

Example of Long Range Interactions discovered in the hMGL enzyme
Figure 29

Reduction of KA-induced seizure severity by AM6701(FAAH, MGL inhibitor) and AM6702 (FAAH inhibitor). Seizures were initiated in rats with an intraperitoneal injection of 9.8 mg/kg KA. Immediately following the KA administration, animals were injected 
Figure 30

(A) Putative mechanism of irreversible inhibition of hNAAA by AM 6701 via thiocarbamylation of Cy126. (B)Representation of the active site of hNAAA after treatment of AM6701. Homology model illustrates thiocarbamylation of catalytic nucleophile Cys126 

Acknowledgments

The work described here has spanned over three decades and has involved a large number of coworkers, post-doctoral fellows, and students, to whom I am eternally grateful. Also, my great thanks to Jessica Ehinger for her major help in producing this perspective and to Drs. David Janero, Spyros Nikas, and Kumar Subramanian Vadivel for reading and assisting in the preparation of the manuscript. The work included here was supported by grants from the National Institute of Drug Abuse (DA3801, DA7215, DA9158, and DA26795).

Nonstandard Abbreviations Used

2AG
2-arachidonylglycerol
AEA
endocannabinoid anandamide
DMPC
dimyristoylphosphatidyl choline
DPPC
dipalmitoylphosphatidyl choline
FABP
fatty acid binding protein
MGL
monoacylglycerol lipase
NAAA
N-acylethanolamine-hydrolyzing acid amidase
TRVP
transient receptor potential vanilloid
VR1
vanilloid receptor 1

Biography

Alexandros Makriyannis is the George Behrakis Trustee Chair in Pharmaceutical Biotechnology at Northeastern University, Professor of Pharmaceutical Sciences, Chemistry, and Chemical Biology, and Director of the Center for Drug Discovery. He received his Ph.D. in medicinal chemistry at the University of Kansas and postdoctoral training in synthetic organic chemistry at the University of California, Berkeley. At the University of Connecticut, he rose to the rank of Distinguished Professor, before joining Northeastern University and establishing its Center for Drug Discovery. His work is characterized by being at the interface of chemistry and biology, encompassing over 450 publications, 40 patents, and numerous awards, most recently the Northeastern Award for Excellence in Research and Creativity (2012) and the ACS Medicinal Chemistry Hall of Fame (2013).

References

1. Charalambous A, Yan G, Houston DB, Howlett AC, Compton DR, Martin BR, Makriyannis A. 5′-Azido-delta 8-THC: a novel photoaffinity label for the cannabinoid receptor. J Med Chem. 1992;35:3076–3079. [PubMed]
2. Burstein SH, Audette CA, Charalambous A, Doyle SA, Guo Y, Hunter SA, Makriyannis A. Detection of cannabinoid receptors by photoaffinity labelling. Biochem Biophys Res Commun. 1991;176:492–497. [PubMed]
3. Pertwee R, Griffin G, Fernando S, Li X, Hill A, Makriyannis A. AM630, a competitive cannabinoid receptor antagonist. Life Sci. 1995;56:1949–1955. [PubMed]
4. Guo J, Williams MK, Makriyannis A. Therapeutic Opportunities through the Modulation of Endocannabinoid Transport. Pharmaceut. Reg. Affairs. 2013;3 In press, doi:10.4172/2167-7689.1000e4123.
5. Picone RP, Khanolkar AD, Xu W, Ayotte LA, Thakur GA, Hurst DP, Abood ME, Reggio PH, Fournier DJ, Makriyannis A. (−)-7′-Isothiocyanato-11-hydroxy-1′,1′-dimethylheptylhexahydrocannabinol (AM841), a high-affinity electrophilic ligand, interacts covalently with a cysteine in helix six and activates the CB1 cannabinoid receptor. Mol Pharmacol. 2005;68:1623–1635. [PubMed]
6. Li C, Xu W, Vadivel SK, Fan P, Makriyannis A. High affinity electrophilic and photoactivatable covalent endocannabinoid probes for the CB1 receptor. J Med Chem.2005;48:6423–6429. [PubMed]
7. Papahatjis DP, Nahmias VR, Nikas SP, Andreou T, Alapafuja SO, Tsotinis A, Guo J, Fan P, Makriyannis A. C1′-cycloalkyl side chain pharmacophore in tetrahydrocannabinols. J Med Chem. 2007;50:4048–4060. [PubMed]
8. Lu D, Meng Z, Thakur GA, Fan P, Steed J, Tartal CL, Hurst DP, Reggio PH, Deschamps JR, Parrish DA, George C, Jarbe TU, Lamb RJ, Makriyannis A. Adamantyl cannabinoids: a novel class of cannabinergic ligands. J Med Chem. 2005;48:4576–4585. [PubMed]
9. Xie XQ, Melvin LS, Makriyannis A. The conformational properties of the highly selective cannabinoid receptor ligand CP-55,940. J Biol Chem. 1996;271:10640–10647. [PubMed]
10. 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;277:1094–1097. [PubMed]
11. Thakur GA, Bajaj S, Paronis C, Peng Y, Bowman AL, Barak LS, Caron MG, Parrish D, Deschamps JR, Makriyannis A. Novel adamantyl cannabinoids as CB1 receptor probes. J Med Chem. 2013;56:3904–3921. [PMC free article] [PubMed]
12. Alapafuja SO, Nikas SP, Bharathan IT, Shukla VG, Nasr ML, Bowman AL, Zvonok N, Li J, Shi X, Engen JR, Makriyannis A. Sulfonyl fluoride inhibitors of fatty acid amide hydrolase. J Med Chem. 2012;55:10074–10089. [PMC free article] [PubMed]
13. Nikas SP, Alapafuja SO, Papanastasiou I, Paronis CA, Shukla VG, Papahatjis DP, Bowman AL, Halikhedkar A, Han X, Makriyannis A. Novel 1′,1′-chain substituted hexahydrocannabinols: 9beta-hydroxy-3-(1-hexyl-cyclobut-1-yl)-hexahydrocannabinol (AM2389) a highly potent cannabinoid receptor 1 (CB1) agonist. J Med Chem.2010;53:6996–7010. [PMC free article] [PubMed]
14. Karageorgos I, Tyukhtenko S, Zvonok N, Janero DR, Sallum C, Makriyannis A. Identification by nuclear magnetic resonance spectroscopy of an active-site hydrogen-bond network in human monoacylglycerol lipase (hMGL): implications for hMGL dynamics, pharmacological inhibition, and catalytic mechanism. Mol Biosyst. 2010;6:1381–1388.[PMC free article] [PubMed]
15. Naidoo V, Karanian DA, Vadivel SK, Locklear JR, Wood JT, Nasr M, Quizon PM, Graves EE, Shukla V, Makriyannis A, Bahr BA. Equipotent inhibition of fatty acid amide hydrolase and monoacylglycerol lipase – dual targets of the endocannabinoid system to protect against seizure pathology. Neurotherapeutics. 2012;9:801–813. [PMC free article][PubMed]
16. Szymanski DW, Papanastasiou M, Melchior K, Zvonok N, Mercier RW, Janero DR, Thakur GA, Cha S, Wu B, Karger B, Makriyannis A. Mass spectrometry-based proteomics of human cannabinoid receptor 2: covalent cysteine 6.47(257)-ligand interaction affording megagonist receptor activation. J Proteome Res. 2011;10:4789–4798. [PMC free article][PubMed]
