Design and Synthesis of Highly Potent and Specific ABHD6 Inhibitors.

Fine-tuning than complete disruption of 2-arachidonoylglycerol (2-AG) metabolism in the brain represents a promising pharmacological approach to limit potential untoward effects associated with complete blockade of monoacylglycerol lipase (MGL), the primary hydrolase of 2-AG. This could be achieved through a/b-hydrolase domain containing 6 (ABHD6) inhibition, which will provide a smaller and safer contribution to 2-AG regulation in the brain. Pharmacological studies with ABHD6 inhibitors have recently been reported, where modulation of ABHD6 activity either through CB1R-dependent or CB1R-independent processes showed promise in preclinical models of epilepsy, neuropathic pain and inflammation. Furthermore in the periphery, ABHD6 modulates 2-AG and other fatty acid monoacylglycerols (MAGs) and is implicated in Type-2 diabetes, metabolic syndrome and potentially other diseases. Herein, we report the discovery of single-digit nanomolar potent and highly specific ABHD6 inhibitors with >1000-fold selectivity against MGL and FAAH. The new ABHD6 inhibitors provide early leads to develop therapeutics for neuroprotection and the treatment of inflammation and diabetes.

Improved cyclobutyl nabilone analogs as potent CB1 receptor agonists.

In earlier work, we explored the SAR for the C3 side chain pharmacophore in the hexahydrocannabinol template represented by the drug nabilone, which resulted in the development of AM2389. In an effort for further optimization, we have merged features of nabilone and AM2389 and explored the C3 side chain with varying chain lengths and terminal substitutions. Of the compounds described here, a nabilone analog, AM8936, with the C6'-cyano-substituted side chain, was identified as the most successful analog capable of serving as a potential candidate for further development and a valuable tool for further in vivo studies. AM8936 behaved as a balanced and potent CB1 agonist in functional assays and was a potent and efficacious CB1 agonist in vivo. Our SAR studies are highlighted with the docking of AM8936 on the crystal structure of the hCB1 receptor.

1-, 2- and 3-AG as substrates of the endocannabinoid enzymes and endogenous ligands of the cannabinoid receptor 1.

2-Arachidonoylglycerol (2-AG) is the most potent and abundant endocannabinoid that acts as a full agonist at the cannabinoid 1 (CB1) and 2 (CB2) receptors. It serves as a substrate for several serine hydrolases, including monoacylglycerol lipase (MGL), α/ß hydrolase domain 6 (ABHD6) and fatty acid amide hydrolase (FAAH). However, 2-AG's rapid conversion to 1-AG (the S stereoisomer) and 3-AG (the R stereoisomer) complicates in vivo signaling. Here, we present the interaction profiles of 2-AG and its isomerization products, 1- and 3-AG, with the endocannabinoid MGL, ABHD6 and FAAH enzymes as well as the CB1 receptor. The 1- and 3-AG enantiomers are less prone to isomerization, and their affinities to endocannabinoid enzymes and potencies at CB1 receptor are quite different compared to 2-AG. Although MGL is the principal hydrolytic enzyme of 2-AG, 3-AG (the R isomer) appears to be the best substrate for hMGL. Contrarily, 1-AG (the S isomer) demonstrates the worst substrate profile, indicating that the stereochemistry of 1(3)-monoacylglycerols is very important for MGL enzyme. On the other hand, both 1- and 3-AG (the sn1 monoacylglycerols) are efficiently hydrolyzed by hABHD6 without preference, while 2-AG (the sn2 monoacylglycerol) has the lowest rate of hydrolysis. FAAH, the principal hydrolytic enzyme for arachidonoylethanolamide (anandamide, AEA), catalyzes the hydrolysis of all three isomers with similar efficiencies. In a functional cAMP assay at CB1 receptor, all three isomers behaved as agonists, with 2-AG being the most potent, followed by 3-AG then 1-AG. The presented data provides stereochemical insights to design chemically stable AG analogs with preferential stability against enzymes of interest.

Synthon-based ligand discovery in virtual libraries of over 11 billion compounds.

