Unique pharmacodynamic properties and low abuse liability of the µ-opioid receptor ligand (S)-methadone.

(R,S)-methadone ((R,S)-MTD) is a µ-opioid receptor (MOR) agonist comprised of (R)-MTD and (S)-MTD enantiomers. (S)-MTD is being developed as an antidepressant and is considered an N-methyl-D-aspartate receptor (NMDAR) antagonist. We compared the pharmacology of (R)-MTD and (S)-MTD and found they bind to MORs, but not NMDARs, and induce full analgesia. Unlike (R)-MTD, (S)-MTD was a weak reinforcer that failed to affect extracellular dopamine or induce locomotor stimulation. Furthermore, (S)-MTD antagonized motor and dopamine releasing effects of (R)-MTD. (S)-MTD acted as a partial agonist at MOR, with complete loss of efficacy at the MOR-galanin Gal1 receptor (Gal1R) heteromer, a key mediator of the dopaminergic effects of opioids. In sum, we report novel and unique pharmacodynamic properties of (S)-MTD that are relevant to its potential mechanism of action and therapeutic use. One-sentence summary: (S)-MTD, like (R)-MTD, binds to and activates MORs in vitro, but (S)-MTD antagonizes the MOR-Gal1R heteromer, decreasing its abuse liability.

The Leu/Val6.51 Side Chain of Cannabinoid Receptors Regulates the Binding Mode of the Alkyl Chain of Δ9-Tetrahydrocannabinol.

(-)-Δ9-trans-tetrahydrocannabinol (THC), which is the principal psychoactive constituent of Cannabis, mediates its action by binding to two members of the G-protein-coupled receptor (GPCR) family: the cannabinoid CB1 (CB1R) and CB2 (CB2R) receptors. Molecular dynamics simulations showed that the pentyl chain of THC could adopts an I-shape conformation, filling an intracellular cavity between Phe3.36 and Trp6.48 for initial agonist-induced receptor activation, in CB1R but not in CB2R. This cavity opens to the five-carbon chain of THC by the conformational change of the γ-branched, flexible, Leu6.51 side chain of CB1R, which is not feasible by the ß-branched, mode rigid, Val6.51 side chain of CB2R. In agreement with our computational results, THC could not decrease the forskolin-induced cAMP levels in cells expressing mutant CB1RL6.51V receptor but could activate the mutant CB2RV6.51L receptor as efficiently as wild-type CB1R. Additionally, JWH-133, a full CB2R agonist, contains a branched dimethyl moiety in the ligand chain that bridges Phe3.36 and Val6.51 for receptor activation. In this case, the substitution of Val6.51 to Leu in CB2R makes JWH-133 unable to activate CB2RV6.51L. In conclusion, our combined computational and experimental results have shown that the amino acid at position 6.51 is a key additional player in the initial mechanism of activation of GPCRs that recognize signaling molecules derived from lipid species.

Unique pharmacodynamic properties and low abuse liability of the µ-opioid receptor ligand (S)-methadone.

(R,S)-methadone ((R,S)-MTD) is a racemic µ-opioid receptor (MOR) agonist comprised of (R)-MTD and (S)-MTD enantiomers used for the treatment of opioid use disorder (OUD) and pain. (R)-MTD is used as an OUD treatment, has high MOR potency, and is believed to mediate (R,S)-MTD's therapeutic efficacy. (S)-MTD is in clinical development as an antidepressant and is considered an N-methyl-D-aspartate receptor (NMDAR) antagonist. In opposition to this purported mechanism of action, we found that (S)-MTD does not occupy NMDARs in vivo in rats. Instead, (S)-MTD produced MOR occupancy and induced analgesia with similar efficacy as (R)-MTD. Unlike (R)-MTD, (S)-MTD was not self-administered and failed to increase locomotion or extracellular dopamine levels indicating low abuse liability. Moreover, (S)-MTD antagonized the effects of (R)-MTD in vivo and exhibited unique pharmacodynamic properties, distinct from those of (R)-MTD. Specifically, (S)-MTD acted as a MOR partial agonist with a specific loss of efficacy at the MOR-galanin 1 receptor (Gal1R) heteromer, a key mediator of the dopaminergic effects of opioids. In sum, we report novel and unique pharmacodynamic properties of (S)-MTD that are relevant to its potential mechanism of action and therapeutic use, as well as those of (R,S)-MTD.

Unique effect of clozapine on adenosine A2A-dopamine D2 receptor heteromerization.