17. Rapaka R, A. M. Structure-Activity Relationship of the Cannabinoids. Vol. 79. DHHS Publication; Rockville, MD: 1987.
18. Naidoo V, Nikas SP, Karanian DA, Hwang J, Zhao J, Wood JT, Alapafuja SO, Vadivel SK, Butler D, Makriyannis A, Bahr BA. A new generation fatty acid amide hydrolase inhibitor protects against kainate-induced excitotoxicity. J Mol Neurosci. 2011;43:493–502.[PMC free article] [PubMed]
19. Fegley D, Kathuria S, Mercier R, Li C, Goutopoulos A, Makriyannis A, Piomelli D. Anandamide transport is independent of fatty-acid amide hydrolase activity and is blocked by the hydrolysis-resistant inhibitor AM1172. Proc Natl Acad Sci U S A. 2004;101:8756–8761.[PMC free article] [PubMed]
20. Pei Y, Mercier RW, Anday JK, Thakur GA, Zvonok AM, Hurst D, Reggio PH, Janero DR, Makriyannis A. Ligand-binding architecture of human CB2 cannabinoid receptor: evidence for receptor subtype-specific binding motif and modeling GPCR activation. Chem Biol. 2008;15:1207–1219. [PMC free article] [PubMed]
21. Tyukhtenko S, Karageorgos I, Zvonok N, Makriyannis A. Direct Observation of the pH-Dependent Equilibrium between Open and Closed Conformations of hMGL by NMR Spectroscopy.. In 53th Experimental NMR Conference; Miami, FL. 2012.
22. Makriyannis A, Deng H. Cannabimimetic indole derivatives. 2001
23. Tian X. PhD Thesis: Solid state NMR studies of interactions of cannabinergic ligands with lipid membrane systems. University of Connecticut; Storrs, CT: 2009.
24. Tyukhtenko S, Jiang R, Makriyannis A, Guo J. NMR Characterization of Fatty Acid Binding Proteins (FABPs) Interactions with Anandamide and Novel Selective Inhibitors.. 54th Experimental NMR Conference; Asilomar, CA. 2013.
25. Karageorgas I. PhD Thesis: Towards the Mechanism of Human Monoacylglycerol Lipase (hMGL) Inactivation: A Combination of Mutational Analysis, MS and NMR approaches.Northeastern University; Boston: 2012.
26. Nasr ML, Shi X, Bowman AL, Johnson M, Zvonok N, Janero DR, Vemuri VK, Wales TE, Engen JR, Makriyannis A. Membrane phospholipid bilayer as a determinant of monoacylglycerol lipase kinetic profile and conformational repertoire. Protein Sci.2013;22:774–787. [PMC free article] [PubMed]
27. Makriyannis A, Guo J, Tian X. Albumin enhances the diffusion of lipophilic drugs into the membrane bilayer. Life Sci. 2005;77:1605–1611. [PubMed]
28. Herkenham M, Lynn AB, Little MD, Johnson MR, Melvin LS, de Costa BR, Rice KC. Cannabinoid receptor localization in brain. Proc Natl Acad Sci U S A. 1990;87:1932–1936.[PMC free article] [PubMed]
29. Yang DP, Banijamali A, Charalambous A, Marciniak G, Makriyannis A. Solid state 2H-NMR as a method for determining the orientation of cannabinoid analogs in membranes.Pharmacol Biochem Behav. 1991;40:553–557. [PubMed]
30. West JM, Zvonok N, Whitten KM, Vadivel SK, Bowman AL, Makriyannis A. Biochemical and mass spectrometric characterization of human N-acylethanolamine-hydrolyzing acid amidase inhibition. PLoS One. 2012;7:e43877. [PMC free article] [PubMed]
31. 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;1024:336–344. [PubMed]
32. Dixon DD, Tius MA, Thakur GA, Zhou H, Bowman AL, Shukla VG, Peng Y, Makriyannis A. C3-heteroaroyl cannabinoids as photolabeling ligands for the CB2 cannabinoid receptor. Bioorg Med Chem Lett. 2012;22:5322–5325. [PMC free article][PubMed]
33. Tian X, Guo J, Yao F, Yang DP, Makriyannis A. The conformation, location, and dynamic properties of the endocannabinoid ligand anandamide in a membrane bilayer. J Biol Chem.2005;280:29788–29795. [PubMed]
34. Guo Y, Abadji V, Morse KL, Fournier DJ, Li X, Makriyannis A. (−)-11-Hydroxy-7′-isothiocyanato-1′,1′-dimethylheptyl-delta 8-THC: a novel, high-affinity irreversible probe for the cannabinoid receptor in the brain. J Med Chem. 1994;37:3867–3870. [PubMed]
35. Ghosh R, Todd AR, Wilkinson S. Cannabis indica. IV. The synthesis of some tetrahydrodibenzopyran derivatives. J. Chem. Soc. 1940:1121–1125.