Structure-based virtual ligand screening is emerging as a key paradigm for early drug discovery owing to the availability of high-resolution target structures1-4 and ultra-large libraries of virtual compounds5,6. However, to keep pace with the rapid growth of virtual libraries, such as readily available for synthesis (REAL) combinatorial libraries7, new approaches to compound screening are needed8,9. Here we introduce a modular synthon-based approach-V-SYNTHES-to perform hierarchical structure-based screening of a REAL Space library of more than 11 billion compounds. V-SYNTHES first identifies the best scaffold-synthon combinations as seeds suitable for further growth, and then iteratively elaborates these seeds to select complete molecules with the best docking scores. This hierarchical combinatorial approach enables the rapid detection of the best-scoring compounds in the gigascale chemical space while performing docking of only a small fraction (<0.1%) of the library compounds. Chemical synthesis and experimental testing of novel cannabinoid antagonists predicted by V-SYNTHES demonstrated a 33% hit rate, including 14 submicromolar ligands, substantially improving over a standard virtual screening of the Enamine REAL diversity subset, which required approximately 100 times more computational resources. Synthesis of selected analogues of the best hits further improved potencies and affinities (best inhibitory constant (Ki) = 0.9 nM) and CB2/CB1 selectivity (50-200-fold). V-SYNTHES was also tested on a kinase target, ROCK1, further supporting its use for lead discovery. The approach is easily scalable for the rapid growth of combinatorial libraries and potentially adaptable to any docking algorithm.

Novel Functionalized Cannabinoid Receptor Probes: Development of Exceptionally Potent Agonists.

We report the development of novel cannabinergic probes that can stabilize the cannabinoid receptors (CBRs) through tight binding interactions. Ligand design involves the introduction of select groups at a judiciously chosen position within the classical hexahydrocannabinol template (monofunctionalized probes). Such groups include the electrophilic isothiocyanato, the photoactivatable azido, and the polar cyano moieties. These groups can also be combined to produce bifunctionalized probes potentially capable of interacting at two distinct sites within the CBR-binding domains. These novel compounds display remarkably high binding affinities for CBRs and are exceptionally potent agonists. A key ligand (27a, AM11245) exhibits exceptionally high potency in both in vitro and in vivo assays and was designated as "megagonist," a property attributed to its tight binding profile. By acting both centrally and peripherally, 27a distinguishes itself from our previously reported "megagonist" AM841, whose functions are restricted to the periphery.

Oxa-adamantyl cannabinoids.

As a continuation of earlier work on classical cannabinoids bearing bulky side chains we report here the design, synthesis, and biological evaluation of 3'-functionalized oxa-adamantyl cannabinoids as a novel class of cannabinergic ligands. Key synthetic steps involve nucleophilic addition/transannular cyclization of aryllithium to epoxyketone in the presence of cerium chloride and stereoselective construction of the tricyclic cannabinoid nucleus. The synthesis of the oxa-adamantyl cannabinoids is convenient, and amenable to scale up allowing the preparation of these analogs in sufficient quantities for detailed in vitro evaluation. The novel oxa-adamantyl cannabinoids reported here were found to be high affinity ligands for the CB1 and CB2 cannabinoid receptors. In the cyclase assay these compounds were found to behave as potent and efficacious CB1 receptor agonists. Isothiocyanate analog AM10504 is capable of irreversibly labeling both the CB1 and CB2 receptors.

Conformational gating, dynamics and allostery in human monoacylglycerol lipase.

Inhibition of human Monoacylglycerol Lipase (hMGL) offers a novel approach for treating neurological diseases. The design of inhibitors, targeting active-inactive conformational transitions of the enzyme, can be aided by understanding the interplay between structure and dynamics. Here, we report the effects of mutations within the catalytic triad on structure, conformational gating and dynamics of hMGL by combining kinetics, NMR, and HDX-MS data with metadynamics simulations. We found that point mutations alter delicate conformational equilibria between active and inactive states. HDX-MS reveals regions of the hMGL that become substantially more dynamic upon substitution of catalytic acid Asp-239 by alanine. These regions, located far from the catalytic triad, include not only loops but also rigid α-helixes and ß-strands, suggesting their involvement in allosteric regulation as channels for long-range signal transmission. The results identify the existence of a preorganized global communication network comprising of tertiary (residue-residue contacts) and quaternary (rigid-body contacts) networks that mediate robust, rapid intraprotein signal transmission. Catalytic Asp-239 controls hMGL allosteric communications and may be considered as an essential residue for the integration and transmission of information to enzymes' remote regions, in addition to its well-known role to facilitate Ser-122 activation. Our findings may assist in the identification of new druggable sites in hMGL.

