The human cannabinoid 1 receptor (hCB1) is involved in numerous physiological processes and therefore provides a wide scope of potential therapeutic opportunities to treat maladies such as obesity, cardio-metabolic disorders, substance abuse, neuropathic pain, and multiple sclerosis. Structure-based drug design using the current knowledge of the hCB1 receptor binding site is limited and requires purified active protein. Heterologous expression and purification of functional hCB1 has been the bottleneck for ligand binding structural studies using biophysical methods such as mass spectrometry, x-ray crystallography and NMR. We constructed several plasmids for in-cell or in vitro Escherichia coli (E. coli) based expression of truncated and stabilized hCB1 receptor (hΔCB1 and hΔCB1T4L) variants and evaluated their competency to bind the CP-55,940 ligand. MALDI-TOF MS analysis of in vitro expressed and purified hΔCB1T4Lhis6 variants, following trypsin digestion, generated ~80% of the receptor sequence coverage. Our data demonstrate the feasibility of a cell-free expression system as a promising part of the strategy for the elucidation of ligand binding sites of the hCB1 receptor using a "Ligand Assisted Protein Structure" (LAPS) approach.

New oximes short-acting CB1 agonists were explored by the introduction of an internal oxime and polar groups at the C3 alkyl tail of Delta(8)-THC. The scope of the research was to drastically alter two important physicochemical properties hydrophobicity (log P) and topological surface area (tPSA) of the compound, which play a critical role in tissue distribution and sequestration (depot effect). Key synthesized analogs demonstrated sub-nanomolar affinity for CB1, marked reduction in hydrophobicity (ClogP similar to 2.5-3.5 vs 9.09 of Delta(8)-THC-DMH), and found to function as either agonists (trans-oximes) or neutral antagonists (cis-oximes) in a cAMP functional assay. All oxime analogs showed comparable affinity at the CB2 receptor, but surprisingly they were found to function as inverse agonists for CB2. In behavioral studies (i.e. analgesia, hypothermia) trans-oxime 8a exhibited a predictable fast onset (similar to 20 min) and short duration of pharmacological action (similar to 180 min), in contrast to the very prolonged duration of Delta(8)-THC-DMH (> 24 h), thus limiting the potential for severe psychotropic side-effects associated with persistent activation of the CB1 receptor. We have conducted 100 ns molecular dynamic (MD) simulations of CB1 complexes with AM11542 (CB1 agonist) and both trans-8a and cis-8b isomeric oximes. These studies revealed that the C3 alkyl tail of cis-8b orientated within the CB1 binding pocket in a manner that triggered a conformational change that stabilized the CB1 receptor at its inactive-state (antagonistic functional effect). In contrast, the trans-8a isomer's conformation was coincided with that of the AM11542 CB1 agonistbound structure, stabilizing the CB1 receptor at the active-state (agonistic functional effect). We have selected oxime trans-8a based on its potency for CB1, and favorable pharmacodynamic profile, such as fast onset and predictable duration of pharmacological action, for evaluation in pre-clinical models of anorexia nervosa.

Cannabinoid 2 receptor (CB2R), a Class-A G-protein coupled receptor (GPCR), is a promising drug target under a wide array of pathological conditions. Rational drug design has been hindered due to our poor understanding of the structural features involved in ligand binding. Binding of a high-affinity biarylpyrazole inverse agonist AM1336 to a library of the human CB2 receptor (hCB2R) cysteine-substituted mutants provided indirect evidence that two cysteines in transmembrane helix-7 (H7) were critical for the covalent attachment. We used proteomics analysis of the hCB2R with bound AM1336 to directly identify peptides with covalently attached ligand and applied in silico modeling for visualization of the ligand-receptor interactions. The hCB2R, with affinity tags (FlaghCB2His6), was produced in a baculovirus-insect cell expression system and purified as a functional receptor using immunoaffinity chromatography. Using mass spectrometry-based bottom-up proteomic analysis of the hCB2R-AM1336, we identified a peptide with AM1336 attached to the cysteine C284(7.38) in H7. The hCB2R homology model in lipid bilayer accommodated covalent attachment of AM1336 to C284(7.38), supporting both biochemical and mass spectrometric data. This work consolidates proteomics data and in silico modeling and integrates with our ligand-assisted protein structure (LAPS) experimental paradigm to assist in structure-based design of cannabinoid antagonist/inverse agonists.

The phenomenon of functional selectivity, whereby a ligand preferentially directs the information output of a G-protein coupled receptor (GPCR) along (a) particular effector pathway(s) and away from others, has redefined traditional GPCR signaling paradigms to provide a new approach to structure-based drug design. The two principal cannabinoid receptors (CBRs) 1 and 2 belong to the class-A GPCR subfamily and are considered tenable therapeutic targets for several indications. Yet conventional orthosteric ligands (agonists, antagonists/inverse agonists) for these receptors have had very limited clinical utility due to their propensity to incite on-target adverse events. Chemically distinct classes of cannabinergic ligands exhibit signaling bias at CBRs towards individual subsets of signal transduction pathways. In this review, we discuss the known signaling pathways regulated by CBRs and examine the current evidence for functional selectivity at CBRs in response to endogenous and exogenous cannabinergic ligands as biased agonists. We further discuss the receptor and ligand structural features allowing for selective activation of CBR-dependent functional responses. The design and development of biased ligands may offer a pathway to therapeutic success for novel CBR-targeted drugs.