36. Mercier RW, Pei Y, Pandarinathan L, Janero DR, Zhang J, Makriyannis A. hCB2 ligand-interaction landscape: cysteine residues critical to biarylpyrazole antagonist binding motif and receptor modulation. Chem Biol. 2010;17:1132–1142. [PMC free article] [PubMed]
37. Adams R, MacKenzie S, Loewe S. Tetrahydrocannabinol homologs. XVIII. Tetrahydrocannabinol homologs with doubly branched alkyl groups in the 3-position. J. Am. Chem. Soc. 1948;70:664–668. [PubMed]
38. Gaoni Y MR, Hashish Isolation, structure, and partial synthesis of an active constituent of hashish. J. Am. Chem. Soc. 1964;86:1646–1647.
39. Mechoulam R, Braun P, Gaoni Y. Syntheses of .DELTA.1-tetrahydrocannabinol and related cannabinoids. J. Am. Chem. Soc. 1972;94:6159–6165. [PubMed]
40. Razdan RK. Structure-activity relationships in cannabinoids. Pharmacol Rev.1986;38:75–149. [PubMed]
41. Archer RA, Blanchard WB, Day WA, Johnson DW, Lavagnino ER, Ryan CW, Baldwin JE. Cannabinoids. 3. Synthetic approaches to 9-ketocannabinoids. Total synthesis of nabilone.J Org Chem. 1977;42:2277–2284. [PubMed]
42. Milne GM, Johnson MR. Levonantradol – a Role for Central Prostanoid Mechanisms. J. Clin. Pharmacol. 1981;21:S367–S374. [PubMed]
43. Devane WA, Dysarz FA, 3rd, Johnson MR, Melvin LS, Howlett AC. Determination and characterization of a cannabinoid receptor in rat brain. Mol Pharmacol. 1988;34:605–613.[PubMed]
44. Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner TI. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature. 1990;346:561–564. [PubMed]
45. Munro S, Thomas KL, Abu-Shaar M. Molecular characterization of a peripheral receptor for cannabinoids. Nature. 1993;365:61–65. [PubMed]
46. Van Sickle MD, Duncan M, Kingsley PJ, Mouihate A, Urbani P, Mackie K, Stella N, Makriyannis A, Piomelli D, Davison JS, Marnett LJ, Di Marzo V, Pittman QJ, Patel KD, Sharkey KA. Identification and functional characterization of brainstem cannabinoid CB2 receptors. Science. 2005;310:329–332. [PubMed]
47. Gong JP, Onaivi ES, Ishiguro H, Liu QR, Tagliaferro PA, Brusco A, Uhl GR. Cannabinoid CB2 receptors: immunohistochemical localization in rat brain. Brain Res.2006;1071:10–23. [PubMed]
48. Devane WA, Hanuš L, Breuer A, Pertwee RG, Stevenson LA, Griffin G, Gibson D, Mandelbaum A, Etinger A, Mechoulam R. Isolation and Structure of a Brain Constituent That Binds to the Cannabinoid Receptor. Science. 1992;258:1946–1949. [PubMed]
49. Sugiura T, Kondo S, Sukagawa A, Nakane S, Shinoda A, Itoh K, Yamashita A, Waku K. 2-Arachidonoylglycerol: a possible endogenous cannabinoid receptor ligand in brain.Biochem Biophys Res Commun. 1995;215:89–97. [PubMed]
50. Mechoulam R, Ben-Shabat S, Hanus L, Ligumsky M, Kaminski NE, Schatz AR, Gopher A, Almog S, Martin BR, Compton DR, Pertwee RG, Griffin G, Bayewitch M, Barg J, Vogel Z. Identification of an endogenous 2-monoglyceride, present in canine gut, that binds to cannabinoid receptors. Biochem Pharmacol. 1995;50:83–90. [PubMed]
51. Williams J, Pandarinathan L, Wood J, Vouros P, Makriyannis A. Endocannabinoid metabolomics: a novel liquid chromatography-mass spectrometry reagent for fatty acid analysis. AAPS J. 2006;8:E655–660. [PMC free article] [PubMed]
52. Williams J, Wood J, Pandarinathan L, Karanian DA, Bahr BA, Vouros P, Makriyannis A. Quantitative method for the profiling of the endocannabinoid metabolome by LC-atmospheric pressure chemical ionization-MS. Anal Chem. 2007;79:5582–5593. [PubMed]
53. Wood JT, Williams JS, Pandarinathan L, Courville A, Keplinger MR, Janero DR, Vouros P, Makriyannis A, Lammi-Keefe CJ. Comprehensive profiling of the human circulating endocannabinoid metabolome: clinical sampling and sample storage parameters. Clin Chem Lab Med. 2008;46:1289–1295. [PMC free article] [PubMed]
54. Wood JT, Williams JS, Pandarinathan L, Janero DR, Lammi-Keefe CJ, Makriyannis A. Dietary docosahexaenoic acid supplementation alters select physiological endocannabinoid-system metabolites in brain and plasma. J Lipid Res. 2010;51:1416–1423. [PMC free article][PubMed]
55. Abadji V, Lin S, Taha G, Griffin G, Stevenson LA, Pertwee RG, Makriyannis A. (R)-methanandamide: a chiral novel anandamide possessing higher potency and metabolic stability. J Med Chem. 1994;37:1889–1893. [PubMed]
56. Khanolkar AD, Abadji V, Lin S, Hill WA, Taha G, Abouzid K, Meng Z, Fan P, Makriyannis A. Head group analogs of arachidonylethanolamide, the endogenous cannabinoid ligand. J Med Chem. 1996;39:4515–4519. [PubMed]
57. Lin S, Khanolkar AD, Fan P, Goutopoulos A, Qin C, Papahadjis D, Makriyannis A. Novel analogues of arachidonylethanolamide (anandamide): affinities for the CB1 and CB2 cannabinoid receptors and metabolic stability. J Med Chem. 1998;41:5353–5361. [PubMed]
58. Jarbe TU, DiPatrizio NV, Li C, Makriyannis A. Effects of AM1346, a high-affinity CB1 receptor selective anandamide analog, on open-field behavior in rats. Behav Pharmacol.2007;18:673–680. [PubMed]
59. Jarbe TU, Li C, Liu Q, Makriyannis A. Discriminative stimulus functions in rats of AM1346, a high-affinity CB1R selective anandamide analog. Psychopharmacology (Berl)2009;203:229–239. [PMC free article] [PubMed]
60. Xie XQ, Yang DP, Melvin LS, Makriyannis A. Conformational analysis of the prototype nonclassical cannabinoid CP-47,497, using 2D NMR and computer molecular modeling. J Med Chem. 1994;37:1418–1426. [PubMed]
61. 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;41:167–174. [PubMed]
62. Yang DP, Mavromoustakos T, Beshah K, Makriyannis A. Amphipathic interactions of cannabinoids with membranes. A comparison between delta 8-THC and its O-methyl analog using differential scanning calorimetry, X-ray diffraction and solid state 2H-NMR. Biochim Biophys Acta. 1992;1103:25–36. [PubMed]
63. Mavromoustakos T, Yang DP, Makriyannis A. Effects of the anesthetic steroid alphaxalone and its inactive delta 16-analog on the thermotropic properties of membrane bilayers. A model for membrane perturbation. Biochim Biophys Acta. 1995;1239:257–264.[PubMed]
64. Makriyannis A. The Role of Cell Membranes in Cannabinoid Activity. In: Pertwee RG, editor. Cannabinoid Receptors. Academic Press; New York: 1995. pp. 87–116.