Activation and Signaling Mechanism Revealed by Cannabinoid Receptor-Gi Complex Structures.

Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB1 and CB2. Their high sequence similarity, low agonist selectivity, and lack of activation and G protein-coupling knowledge have hindered the development of therapeutic applications. Importantly, missing structural information has significantly held back the development of promising CB2-selective agonist drugs for treating inflammatory and neuropathic pain without the psychoactivity of CB1. Here, we report the cryoelectron microscopy structures of synthetic cannabinoid-bound CB2 and CB1 in complex with Gi, as well as agonist-bound CB2 crystal structure. Of important scientific and therapeutic benefit, our results reveal a diverse activation and signaling mechanism, the structural basis of CB2-selective agonists design, and the unexpected interaction of cholesterol with CB1, suggestive of its endogenous allosteric modulating role.

Biochemical and Proteomic Characterization of Recombinant Human α/ß Hydrolase Domain 6.

Human alpha/beta hydrolase domain 6 (hABHD6) is an enzyme that hydrolyzes 2-arachidonoylglycerol (2-AG), a potent agonist at both cannabinoid CB1 and CB2 receptors. In vivo modulation of ABHD6 expression has been shown to have potential therapeutic applications, making the enzyme a promising drug target. However, the lack of structural information on hABHD6 limits the discovery and design of selective inhibitors. We have performed E. coli expression, purification and activity profiling screening of different hABHD6 constructs and identified a truncated variant without N-terminal transmembrane (TM) domain, hΔ29-3-ABHD6, as the most promising protein for further characterization. The elimination of the TM domain did not affect 2-AG or fluorogenic arachidonoyl, 7-hydroxy-6-methoxy-4-methylcoumarin ester (AHMMCE) substrates hydrolysis, suggesting that the TM is not essential for enzyme catalytic activity. The hΔ29-3-ABHD6 variant was purified in a single step using Immobilized Metal Affinity Chromatography (IMAC), in-solution trypsin digested, and proteomically characterized by Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). The N-terminal peptide without methionine was identified indicating on a post-translational modification of the recombinant protein. The mechanism of inhibition of hABHD6 with AM6701 and WWL70 covalent probes was elucidated based on MS analysis of trypsin digested hABHD6 following the Ligand Assisted Protein Structure (LAPS) approach. We identified the carbamylated peptides containing catalytic serine (Ser148) suggesting a selective carbamylation of the enzyme by AM6701 or WWL70 and confirming an essential role of this residue in catalysis. The ability to produce substantial quantities of functional, pure hABHD6 will aid in the downstream structural characterization, and development of potent, selective inhibitors.

Crystal Structure of the Human Cannabinoid Receptor CB2.

The cannabinoid receptor CB2 is predominately expressed in the immune system, and selective modulation of CB2 without the psychoactivity of CB1 has therapeutic potential in inflammatory, fibrotic, and neurodegenerative diseases. Here, we report the crystal structure of human CB2 in complex with a rationally designed antagonist, AM10257, at 2.8 Å resolution. The CB2-AM10257 structure reveals a distinctly different binding pose compared with CB1. However, the extracellular portion of the antagonist-bound CB2 shares a high degree of conformational similarity with the agonist-bound CB1, which led to the discovery of AM10257's unexpected opposing functional profile of CB2 antagonism versus CB1 agonism. Further structural analysis using mutagenesis studies and molecular docking revealed the molecular basis of their function and selectivity for CB2 and CB1. Additional analyses of our designed antagonist and agonist pairs provide important insight into the activation mechanism of CB2. The present findings should facilitate rational drug design toward precise modulation of the endocannabinoid system.

Aliphatic Azides as Selective Cysteine Labeling Reagents for Integral Membrane Proteins.