The striatal dopamine D2 receptor (D2R) is generally accepted to be involved in positive symptoms of schizophrenia and is a main target for clinically used antipsychotics. D2R are highly expressed in the striatum, where they form heteromers with the adenosine A2A receptor (A2AR). Changes in the density of A2AR-D2R heteromers have been reported in postmortem tissue from patients with schizophrenia, but the degree to which A2R are involved in schizophrenia and the effect of antipsychotic drugs is unknown. Here, we examine the effect of exposure to three prototypical antipsychotic drugs on A2AR-D2R heteromerization in mammalian cells using a NanoBiT assay. After 16 h of exposure, a significant increase in the density of A2AR-D2R heteromers was found with haloperidol and aripiprazole, but not with clozapine. On the other hand, clozapine, but not haloperidol or aripiprazole, was associated with a significant decrease in A2AR-D2R heteromerization after 2 h of treatment. Computational binding models of these compounds revealed distinctive molecular signatures that explain their different influence on heteromerization. The bulky tricyclic moiety of clozapine displaces TM 5 of D2R, inducing a clash with A2AR, while the extended binding mode of haloperidol and aripiprazole stabilizes a specific conformation of the second extracellular loop of D2R that enhances the interaction with A2AR. It is proposed that an increase in A2AR-D2R heteromerization is involved in the extrapyramidal side effects (EPS) of antipsychotics and that the specific clozapine-mediated destabilization of A2AR-D2R heteromerization can explain its low EPS liability.

The ADORA1 mutation linked to early-onset Parkinson's disease alters adenosine A1-A2A receptor heteromer formation and function.

Adenosine modulates neurotransmission through inhibitory adenosine A1 receptors (A1Rs) and stimulatory A2A receptors (A2ARs). These G protein-coupled receptors are involved in motor function and related to neurodegenerative diseases such as Parkinson's disease (PD). An autosomal-recessive mutation (G2797.44S) within the transmembrane helix (TM) 7 of A1R (A1RG279S) has been associated with the development of early onset PD (EOPD). Here, we aimed at investigating the impact of this mutation on the structure and function of the A1R and the A1R-A2AR heteromer. Our results revealed that the G2797.44S mutation does not alter A1R expression, ligand binding, constitutive activity or coupling to transducer proteins (Gαi, Gαq, Gα12/13, Gαs, ß-arrestin2 and GRK2) in transfected HEK-293 T cells. However, A1RG279S weakened the ability of A1R to heteromerize with A2AR, as shown in a NanoBiT assay, which led to the disappearance of the heteromerization-dependent negative allosteric modulation that A1R imposes on the constitutive activity and agonist-induced activation of the A2AR. Molecular dynamic simulations allowed to propose an indirect mechanism by which the G2797.44S mutation in TM 7 of A1R weakens the TM 5/6 interface of the A1R-A2AR heteromer. Therefore, it is demonstrated that a PD linked ADORA1 mutation is associated with dysfunction of adenosine receptor heteromerization. We postulate that a hyperglutamatergic state secondary to increased constitutive activity and sensitivity to adenosine of A2AR not forming heteromers with A1R could represent a main pathogenetic mechanism of the EOPD associated with the G2797.44S ADORA1 mutation.

A Single Point Mutation Blocks the Entrance of Ligands to the Cannabinoid CB2 Receptor via the Lipid Bilayer.

Molecular dynamic (MD) simulations have become a common tool to study the pathway of ligand entry to the orthosteric binding site of G protein-coupled receptors. Here, we have combined MD simulations and site-directed mutagenesis to study the binding process of the potent JWH-133 agonist to the cannabinoid CB2 receptor (CB2R). In CB2R, the N-terminus and extracellular loop 2 fold over the ligand binding pocket, blocking access to the binding cavity from the extracellular environment. We, thus, hypothesized that the binding pathway is a multistage process consisting of the hydrophobic ligand diffusing in the lipid bilayer to contact a lipid-facing vestibule, from which the ligand enters an allosteric site inside the transmembrane bundle through a tunnel formed between TMs 1 and 7 and finally moving from the allosteric to the orthosteric binding cavity. This pathway was experimentally validated by the Ala2827.36Phe mutation that blocks the entrance of the ligand, as JWH-133 was not able to decrease the forskolin-induced cAMP levels in cells expressing the mutant receptor. This proposed ligand entry pathway defines transient binding sites that are potential cavities for the design of synthetic modulators.

Pharmacological targeting of G protein-coupled receptor heteromers.