65. Martel P, Makriyannis A, Mavromoustakos T, Kelly K, Jeffrey KR. Topography of tetrahydrocannabinol in model membranes using neutron diffraction. Biochim Biophys Acta.1993;1151:51–58. [PubMed]
66. Guo J, Pavlopoulos S, Tian X, Lu D, Nikas SP, Yang DP, Makriyannis A. Conformational study of lipophilic ligands in phospholipid model membrane systems by solution NMR. J Med Chem. 2003;46:4838–4846. [PubMed]
67. Makriyannis A, Tian X, Guo J. How lipophilic cannabinergic ligands reach their receptor sites. Prostaglandins Other Lipid Mediators. 2005;77:210–218. [PubMed]
68. Xie XQ, Han XW, Chen JZ, Eissenstat M, Makriyannis A. High-resolution NMR and computer modeling studies of the cannabimimetic aminoalkylindole prototype WIN-55212-2.J Med Chem. 1999;42:4021–4027. [PubMed]
69. Makriyannis A, Yang DP. Solid-State NMR Spectroscopy in the Study of Drug Membrane Interactions; Potential Applications with Antiarrhythmic Agents. In: Hondeghem L, editor. Molecular and Cellular Mechanisms of Antiarrhythmic Agents. Vol. 20. Futura Publishing Co.; Mount Kisco, NY: 1989. pp. 293–305.
70. Makriyannis A, Banijamali A, Jarrell HC, Yang DP. The orientation of (−)-delta 9-tetrahydrocannabinol in DPPC bilayers as determined from solid-state 2H-NMR. Biochim Biophys Acta. 1989;986:141–145. [PubMed]
71. Mavromoustakos T, Yang DP, Makriyannis A. Small angle X-ray diffraction and differential scanning calorimetric studies on O-methyl-(−)-delta 8-tetrahydrocannabinol and its 5′ iodinated derivative in membrane bilayers. Biochim Biophys Acta. 1995;1237:183–188.[PubMed]
72. 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;40:547–552. [PubMed]
73. Guo J, Yang DP, Chari R, Tian X, Pavlopoulos S, Lu D, Makriyannis A. Magnetically aligned bicelles to study the orientation of lipophilic ligands in membrane bilayers. J Med Chem. 2008;51:6793–6799. [PMC free article] [PubMed]
74. Mavromoustakos T, Yang DP, Makriyannis A. Topography of alphaxalone and delta 16-alphaxalone in membrane bilayers containing cholesterol. Biochim Biophys Acta.1994;1194:69–74. [PubMed]
75. Mavromoustakos T, Yang DP, Makriyannis A. Topography and thermotropic properties of cannabinoids in brain sphingomyelin bilayers. Life Sci. 1996;59:1969–1979. [PubMed]
76. Makriyannis A, Fesik S. Mechanism of steroid anesthetic action: interactions of alphaxalone and delta 16-alphaxalone with bilayer vesicles. J Med Chem. 1983;26:463–465.[PubMed]
77. Piomelli D, Beltramo M, Glasnapp S, Lin SY, Goutopoulos A, Xie XQ, Makriyannis A. Structural determinants for recognition and translocation by the anandamide transporter. Proc Natl Acad Sci U S A. 1999;96:5802–5807. [PMC free article] [PubMed]
78. Calignano A, La Rana G, Beltramo M, Makriyannis A, Piomelli D. Potentiation of anandamide hypotension by the transport inhibitor, AM404. Eur J Pharmacol. 1997;337:R1–2. [PubMed]
79. Fowler CJ. The cannabinoid system and its pharmacological manipulation–a review, with emphasis upon the uptake and hydrolysis of anandamide. Fundam Clin Pharmacol.2006;20:549–562. [PubMed]
80. Zhuang J, Yang D-P, Tian X, Nikas SP, Sharma R, Guo JJ, Makriyannis A. Targeting the Endocannabinoid System for Neuroprotection: A 19F-NMR Study of a Selective FAAH Inhibitor Binding with an Anandamide Carrier Protein, HAS. J Pharmaceutics Pharmacol.2013 In press, DOI: 10.13188/12327-13204X.1000002. [PMC free article] [PubMed]
81. Zhuang J, Yang DP, Nikas SP, Zhao J, Guo J, Makriyannis A. The interaction of fatty acid amide hydrolase (FAAH) inhibitors with an anandamide carrier protein using (19)F-NMR.AAPS J. 2013;15:477–482. [PMC free article] [PubMed]
82. Malan TP, Jr., Ibrahim MM, Vanderah TW, Makriyannis A, Porreca F. Inhibition of pain responses by activation of CB2 cannabinoid receptors. Chem Phys Lipids. 2002;121:191–200. [PubMed]
83. Hanus L, Breuer A, Tchilibon S, Shiloah S, Goldenberg D, Horowitz M, Pertwee RG, Ross RA, Mechoulam R, Fride E. HU-308: a specific agonist for CB2, a peripheral cannabinoid receptor. Proc Natl Acad Sci U S A. 1999;96:14228–14233. [PMC free article][PubMed]
84. Ibrahim MM, Deng H, Zvonok A, Cockayne DA, Kwan J, Mata HP, Vanderah TW, Lai J, Porreca F, Makriyannis A, Malan TP., Jr. Activation of CB2 cannabinoid receptors by AM1241 inhibits experimental neuropathic pain: pain inhibition by receptors not present in the CNS. Proc Natl Acad Sci U S A. 2003;100:10529–10533. [PMC free article] [PubMed]
85. Nackley AG, Makriyannis A, Hohmann AG. Selective activation of cannabinoid CB2 receptors suppresses spinal fos protein expression and pain behavior in a rat model of inflammation. Neuroscience. 2003;119:747–757. [PubMed]
86. Rahn EJ, Zvonok AM, Makriyannis A, Hohmann AG. Antinociceptive effects of racemic AM1241 and its chirally synthesized enantiomers: lack of dependence upon opioid receptor activation. AAPS J. 2010;12:147–157. [PMC free article] [PubMed]
87. Quartilho A, Mata HP, Ibrahim MM, Vanderah TW, Porreca F, Makriyannis A, Malan TP., Jr. Inhibition of inflammatory hyperalgesia by activation of peripheral CB2 cannabinoid receptors. Anesthesiology. 2003;99:955–960. [PubMed]
