Bioisosteric analogs of MDMA with improved pharmacological profile.

3,4-Methylenedioxymethamphetamine (MDMA, ' ecstasy' ) is re-emerging in clinical settings as a candidate for the treatment of specific psychiatric disorders (e.g. post-traumatic stress disorder) in combination with psychotherapy. MDMA is a psychoactive drug, typically regarded as an empathogen or entactogen, which leads to transporter-mediated monoamine release. Despite its therapeutic potential, MDMA can induce dose-, individual-, and context-dependent untoward effects outside safe settings. In this study, we investigated whether three new methylenedioxy bioisosteres of MDMA improve its off-target profile. In vitro methods included radiotracer assays, transporter electrophysiology, bioluminescence resonance energy transfer and fluorescence-based assays, pooled human liver microsome/S9 fraction incubation with isozyme mapping, and liquid chromatography coupled to high-resolution mass spectrometry. In silico methods included molecular docking. Compared with MDMA, all three MDMA bioisosteres (ODMA, TDMA, and SeDMA) showed similar pharmacological activity at human serotonin and dopamine transporters (hSERT and hDAT, respectively) but decreased activity at 5-HT 2A/2B/2C receptors. Regarding their hepatic metabolism, they differed from MDMA, with N -demethylation being the only metabolic route shared, and without forming phase II metabolites. Additional screening for their interaction with human organic cation transporters (hOCTs) and plasma membrane transporter (hPMAT) revealed a weaker interaction of the MDMA analogs with hOCT1, hOCT2, and hPMAT. Our findings suggest that these new MDMA analogs might constitute appealing therapeutic alternatives to MDMA, sparing the primary pharmacological activity at hSERT and hDAT, but displaying a reduced activity at 5-HT 2A/2B/2C receptors and reduced hepatic metabolism. Whether these MDMA bioisosteres may pose lower risk alternatives to the clinically re-emerging MDMA warrants further studies.

Functional profiling of the G protein-coupled receptor C3aR1 reveals ligand-mediated biased agonism.

G protein-coupled receptors (GPCRs) are leading druggable targets for several medicines, but many GPCRs are still untapped for their therapeutic potential due to poor understanding of specific signaling properties. The complement C3a receptor 1 (C3aR1) has been extensively studied for its physiological role in C3a-mediated anaphylaxis/inflammation, and in TLQP-21-mediated lipolysis, but direct evidence for the functional relevance of the C3a and TLQP-21 ligands and signal transduction mechanisms are still limited. In addition, C3aR1 G protein coupling specificity is still unclear, and whether endogenous ligands, or drug-like compounds, show ligand-mediated biased agonism is unknown. Here, we demonstrate that C3aR1 couples preferentially to Gi/o/z proteins and can recruit ß-arrestins to cause internalization. Furthermore, we showed that in comparison to C3a63-77, TLQP-21 exhibits a preference toward Gi/o-mediated signaling compared to ß-arrestin recruitment and internalization. We also show that the purported antagonist SB290157 is a very potent C3aR1 agonist, where antagonism of ligand-stimulated C3aR1 calcium flux is caused by potent ß-arrestin-mediated internalization. Finally, ligand-mediated signaling bias impacted cell function as demonstrated by the regulation of calcium influx, lipolysis in adipocytes, phagocytosis in microglia, and degranulation in mast cells. Overall, we characterize C3aR1 as a Gi/o/z-coupled receptor and demonstrate the functional relevance of ligand-mediated signaling bias in key cellular models. Due to C3aR1 and its endogenous ligands being implicated in inflammatory and metabolic diseases, these results are of relevance toward future C3aR1 drug discovery.

Psilocybin analog 4-OH-DiPT enhances fear extinction and GABAergic inhibition of principal neurons in the basolateral amygdala.

Psychedelics such as psilocybin show great promise for the treatment of depression and PTSD, but their long duration of action poses practical limitations for patient access. 4-OH-DiPT is a fast-acting and shorter-lasting derivative of psilocybin. Here we characterized the pharmacological profile of 4-OH-DiPT and examined its impact on fear extinction learning as well as a potential mechanism of action. First, we profiled 4-OH-DiPT at all 12 human 5-HT GPCRs. 4-OH-DiPT showed strongest agonist activity at all three 5-HT2A/2B/2C receptors with near full agonist activity at 5-HT2A. Notably, 4-OH-DiPT had comparable activity at mouse and human 5-HT2A/2B/2C receptors. In a fear extinction paradigm, 4-OH-DiPT significantly reduced freezing responses to conditioned cues in a dose-dependent manner with a greater potency in female mice than male mice. Female mice that received 4-OH-DiPT before extinction training had reduced avoidance behaviors several days later in the light dark box, elevated plus maze and novelty-suppressed feeding test compared to controls, while male mice did not show significant differences. 4-OH-DiPT produced robust increases in spontaneous inhibitory postsynaptic currents (sIPSCs) in basolateral amygdala (BLA) principal neurons and action potential firing in BLA interneurons in a 5-HT2A-dependent manner. RNAscope demonstrates that Htr2a mRNA is expressed predominantly in BLA GABA interneurons, Htr2c mRNA is expressed in both GABA interneurons and principal neurons, while Htr2b mRNA is absent in the BLA. Our findings suggest that 4-OH-DiPT activates BLA interneurons via the 5-HT2A receptor to enhance GABAergic inhibition of BLA principal neurons, which provides a potential mechanism for suppressing learned fear.