A main rationale for the role of G protein-coupled receptor (GPCR) heteromers as targets for drug development is the putative ability of selective ligands for specific GPCRs to change their pharmacological properties upon GPCR heteromerization. The present study provides a proof of concept for this rationale by demonstrating that heteromerization of dopamine D1 and D3 receptors (D1R and D3R) influences the pharmacological properties of three structurally similar selective dopamine D3R ligands, the phenylpiperazine derivatives PG01042, PG01037 and VK4-116. By using D1R-D3R heteromer-disrupting peptides, it could be demonstrated that the three D3R ligands display different D1R-D3R heteromer-dependent pharmacological properties: PG01042, acting as G protein-biased agonist, counteracted D1R-mediated signaling in the D1R-D3R heteromer; PG01037, acting as a D3R antagonist cross-antagonized D1R-mediated signaling in the D1R-D3R heteromer; and VK4-116 specifically acted as a ß-arrestin-biased agonist in the D1R-D3R heteromer. Molecular dynamics simulations predicted potential molecular mechanisms mediating these qualitatively different pharmacological properties of the selective D3R ligands that are dependent on D1R-D3R heteromerization. The results of in vitro experiments were paralleled by qualitatively different pharmacological properties of the D3R ligands in vivo. The results supported the involvement of D1R-D3R heteromers in the locomotor activation by D1R agonists in reserpinized mice and L-DOPA-induced dyskinesia in rats, highlighting the D1R-D3R heteromer as a main pharmacological target for L-DOPA-induced dyskinesia in Parkinson's disease. More generally, the present study implies that when suspecting its pathogenetic role, a GPCR heteromer, and not its individual GPCR units, should be considered as main target for drug development.

Preferential Gs protein coupling of the galanin Gal1 receptor in the µ-opioid-Gal1 receptor heterotetramer.

Recent studies have proposed that heteromers of µ-opioid receptors (MORs) and galanin Gal1 receptors (Gal1Rs) localized in the mesencephalon mediate the dopaminergic effects of opioids. The present study reports converging evidence, using a peptide-interfering approach combined with biophysical and biochemical techniques, including total internal reflection fluorescence microscopy, for a predominant homodimeric structure of MOR and Gal1R when expressed individually, and for their preference to form functional heterotetramers when co-expressed. Results show that a heteromerization-dependent change in the Gal1R homodimeric interface leads to a switch in G-protein coupling from inhibitory Gi to stimulatory Gs proteins. The MOR-Gal1R heterotetramer, which is thus bound to Gs via the Gal1R homodimer and Gi via the MOR homodimer, provides the framework for a canonical Gs-Gi antagonist interaction at the adenylyl cyclase level. These novel results shed light on the intense debate about the oligomeric quaternary structure of G protein-coupled receptors, their predilection for heteromer formation, and the resulting functional significance.

Heterobivalent Ligand for the Adenosine A2A-Dopamine D2 Receptor Heteromer.

A G protein-coupled receptor heteromer that fulfills the established criteria for its existence in vivo is the complex between adenosine A2A (A2AR) and dopamine D2 (D2R) receptors. Here, we have designed and synthesized heterobivalent ligands for the A2AR-D2R heteromer with various spacer lengths. The indispensable simultaneous binding of these ligands to the two different orthosteric sites of the heteromer has been evaluated by radioligand competition-binding assays in the absence and presence of specific peptides that disrupt the formation of the heteromer, label-free dynamic mass redistribution assays in living cells, and molecular dynamic simulations. This combination of techniques has permitted us to identify compound 26 [KDB1 (A2AR) = 2.1 nM, KDB1 (D2R) = 0.13 nM], with a spacer length of 43-atoms, as a true bivalent ligand that simultaneously binds to the two different orthosteric sites. Moreover, bioluminescence resonance energy transfer experiments indicate that 26 favors the stabilization of the A2AR-D2R heteromer.

G protein-coupled receptor-effector macromolecular membrane assemblies (GEMMAs).

G protein-coupled receptors (GPCRs) are the largest group of receptors involved in cellular signaling across the plasma membrane and a major class of drug targets. The canonical model for GPCR signaling involves three components - the GPCR, a heterotrimeric G protein and a proximal plasma membrane effector - that have been generally thought to be freely mobile molecules able to interact by 'collision coupling'. Here, we synthesize evidence that supports the existence of GPCR-effector macromolecular membrane assemblies (GEMMAs) comprised of specific GPCRs, G proteins, plasma membrane effector molecules and other associated transmembrane proteins that are pre-assembled prior to receptor activation by agonists, which then leads to subsequent rearrangement of the GEMMA components. The GEMMA concept offers an alternative and complementary model to the canonical collision-coupling model, allowing more efficient interactions between specific signaling components, as well as the integration of the concept of GPCR oligomerization as well as GPCR interactions with orphan receptors, truncated GPCRs and other membrane-localized GPCR-associated proteins. Collision-coupling and pre-assembled mechanisms are not exclusive and likely both operate in the cell, providing a spectrum of signaling modalities which explains the differential properties of a multitude of GPCRs in their different cellular environments. Here, we explore the unique pharmacological characteristics of individual GEMMAs, which could provide new opportunities to therapeutically modulate GPCR signaling.