Upon ultraviolet activation, cannabinergic aliphatic azido (N3) ligands covalently label cannabinoid receptors, prominent G-protein-coupled receptor (GPCR) drug targets. We report here the mechanism of covalent attachment to selected substrates of the high-affinity CBR inverse agonist AM1335 and its deuterated analog AM1335(d10), arylpyrazole compounds with an azide moiety at their n-pentyl side chain. To model the receptor interaction, we utilized the human cannabinoid 2 receptor (hCB2R) transmembrane helix 6 (TMH6) peptide and an N-acyl-protected cysteine (NAC). The photochemical reaction products of model substrates with AM1335 and AM1335(d10) were analyzed with tandem electrospray ionization mass spectrometry fragmentation and deuterium exchange mass spectrometry. The nitrene initially formed after photoreaction undergoes rearrangement to an imine which then interacts with the cysteine sulfhydryl group, resulting in ligand attachment. Our results demonstrate that covalent probes carrying aliphatic azides behave more selectively than originally thought and can be used to label protein cysteine residues preferentially.

( R)- N-(1-Methyl-2-hydroxyethyl)-13-( S)-methyl-arachidonamide (AMG315): A Novel Chiral Potent Endocannabinoid Ligand with Stability to Metabolizing Enzymes.

The synthesis of potent metabolically stable endocannabinoids is challenging. Here we report a chiral arachidonoyl ethanolamide (AEA) analogue, namely, (13 S,1' R)-dimethylanandamide (AMG315, 3a), a high affinity ligand for the CB1 receptor ( Ki of 7.8 ± 1.4 nM) that behaves as a potent CB1 agonist in vitro (EC50 = 0.6 ± 0.2 nM). (13 S,1' R)-dimethylanandamide is the first potent AEA analogue with significant stability for all endocannabinoid hydrolyzing enzymes as well as the oxidative enzymes COX-2. When tested in vivo using the CFA-induced inflammatory pain model, 3a behaved as a more potent analgesic when compared to endogenous AEA or its hydrolytically stable analogue AM356. This novel analogue will serve as a very useful endocannabinoid probe.

Expression, Purification and Characterization of the Human Cannabinoid 1 Receptor.

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.

Effects of Distal Mutations on the Structure, Dynamics and Catalysis of Human Monoacylglycerol Lipase.

An understanding of how conformational dynamics modulates function and catalysis of human monoacylglycerol lipase (hMGL), an important pharmaceutical target, can facilitate the development of novel ligands with potential therapeutic value. Here, we report the discovery and characterization of an allosteric, regulatory hMGL site comprised of residues Trp-289 and Leu-232 that reside over 18 Å away from the catalytic triad. These residues were identified as critical mediators of long-range communication and as important contributors to the integrity of the hMGL structure. Nonconservative replacements of Trp-289 or Leu-232 triggered concerted motions of structurally distinct regions with a significant conformational shift toward inactive states and dramatic loss in catalytic efficiency of the enzyme. Using a multimethod approach, we show that the dynamically relevant Trp-289 and Leu-232 residues serve as communication hubs within an allosteric protein network that controls signal propagation to the active site, and thus, regulates active-inactive interconversion of hMGL. Our findings provide new insights into the mechanism of allosteric regulation of lipase activity, in general, and may provide alternative drug design possibilities.

Secretion, isotopic labeling and deglycosylation of N-acylethanolamine acid amidase for biophysical studies.

N-acylethanolamine acid amidase (NAAA) is an N-terminal nucleophile (Ntn) enzyme with a catalytic cysteine residue that has highest activity at acidic pH. The most prominent substrate hydrolyzed is palmitoylethanolamine (PEA), which regulates inflammation. Inhibitors of NAAA have been shown to increase endogenous levels of PEA, and are of interest as potential treatments for inflammatory disorders and other maladies. Currently, there are no X-ray or NMR structures of NAAA available to inform medicinal chemistry. Additionally, there are a limited number of enzyme structures available that are within the Ntn-hydrolase family, have a catalytic cysteine residue, and have a high sequence homology. For these reasons, we developed expression and purification methods for the production of enzyme samples amenable to structural characterization. Mammalian cells are necessary for post-translational processing, including signal sequence cleavage and glycosylation, that are required for a correctly folded zymogen before conversion to active, and mature enzyme. We have identified an expression construct, mammalian cell line, specific media and additives to express and secrete hNAAA zymogen and we further optimized propagation conditions and show this secretion method is suitable for isotopic labeling of the protein. We refined purification methods to achieve a high degree of protein purity potentially suited to crystallography. Glycosylated proteins can present challenges to biophysical methods. Therefore we deglycosylate the enzyme and show that the activity of the mature enzyme is not affected by deglycosylation.