88. Hohmann AG, Farthing JN, Zvonok AM, Makriyannis A. Selective activation of cannabinoid CB2 receptors suppresses hyperalgesia evoked by intradermal capsaicin. J Pharmacol Exp Ther. 2004;308:446–453. [PubMed]
89. Nackley AG, Zvonok AM, Makriyannis A, Hohmann AG. Activation of cannabinoid CB2 receptors suppresses C-fiber responses and windup in spinal wide dynamic range neurons in the absence and presence of inflammation. J Neurophysiol. 2004;92:3562–3574.[PubMed]
90. Ibrahim MM, Porreca F, Lai J, Albrecht PJ, Rice FL, Khodorova A, Davar G, Makriyannis A, Vanderah TW, Mata HP, Malan TP., Jr. CB2 cannabinoid receptor activation produces antinociception by stimulating peripheral release of endogenous opioids. Proc Natl Acad Sci U S A. 2005;102:3093–3098. [PMC free article] [PubMed]
91. Ibrahim MM, Rude ML, Stagg NJ, Mata HP, Lai J, Vanderah TW, Porreca F, Buckley NE, Makriyannis A, Malan TP., Jr. CB2 cannabinoid receptor mediation of antinociception. Pain.2006;122:36–42. [PubMed]
92. Khanolkar AD, Lu D, Ibrahim M, Duclos RI, Jr., Thakur GA, Malan TP, Jr., Porreca F, Veerappan V, Tian X, George C, Parrish DA, Papahatjis DP, Makriyannis A. Cannabilactones: a novel class of CB2 selective agonists with peripheral analgesic activity. J Med Chem. 2007;50:6493–6500. [PubMed]
93. Rahn EJ, Thakur GA, Wood JA, Zvonok AM, Makriyannis A, Hohmann AG. Pharmacological characterization of AM1710, a putative cannabinoid CB2 agonist from the cannabilactone class: antinociception without central nervous system side-effects. Pharmacol Biochem Behav. 2011;98:493–502. [PMC free article] [PubMed]
94. Wilkerson JL, Gentry KR, Dengler EC, Wallace JA, Kerwin AA, Kuhn MN, Zvonok AM, Thakur GA, Makriyannis A, Milligan ED. Immunofluorescent spectral analysis reveals the intrathecal cannabinoid agonist, AM1241, produces spinal anti-inflammatory cytokine responses in neuropathic rats exhibiting relief from allodynia. Brain Behav. 2012;2:155–177.[PMC free article] [PubMed]
95. Rahn EJ, Makriyannis A, Hohmann AG. Activation of cannabinoid CB1 and CB2 receptors suppresses neuropathic nociception evoked by the chemotherapeutic agent vincristine in rats. Br J Pharmacol. 2007;152:765–777. [PMC free article] [PubMed]
96. Rahn EJ, Zvonok AM, Thakur GA, Khanolkar AD, Makriyannis A, Hohmann AG. Selective activation of cannabinoid CB2 receptors suppresses neuropathic nociception induced by treatment with the chemotherapeutic agent paclitaxel in rats. J Pharmacol Exp Ther. 2008;327:584–591. [PMC free article] [PubMed]
97. Deng L, Guindon J, Vemuri VK, Thakur GA, White FA, Makriyannis A, Hohmann AG. The maintenance of cisplatin- and paclitaxel-induced mechanical and cold allodynia is suppressed by cannabinoid CB2 receptor activation and independent of CXCR4 signaling in models of chemotherapy-induced peripheral neuropathy. Mol Pain. 2012;8:71.[PMC free article] [PubMed]
98. Wilkerson JL, Gentry KR, Dengler EC, Wallace JA, Kerwin AA, Armijo LM, Kuhn MN, Thakur GA, Makriyannis A, Milligan ED. Intrathecal cannabilactone CB(2)R agonist, AM1710, controls pathological pain and restores basal cytokine levels. Pain. 2012;153:1091–1106. [PMC free article] [PubMed]
99. Bab I, Zimmer A. Cannabinoid receptors and the regulation of bone mass. Br J Pharmacol. 2008;153:182–188. [PMC free article] [PubMed]
100. Kim K, Moore DH, Makriyannis A, Abood ME. AM1241, a cannabinoid CB2 receptor selective compound, delays disease progression in a mouse model of amyotrophic lateral sclerosis. Eur J Pharmacol. 2006;542:100–105. [PubMed]
101. Storr MA, Keenan CM, Zhang H, Patel KD, Makriyannis A, Sharkey KA. Activation of the cannabinoid 2 receptor (CB2) protects against experimental colitis. Inflamm Bowel Dis.2009;15:1678–1685. [PubMed]
102. Xi ZX, Peng XQ, Li X, Song R, Zhang HY, Liu QR, Yang HJ, Bi GH, Li J, Gardner EL. Brain cannabinoid CB(2) receptors modulate cocaine’s actions in mice. Nat Neurosci.2011;14:1160–1166. [PMC free article] [PubMed]
103. Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, Trong IL, Teller DC, Okada T, Stenkamp RE, Yamamoto M, Miyano M. Crystal Structure of Rhodopsin: A G Protein-Coupled Receptor. Science. 2000;289:739–745. [PubMed]
104. Rosenbaum DM, Cherezov V, Hanson MA, Rasmussen SGF, Thian FS, Kobilka TS, Choi HJ, Yao XJ, Weis WI, Stevens RC, Kobilka BK. GPCR engineering yields high-resolution structural insights into beta(2)-adrenergic receptor function. Science.2007;318:1266–1273. [PubMed]
105. Venkatakrishnan AJ, Deupi X, Lebon G, Tate CG, Schertler GF, Babu MM. Molecular signatures of G-protein-coupled receptors. Nature. 2013;494:185–194. [PubMed]
106. Morse KL, Fournier DJ, Li XY, Grzybowska J, Makriyannis A. A Novel Electrophilic High-Affinity Irreversible Probe for the Cannabinoid Receptor. Life Sci. 1995;56:1957–1962.[PubMed]
107. Picone RP, Khanolkar AD, Morse KL, Reggio PH, Fournier DJ, Makriyannis A. Classical cannabinoid affinity labels suggest different binding motifs on CB1 receptors.. Symposium on the Cannabinoids; Burlington VT. 1999.