Design of Negative and Positive Allosteric Modulators of the Cannabinoid CB2 Receptor Derived from the Natural Product Cannabidiol.

Cannabidiol (CBD), the second most abundant of the active compounds found in the Cannabis sativa plant, is of increasing interest because it is approved for human use and is neither euphorizing nor addictive. Here, we design and synthesize novel compounds taking into account that CBD is both a partial agonist, when it binds to the orthosteric site, and a negative allosteric modulator, when it binds to the allosteric site of the cannabinoid CB2 receptor. Molecular dynamic simulations and site-directed mutagenesis studies have identified the allosteric site near the receptor entrance. This knowledge has permitted to perform structure-guided design of negative and positive allosteric modulators of the CB2 receptor with potential therapeutic utility.

Orally Active Peptide Vector Allows Using Cannabis to Fight Pain While Avoiding Side Effects.

The activation of cannabinoid CB1 receptors (CB1R) by Δ9-tetrahydrocannabinol (THC), the main component of Cannabis sativa, induces analgesia. CB1R activation, however, also causes cognitive impairment via the serotonin 5HT2A receptor (5HT2AR), a component of a CB1R-5HT2AR heteromer, posing a serious drawback for cannabinoid therapeutic use. We have shown that peptides reproducing CB1R transmembrane (TM) helices 5 and 6, fused to a cell-penetrating sequence (CPP), can alter the structure of the CB1R-5HT2AR heteromer and avert THC cognitive impairment while preserving analgesia. Here, we report the optimization of these prototypes into drug-like leads by (i) shortening the TM5, TM6, and CPP sequences, without losing the ability to disturb the CB1R-5HT2AR heteromer, and (ii) extensive sequence remodeling to achieve protease resistance and blood-brain barrier penetration. Our efforts have culminated in the identification of an ideal candidate for cannabis-based pain management, an orally active 16-residue peptide preserving THC-induced analgesia.

Discovery of a macromolecular complex mediating the hunger suppressive actions of cocaine: Structural and functional properties.

Cocaine not only increases brain dopamine levels but also activates the sigma1 receptor (σ1 R) that in turn regulates orexigenic receptor function. Identification of interactions involving dopamine D1 (D1 R), ghrelin (GHS-R1a ), and σ1 receptors have been addressed by biophysical techniques and a complementation approach using interfering peptides. The effect of cocaine on receptor functionality was assayed by measuring second messenger, cAMP and Ca2+ , levels. The effect of acute or chronic cocaine administration on receptor complex expression was assayed by in situ proximity ligation assay. In silico procedures were used for molecular model building. σ1 R KO mice were used for confirming involvement of this receptor. Upon identification of protomer interaction and receptor functionality, a unique structural model for the macromolecular complex formed by σ1 R, D1 R, and GHS-R1a is proposed. The functionality of the complex, able to couple to both Gs and Gq proteins, is affected by cocaine binding to the σ1 R, as confirmed using samples from σ1 R-/- mice. The expression of the macromolecular complex was differentially affected upon acute and chronic cocaine administration to rats. The constructed 3D model is consistent with biochemical, biophysical, and available structural data. The σ1 R, D1 R, and GHS-R1a complex constitutes a functional unit that is altered upon cocaine binding to the σ1 R. Remarkably, the heteromer can simultaneously couple to two G proteins, thus allowing dopamine to signal via Ca2+ and ghrelin via cAMP. The anorexic action of cocaine is mediated by such complex whose expression is higher after acute than after chronic administration regimens.

Structure and function of adenosine receptor heteromers.

Adenosine is one of the most ancient signaling molecules and has receptors in both animals and plants. In mammals there are four specific receptors, A1, A2A, A2B, and A3, which belong to the superfamily of G-protein-coupled receptors (GPCRs). Evidence accumulated in the last 20 years indicates that GPCRs are often expressed as oligomeric complexes formed by a number of equal (homomers) or different (heteromers) receptors. This review presents the data showing the occurrence of heteromers formed by A1 and A2A, A2A and A2B, and A2A and A3 receptors highlighting (i) their tetrameric structural arrangements, and (ii) the functional diversity that those heteromers provide to adenosinergic signaling.

The mutational landscape of human olfactory G protein-coupled receptors.