The role of human monoacylglycerol lipase (hMAGL) binding pocket in breakup of unsaturated phospholipid membranes.

Human monoacylglycerol lipase (hMAGL) plays a key role in homeostatic tuning of the endocannabinoid signaling system and supports aggressive tumorogenesis, making this enzyme a promising therapeutic target. hMAGL features a membrane-associated lid domain that regulates entry of endocannabinoid lipid substrates into the hydrophobic channel accessing the active site, likely from the membrane bilayer. The present work applied simultaneous surface plasmon resonance and electrochemical impedance spectroscopy measurements to show that, in absence of the substrate, hMAGL can remove phospholipid molecules from the membrane and, thereby, disintegrate pre-formed, intact, tethered phospholipid bilayer membrane mimetics (tBLMs) composed of unsaturated phosphatidylcholines. To probe the mechanism of hMAGL-induced on tBLMs compromise, we investigated the effect of wild type and mutant hMAGLs and hMAGL rendered catalytically inactive, as a function of concentration and in the presence of chemically distinct active-site inhibitors. Our data show that hMAGL's lid domain and hydrophobic substrate-binding pocket play important roles in hMAGL-induced bilayer lipid mobilization, whereas hydrolytic activity of the enzyme does not appear to be a factor.

Binding Site Characterization of AM1336, a Novel Covalent Inverse Agonist at Human Cannabinoid 2 Receptor, Using Mass Spectrometric Analysis.

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.

Functional selectivity at G-protein coupled receptors: Advancing cannabinoid receptors as drug targets.

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.

Crystal Structure of the Human Cannabinoid Receptor CB1.

Cannabinoid receptor 1 (CB1) is the principal target of Δ9-tetrahydrocannabinol (THC), a psychoactive chemical from Cannabis sativa with a wide range of therapeutic applications and a long history of recreational use. CB1 is activated by endocannabinoids and is a promising therapeutic target for pain management, inflammation, obesity, and substance abuse disorders. Here, we present the 2.8 Å crystal structure of human CB1 in complex with AM6538, a stabilizing antagonist, synthesized and characterized for this structural study. The structure of the CB1-AM6538 complex reveals key features of the receptor and critical interactions for antagonist binding. In combination with functional studies and molecular modeling, the structure provides insight into the binding mode of naturally occurring CB1 ligands, such as THC, and synthetic cannabinoids. This enhances our understanding of the molecular basis for the physiological functions of CB1 and provides new opportunities for the design of next-generation CB1-targeting pharmaceuticals.

Novel Electrophilic and Photoaffinity Covalent Probes for Mapping the Cannabinoid 1 Receptor Allosteric Site(s).

Undesirable side effects associated with orthosteric agonists/antagonists of cannabinoid 1 receptor (CB1R), a tractable target for treating several pathologies affecting humans, have greatly limited their translational potential. Recent discovery of CB1R negative allosteric modulators (NAMs) has renewed interest in CB1R by offering a potentially safer therapeutic avenue. To elucidate the CB1R allosteric binding motif and thereby facilitate rational drug discovery, we report the synthesis and biochemical characterization of first covalent ligands designed to bind irreversibly to the CB1R allosteric site. Either an electrophilic or a photoactivatable group was introduced at key positions of two classical CB1R NAMs: Org27569 (1) and PSNCBAM-1 (2). Among these, 20 (GAT100) emerged as the most potent NAM in functional assays, did not exhibit inverse agonism, and behaved as a robust positive allosteric modulator of binding of orthosteric agonist CP55,940. This novel covalent probe can serve as a useful tool for characterizing CB1R allosteric ligand-binding motifs.