108. Chu C, Ramamurthy A, Makriyannis A, Tius MA. Synthesis of covalent probes for the radiolabeling of the cannabinoid receptor. J Org Chem. 2003;68:55–61. [PubMed]
109. Picone RP, Fournier DJ, Makriyannis A. Ligand based structural studies of the CB1 cannabinoid receptor. J Pept Res. 2002;60:348–356. [PubMed]
110. Zvonok N, Pandarinathan L, Williams J, Johnston M, Karageorgos I, Janero DR, Krishnan SC, Makriyannis A. Covalent inhibitors of human monoacylglycerol lipase: ligand-assisted characterization of the catalytic site by mass spectrometry and mutational analysis.Chem Biol. 2008;15:854–862. [PMC free article] [PubMed]
111. Zvonok N, Williams J, Johnston M, Pandarinathan L, Janero DR, Li J, Krishnan SC, Makriyannis A. Full mass spectrometric characterization of human monoacylglycerol lipase generated by large-scale expression and single-step purification. J Proteome Res.2008;7:2158–2164. [PMC free article] [PubMed]
112. West JM, Zvonok N, Whitten KM, Wood JT, Makriyannis A. Mass spectrometric characterization of human N-acylethanolamine-hydrolyzing acid amidase. J Proteome Res.2012;11:972–981. [PMC free article] [PubMed]
113. Filppula S, Yaddanapudi S, Mercier R, Xu W, Pavlopoulos S, Makriyannis A. Purification and mass spectroscopic analysis of human CB2 cannabinoid receptor expressed in the baculovirus system. J Pept Res. 2004;64:225–236. [PubMed]
114. Xu W, Filppula SA, Mercier R, Yaddanapudi S, Pavlopoulos S, Cai J, Pierce WM, Makriyannis A. Purification and mass spectroscopic analysis of human CB1 cannabinoid receptor functionally expressed using the baculovirus system. J Pept Res. 2005;66:138–150.[PubMed]
115. Zvonok N, Xu W, Williams J, Janero DR, Krishnan SC, Makriyannis A. Mass spectrometry-based GPCR proteomics: comprehensive characterization of the human cannabinoid 1 receptor. J Proteome Res. 2010;9:1746–1753. [PMC free article] [PubMed]
116. Zvonok N, Yaddanapudi S, Williams J, Dai S, Dong K, Rejtar T, Karger BL, Makriyannis A. Comprehensive proteomic mass spectrometric characterization of human cannabinoid CB2 receptor. J Proteome Res. 2007;6:2068–2079. [PubMed]
117. Le Foll B, Gorelick DA, Goldberg SR. The future of endocannabinoid-oriented clinical research after CB1 antagonists. Psychopharmacology (Berl) 2009;205:171–174.[PMC free article] [PubMed]
118. Christensen R, Kristensen PK, Bartels EM, Bliddal H, Astrup A. Efficacy and safety of the weight-loss drug rimonabant: a meta-analysis of randomised trials. Lancet.2007;370:1706–1713. [PubMed]
119. Cosenza M, Gifford AN, Gatley SJ, Pyatt B, Liu Q, Makriyannis A, Volkow ND. Locomotor activity and occupancy of brain cannabinoid CB1 receptors by the antagonist/inverse agonist AM281. Synapse. 2000;38:477–482. [PubMed]
120. Gatley SJ, Lan R, Volkow ND, Pappas N, King P, Wong CT, Gifford AN, Pyatt B, Dewey SL, Makriyannis A. Imaging the brain marijuana receptor: development of a radioligand that binds to cannabinoid CB1 receptors in vivo. J Neurochem. 1998;70:417–423.[PubMed]
121. Lan R, Gatley J, Lu Q, Fan P, Fernando SR, Volkow ND, Pertwee R, Makriyannis A. Design and synthesis of the CB1 selective cannabinoid antagonist AM281: a potential human SPECT ligand. AAPS PharmSci. 1999;1:E4. [PMC free article] [PubMed]
122. Lan R, Liu Q, Fan P, Lin S, Fernando SR, McCallion D, Pertwee R, Makriyannis A. Structure-activity relationships of pyrazole derivatives as cannabinoid receptor antagonists. J Med Chem. 1999;42:769–776. [PubMed]
123. McLaughlin PJ, Qian L, Wood JT, Wisniecki A, Winston KM, Swezey LA, Ishiwari K, Betz AJ, Pandarinathan L, Xu W, Makriyannis A, Salamone JD. Suppression of food intake and food-reinforced behavior produced by the novel CB1 receptor antagonist/inverse agonist AM 1387. Pharmacol Biochem Behav. 2006;83:396–402. [PubMed]
124. Le Foll B, Pushparaj A, Pryslawsky Y, Forget B, Vemuri VK, Makriyannis A, Trigo JM. Translational strategies for therapeutic development in nicotine addiction: Rethinking the conventional bench to bedside approach. Prog. Neuro-Psychopharmacol. Biol. Psychiatry.2013 In press, doi: 10.1016/j.pnpbp.2013.1010.1009. [PMC free article] [PubMed]
125. Cluny NL, Chambers AP, Vemuri VK, Wood JT, Eller LK, Freni C, Reimer RA, Makriyannis A, Sharkey KA. The neutral cannabinoid CB(1) receptor antagonist AM4113 regulates body weight through changes in energy intake in the rat. Pharmacol Biochem Behav. 2011;97:537–543. [PMC free article] [PubMed]