BACKGROUND: Olfactory receptors (ORs) constitute a large family of sensory proteins that enable us to recognize a wide range of chemical volatiles in the environment. By contrast to the extensive information about human olfactory thresholds for thousands of odorants, studies of the genetic influence on olfaction are limited to a few examples. To annotate on a broad scale the impact of mutations at the structural level, here we analyzed a compendium of 119,069 natural variants in human ORs collected from the public domain. RESULTS: OR mutations were categorized depending on their genomic and protein contexts, as well as their frequency of occurrence in several human populations. Functional interpretation of the natural changes was estimated from the increasing knowledge of the structure and function of the G protein-coupled receptor (GPCR) family, to which ORs belong. Our analysis reveals an extraordinary diversity of natural variations in the olfactory gene repertoire between individuals and populations, with a significant number of changes occurring at the structurally conserved regions. A particular attention is paid to mutations in positions linked to the conserved GPCR activation mechanism that could imply phenotypic variation in the olfactory perception. An interactive web application (hORMdb, Human Olfactory Receptor Mutation Database) was developed for the management and visualization of this mutational dataset. CONCLUSION: We performed topological annotations and population analysis of natural variants of human olfactory receptors and provide an interactive application to explore human OR mutation data. We envisage that the utility of this information will increase as the amount of available pharmacological data for these receptors grow. This effort, together with ongoing research in the study of genetic changes in other sensory receptors could shape an emerging sensegenomics field of knowledge, which should be considered by food and cosmetic consumer product manufacturers for the benefit of the general population.

Experimental and computational analysis of biased agonism on full-length and a C-terminally truncated adenosine A2A receptor.

Biased agonism, the ability of agonists to differentially activate downstream signaling pathways by stabilizing specific receptor conformations, is a key issue for G protein-coupled receptor (GPCR) signaling. The C-terminal domain might influence this functional selectivity of GPCRs as it engages G proteins, GPCR kinases, ß-arrestins, and several other proteins. Thus, the aim of this paper is to compare the agonist-dependent selectivity for intracellular pathways in a heterologous system expressing the full-length (A2AR) and a C-tail truncated (A2A Δ40R lacking the last 40 amino acids) adenosine A2A receptor, a GPCR that is already targeted in Parkinson's disease using a first-in-class drug. Experimental data such as ligand binding, cAMP production, ß-arrestin recruitment, ERK1/2 phosphorylation and dynamic mass redistribution assays, which correspond to different aspects of signal transduction, were measured upon the action of structurally diverse compounds (the agonists adenosine, NECA, CGS-21680, PSB-0777 and LUF-5834 and the SCH-58261 antagonist) in cells expressing A2AR and A2A Δ40R. The results show that taking cAMP levels and the endogenous adenosine agonist as references, the main difference in bias was obtained with PSB-0777 and LUF-5834. The C-terminus is dispensable for both G-protein and ß-arrestin recruitment and also for MAPK activation. Unrestrained molecular dynamics simulations, at the µs timescale, were used to understand the structural arrangements of the binding cavity, triggered by these chemically different agonists, facilitating G protein binding with different efficacy.

Discovery of Homobivalent Bitopic Ligands of the Cannabinoid CB2 Receptor*.

Single chemical entities with potential to simultaneously interact with two binding sites are emerging strategies in medicinal chemistry. We have designed, synthesized and functionally characterized the first bitopic ligands for the CB2 receptor. These compounds selectively target CB2 versus CB1 receptors. Their binding mode was studied by molecular dynamic simulations and site-directed mutagenesis.

Adenosine/A2B Receptor Signaling Ameliorates the Effects of Aging and Counteracts Obesity.

The combination of aging populations with the obesity pandemic results in an alarming rise in non-communicable diseases. Here, we show that the enigmatic adenosine A2B receptor (A2B) is abundantly expressed in skeletal muscle (SKM) as well as brown adipose tissue (BAT) and might be targeted to counteract age-related muscle atrophy (sarcopenia) as well as obesity. Mice with SKM-specific deletion of A2B exhibited sarcopenia, diminished muscle strength, and reduced energy expenditure (EE), whereas pharmacological A2B activation counteracted these processes. Adipose tissue-specific ablation of A2B exacerbated age-related processes and reduced BAT EE, whereas A2B stimulation ameliorated obesity. In humans, A2B expression correlated with EE in SKM, BAT activity, and abundance of thermogenic adipocytes in white fat. Moreover, A2B agonist treatment increased EE from human adipocytes, myocytes, and muscle explants. Mechanistically, A2B forms heterodimers required for adenosine signaling. Overall, adenosine/A2B signaling links muscle and BAT and has both anti-aging and anti-obesity potential.