In pursuit of safer controlled-deactivation cannabinoids with high potency and short duration of action, we report the design, synthesis, and pharmacological evaluation of novel C9- and C11-hydroxy-substituted hexahydrocannabinol (HHC) and tetrahydrocannabinol (THC) analogues in which a seven atom long side chain, with or without 1'-substituents, carries a metabolically labile 2',3'-ester group. Importantly, in vivo studies validated our controlled deactivation approach in rodents and non-human primates. The lead molecule identified here, namely, butyl-2-[(6aR,9R,10aR)-1-hydroxy-9-(hydroxymethyl)-6,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-3-yl]-2-methylpropanoate (AM7499), was found to exhibit remarkably high in vitro and in vivo potency with shorter duration of action than the currently existing classical cannabinoid agonists.

The aliphatic side chain plays a pivotal role in determining the cannabinergic potency of tricyclic classical cannabinoids, and we have previously shown that this chain could be substituted successfully by adamantyl or other polycyclic groups. In an effort to explore the pharmacophoric features of these conformationally fixed groups, we have synthesized a series of analogues in which the C3 position is substituted directly with an adamantyl group bearing functionality at one of the tertiary carbon atoms. These substituents included the electrophilic isothiocyanate and photoactivatable azido groups, both of which are capable of covalent attachment with the target protein. Our results show that substitution at the 3'-adamantyl position can lead to ligands with improved affinities and CB1/CB2 selectivities. Our work has also led to the development of two successful covalent probes with high affinities for both cannabinoid receptors, namely, the electrophilic isothiocyanate AM994 and the photoactivatable aliphatic azido AM993 analogues.

Cannabinoid 2 receptor (CB2) belongs to the Class A family of G-protein coupled receptors and is expressed predominantly in the periphery and to a lower extent in the CNS. CB2 is an attractive target for pain management and other neurological and peripheral inflammatory disorders. CB2 upon activation by endogenous or synthetic ligands, modulate a diverse set of downstream signaling pathways. These responses are dictated by the structural conformation of CB2 following ligand binding. Therefore, detailed structural studies of CB2 in lipid bilayer membranes in the presence of the cannabinergic ligands are vital for the development of effective drugs. Here, we report the reconstitution of functional, purified human CB2 into novel lipid bilayer membrane mimic (nanodiscs), for structural studies. Using a baculovirus expression system, we expressed functional, full-length human CB2. The radioligand [3H] CP55940 binding assays revealed a Bmax of ~500 pmol/g and KD of 0.68 nM. We were able to purify, using immunoaffinity chromatography, a functional receptor with yields of ~200 ug/L of culture. The receptor was reconstituted into POPC/POPG nanodiscs and separated from empty nanodiscs using additional purification steps. CB2-incorporated nanodiscs were further evaluated using SDS-PAGE, western blotting, radioligand binding assay and proteomic mass spectrometric (MS) analysis. The CB2-nanodisc preparation would further be studied using hydrogen deuterium exchange MS, to elucidate protein structure and dynamics upon ligand binding. The work was supported by grants DA9158 and DA3801 to AM.

As a part of our controlled-deactivation ligand development project, we recently disclosed a series of (-)-Δ(8)-tetrahydrocannabinols (THCs) with a metabolically labile ester group at the 2'-position of the side chain. Now, we have replaced the C-ring in the classical THC structure with a hydrolyzable seven-membered lactone. One of the synthesized analogues binds with high affinity to the CB1 receptor (K i = 4.6 nM) and exhibits much lower affinities for the mCB2 and the hCB2. Also, in vitro functional characterization found the compound to be an agonist at rCB1. Consistent with our rational design, the lead cannabinergic lactone identified here is susceptible to metabolic inactivation by plasma esterases, while the respective acid metabolite is inactive at CB receptors. These results are highlighted with molecular modeling of the two regiosomeric lactones.

The Cannabinoid 1 receptor (CB1) is a type A G-protein coupled receptor (GPCR) expressed most widely in the human brain and has been identified as a key receptor in modulating a number of physiological functions of the human body. Therefore, drugs acting on the CB1 have an enormous pharmacotherapeutic potential in disease conditions such as chronic neuropathic pain, cardio-metabolic diseases, obesity, and even in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. In order to develop CB1-specific, high-affinity ligands, a better understanding of the structural aspects of the receptor in its native state is essential. Using an E. coli-based cell-free (in vitro) expression system in addition with nanolipid bilayers (Nanodiscs), we developed a convenient method for efficient production of the re-engineered human cannabinoid receptor and showed proof of functionality of the expressed receptor using a radioligand-binding assay. The saturation binding curve with CP55,940, a non-classical bicyclic analog of Δ9-tetrahydrocannabinol (Δ9-THC), showed Bmax of 1358 pmoles/g and a Kd of 8.57 nM. The total amount of functional receptor was calculated to be 500 ng/ml of expression reaction.

Cannabinoid receptors are Class A G-protein coupled receptors (GPCRs) and are involved in the modulation of a diverse set of signaling pathways in the human body. The two subtypes of cannabinoid receptors are Cannabinoid 1 receptor (CB1) and Cannabinoid 2 receptor (CB2), which are expressed predominantly in the human brain and in the immune system, respectively. Both receptors are considered to be key drug targets with enormous therapeutic potential in disease conditions ranging from neuropathic pain and neurodegenerative disorders to cardio-metabolic and inflammatory disorders. In order to develop high-affinity, selective Cannabinergic ligands with specific downstream signaling effects, we need to better understand the structural aspects of the receptor in its native environment.