126. Storr MA, Bashashati M, Hirota C, Vemuri VK, Keenan CM, Duncan M, Lutz B, Mackie K, Makriyannis A, Macnaughton WK, Sharkey KA. Differential effects of CB(1) neutral antagonists and inverse agonists on gastrointestinal motility in mice.Neurogastroenterol Motil. 2010;22:787–796. e223. [PMC free article] [PubMed]
127. Salamone JD, McLaughlin PJ, Sink K, Makriyannis A, Parker LA. Cannabinoid CB1 receptor inverse agonists and neutral antagonists: effects on food intake, food-reinforced behavior and food aversions. Physiol Behav. 2007;91:383–388. [PMC free article] [PubMed]
128. Sink KS, McLaughlin PJ, Wood JA, Brown C, Fan P, Vemuri VK, Peng Y, Olszewska T, Thakur GA, Makriyannis A, Parker LA, Salamone JD. The novel cannabinoid CB1 receptor neutral antagonist AM4113 suppresses food intake and food-reinforced behavior but does not induce signs of nausea in rats. Neuropsychopharmacology. 2008;33:946–955.[PMC free article] [PubMed]
129. Sink KS, Segovia KN, Collins LE, Markus EJ, Vemuri VK, Makriyannis A, Salamone JD. The CB1 inverse agonist AM251, but not the CB1 antagonist AM4113, enhances retention of contextual fear conditioning in rats. Pharmacol Biochem Behav. 2010;95:479–484. [PubMed]
130. Sink KS, Segovia KN, Sink J, Randall PA, Collins LE, Correa M, Markus EJ, Vemuri VK, Makriyannis A, Salamone JD. Potential anxiogenic effects of cannabinoid CB1 receptor antagonists/inverse agonists in rats: comparisons between AM4113, AM251, and the benzodiazepine inverse agonist FG-7142. Eur Neuropsychopharmacol. 2010;20:112–122.[PMC free article] [PubMed]
131. Sink KS, Vemuri VK, Wood J, Makriyannis A, Salamone JD. Oral bioavailability of the novel cannabinoid CB1 antagonist AM6527: effects on food-reinforced behavior and comparisons with AM4113. Pharmacol Biochem Behav. 2009;91:303–306.[PMC free article] [PubMed]
132. Limebeer CL, Vemuri VK, Bedard H, Lang ST, Ossenkopp KP, Makriyannis A, Parker LA. Inverse agonism of cannabinoid CB1 receptors potentiates LiCl-induced nausea in the conditioned gaping model in rats. Br J Pharmacol. 2010;161:336–349. [PMC free article][PubMed]
133. Hodge J, Bow JP, Plyler KS, Vemuri VK, Wisniecki A, Salamone JD, Makriyannis A, McLaughlin PJ. The cannabinoid CB1 receptor inverse agonist AM 251 and antagonist AM 4113 produce similar effects on the behavioral satiety sequence in rats. Behav Brain Res.2008;193:298–305. [PubMed]
134. Bergman J, Delatte MS, Paronis CA, Vemuri K, Thakur GA, Makriyannis A. Some effects of CB1 antagonists with inverse agonist and neutral biochemical properties.Physiology & Behavior. 2008;93:666–670. [PMC free article] [PubMed]
135. Chambers AP, Vemuri VK, Peng Y, Wood JT, Olszewska T, Pittman QJ, Makriyannis A, Sharkey KA. A neutral CB1 receptor antagonist reduces weight gain in rat. Am J Physiol Regul Integr Comp Physiol. 2007;293:R2185–2193. [PubMed]
136. Janero DR, Lindsley L, Vemuri VK, Makriyannis A. Cannabinoid 1 G protein-coupled receptor (periphero-)neutral antagonists: emerging therapeutics for treating obesity-driven metabolic disease and reducing cardiovascular risk. Expert Opin Drug Discov. 2011;6:995–1025. [PubMed]
137. Tam J, Vemuri VK, Liu J, Batkai S, Mukhopadhyay B, Godlewski G, Osei-Hyiaman D, Ohnuma S, Ambudkar SV, Pickel J, Makriyannis A, Kunos G. Peripheral CB1 cannabinoid receptor blockade improves cardiometabolic risk in mouse models of obesity. J Clin Invest.2010;120:2953–2966. [PMC free article] [PubMed]
138. Cluny NL, Vemuri VK, Chambers AP, Limebeer CL, Bedard H, Wood JT, Lutz B, Zimmer A, Parker LA, Makriyannis A, Sharkey KA. A novel peripherally restricted cannabinoid receptor antagonist, AM6545, reduces food intake and body weight, but does not cause malaise, in rodents. Br J Pharmacol. 2010;161:629–642. [PMC free article] [PubMed]
139. Randall PA, Vemuri VK, Segovia KN, Torres EF, Hosmer S, Nunes EJ, Santerre JL, Makriyannis A, Salamone JD. The novel cannabinoid CB1 antagonist AM6545 suppresses food intake and food-reinforced behavior. Pharmacol Biochem Behav. 2010;97:179–184.[PMC free article] [PubMed]
140. Sharma R, Nikas SP, Paronis CA, Wood JT, Halikhedkar A, Guo JJ, Thakur GA, Kulkarni S, Benchama O, Raghav JG, Gifford RS, Jarbe TU, Bergman J, Makriyannis A. Controlled-deactivation cannabinergic ligands. J Med Chem. 2013;56:10142–10157.[PMC free article] [PubMed]
141. Sharma R NS, Guo JJ, Mallipeddi S, Wood JT, Makriyannis A. C-Ring Cannabinoid Lactones: A Novel Cannabinergic Chemotype. ACS Med Chem Lett. 2014 In press, doi:org/10.1021/ml4005304. [PMC free article] [PubMed]
142. Deutsch DG, Lin S, Hill WA, Morse KL, Salehani D, Arreaza G, Omeir RL, Makriyannis A. Fatty acid sulfonyl fluorides inhibit anandamide metabolism and bind to the cannabinoid receptor. Biochem Biophys Res Commun. 1997;231:217–221. [PubMed]
143. Deutsch DG, Makriyannis A. Inhibitors of anandamide breakdown. NIDA Res Monogr.1997;173:65–84. [PubMed]
144. Qin C, Lin S, Lang W, Goutopoulos A, Pavlopoulos S, Mauri F, Makriyannis A. Determination of anandamide amidase activity using ultraviolet-active amine derivatives and reverse-phase high-performance liquid chromatography. Anal Biochem. 1998;261:8–15.[PubMed]
145. Lang W, Qin C, Hill WA, Lin S, Khanolkar AD, Makriyannis A. High-performance liquid chromatographic determination of anandamide amidase activity in rat brain microsomes. Anal Biochem. 1996;238:40–45. [PubMed]
146. Qin C. PhD Thesis: Purificaion and charaterization of anandamide amidase and development of novel inhibitors. University of Connecticut; Storrs, CT: 1998.
147. Bracey MH, Hanson MA, Masuda KR, Stevens RC, Cravatt BF. Structural Adaptations in a Membrane Enzyme That Terminates Endocannabinoid Signaling. Science.2002;298:1793–1796. [PubMed]
148. Mor M, Rivara S, Lodola A, Plazzi PV, Tarzia G, Duranti A, Tontini A, Piersanti G, Kathuria S, Piomelli D. Cyclohexylcarbamic acid 3′- or 4′-substituted biphenyl-3-yl esters as fatty acid amide hydrolase inhibitors: synthesis, quantitative structure-activity relationships, and molecular modeling studies. J Med Chem. 2004;47:4998–5008. [PubMed]
149. Karanian DA, Brown QB, Makriyannis A, Kosten TA, Bahr BA. Dual modulation of endocannabinoid transport and fatty acid amide hydrolase protects against excitotoxicity. J Neurosci. 2005;25:7813–7820. [PubMed]
150. Karanian DA, Karim SL, Wood JT, Williams JS, Lin S, Makriyannis A, Bahr BA. Endocannabinoid enhancement protects against kainic acid-induced seizures and associated brain damage. J Pharmacol Exp Ther. 2007;322:1059–1066. [PubMed]
151. Bahr BA, Karanian DA, Makanji SS, Makriyannis A. Targeting the endocannabinoid system in treating brain disorders. Expert Opin Investig Drugs. 2006;15:351–365. [PubMed]
152. Karanian DA, Brown QB, Makriyannis A, Bahr BA. Blocking cannabinoid activation of FAK and ERK1/2 compromises synaptic integrity in hippocampus. Eur J Pharmacol.2005;508:47–56. [PubMed]
153. Johnson DS, Stiff C, Lazerwith SE, Kesten SR, Fay LK, Morris M, Beidler D, Liimatta MB, Smith SE, Dudley DT, Sadagopan N, Bhattachar SN, Kesten SJ, Nomanbhoy TK, Cravatt BF, Ahn K. Discovery of PF-04457845: A Highly Potent, Orally Bioavailable, and Selective Urea FAAH Inhibitor. ACS Med Chem Lett. 2011;2:91–96. [PMC free article][PubMed]
154. Rengachari S, Aschauer P, Gruber K, Dreveny I, Oberer M. Crystal structure of monoacylglycerol lipase from Bacillus sp. H257 in complex with monopalmitoylglycerol analog. J Biol Chem. 2013;288:31093–311104. [PMC free article] [PubMed]
155. Schalk-Hihi C, Schubert C, Alexander R, Bayoumy S, Clemente JC, Deckman I, DesJarlais RL, Dzordzorme KC, Flores CM, Grasberger B, Kranz JK, Lewandowski F, Liu L, Ma H, Maguire D, Macielag MJ, McDonnell ME, Mezzasalma Haarlander T, Miller R, Milligan C, Reynolds C, Kuo LC. Crystal structure of a soluble form of human monoglyceride lipase in complex with an inhibitor at 1.35 A resolution. Protein Sci.2011;20:670–683. [PMC free article] [PubMed]
156. Karageorgos I, Wales TE, Janero DR, Zvonok N, Vemuri VK, Engen JR, Makriyannis A. Active-Site Inhibitors Modulate the Dynamic Properties of Human Monoacylglycerol Lipase: A Hydrogen Exchange Mass Spectrometry Study. Biochemistry. 2013[PMC free article] [PubMed]
157. Ramesh D, Gamage TF, Vanuytsel T, Owens RA, Abdullah RA, Niphakis MJ, Shea-Donohue T, Cravatt BF, Lichtman AH. Dual inhibition of endocannabinoid catabolic enzymes produces enhanced antiwithdrawal effects in morphine-dependent mice.Neuropsychopharmacology. 2013;38:1039–1049. [PMC free article] [PubMed]
158. Long JZ, Nomura DK, Vann RE, Walentiny DM, Booker L, Jin X, Burston JJ, Sim-Selley LJ, Lichtman AH, Wiley JL, Cravatt BF. Dual blockade of FAAH and MAGL identifies behavioral processes regulated by endocannabinoid crosstalk in vivo. Proc Natl Acad Sci U S A. 2009;106:20270–20275. [PMC free article] [PubMed]
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