Identification of 5-HT2A receptor signaling pathways associated with psychedelic potential.

Serotonergic psychedelics possess considerable therapeutic potential. Although 5-HT2A receptor activation mediates psychedelic effects, prototypical psychedelics activate both 5-HT2A-Gq/11 and ß-arrestin2 transducers, making their respective roles unclear. To elucidate this, we develop a series of 5-HT2A-selective ligands with varying Gq efficacies, including ß-arrestin-biased ligands. We show that 5-HT2A-Gq but not 5-HT2A-ß-arrestin2 recruitment efficacy predicts psychedelic potential, assessed using head-twitch response (HTR) magnitude in male mice. We further show that disrupting Gq-PLC signaling attenuates the HTR and a threshold level of Gq activation is required to induce psychedelic-like effects, consistent with the fact that certain 5-HT2A partial agonists (e.g., lisuride) are non-psychedelic. Understanding the role of 5-HT2A Gq-efficacy in psychedelic-like psychopharmacology permits rational development of non-psychedelic 5-HT2A agonists. We also demonstrate that ß-arrestin-biased 5-HT2A receptor agonists block psychedelic effects and induce receptor downregulation and tachyphylaxis. Overall, 5-HT2A receptor Gq-signaling can be fine-tuned to generate ligands distinct from classical psychedelics.

Beyond the 5-HT2A Receptor: Classic and Nonclassic Targets in Psychedelic Drug Action.

Serotonergic psychedelics, such as psilocybin and LSD, have garnered significant attention in recent years for their potential therapeutic effects and unique mechanisms of action. These compounds exert their primary effects through activating serotonin 5-HT2A receptors, found predominantly in cortical regions. By interacting with these receptors, serotonergic psychedelics induce alterations in perception, cognition, and emotions, leading to the characteristic psychedelic experience. One of the most crucial aspects of serotonergic psychedelics is their ability to promote neuroplasticity, the formation of new neural connections, and rewire neuronal networks. This neuroplasticity is believed to underlie their therapeutic potential for various mental health conditions, including depression, anxiety, and substance use disorders. In this mini-review, we will discuss how the 5-HT2A receptor activation is just one facet of the complex mechanisms of action of serotonergic psychedelics. They also interact with other serotonin receptor subtypes, such as 5-HT1A and 5-HT2C receptors, and with neurotrophin receptors (e.g., tropomyosin receptor kinase B). These interactions contribute to the complexity of their effects on perception, mood, and cognition. Moreover, as psychedelic research advances, there is an increasing interest in developing nonhallucinogenic derivatives of these drugs to create safer and more targeted medications for psychiatric disorders by removing the hallucinogenic properties while retaining the potential therapeutic benefits. These nonhallucinogenic derivatives would offer patients therapeutic advantages without the intense psychedelic experience, potentially reducing the risks of adverse reactions. Finally, we discuss the potential of psychedelics as substrates for post-translational modification of proteins as part of their mechanism of action.

Molecular insights into GPCR mechanisms for drugs of abuse.

Substance abuse is on the rise, and while many people may use illicit drugs mainly due to their rewarding effects, their societal impact can range from severe, as is the case for opioids, to promising, as is the case for psychedelics. Common with all these drugs' mechanisms of action are G protein-coupled receptors (GPCRs), which lie at the center of how these drugs mediate inebriation, lethality, and therapeutic effects. Opioids like fentanyl, cannabinoids like tetrahydrocannabinol, and psychedelics like lysergic acid diethylamide all directly bind to GPCRs to initiate signaling which elicits their physiological actions. We herein review recent structural studies and provide insights into the molecular mechanisms of opioids, cannabinoids, and psychedelics at their respective GPCR subtypes. We further discuss how such mechanistic insights facilitate drug discovery, either toward the development of novel therapies to combat drug abuse or toward harnessing therapeutic potential.

Identification of 5-HT2A Receptor Signaling Pathways Responsible for Psychedelic Potential.

Serotonergic psychedelics possess considerable therapeutic potential. Although 5-HT2A receptor activation mediates psychedelic effects, prototypical psychedelics activate both 5-HT2A-Gq/11 and ß-arrestin2 signaling, making their respective roles unclear. To elucidate this, we developed a series of 5-HT2A-selective ligands with varying Gq efficacies, including ß-arrestin-biased ligands. We show that 5-HT2A-Gq but not 5-HT2A-ß-arrestin2 efficacy predicts psychedelic potential, assessed using head-twitch response (HTR) magnitude in male mice. We further show that disrupting Gq-PLC signaling attenuates the HTR and a threshold level of Gq activation is required to induce psychedelic-like effects, consistent with the fact that certain 5-HT2A partial agonists (e.g., lisuride) are non-psychedelic. Understanding the role of 5-HT2A-Gq efficacy in psychedelic-like psychopharmacology permits rational development of non-psychedelic 5-HT2A agonists. We also demonstrate that ß-arrestin-biased 5-HT2A receptor agonists induce receptor downregulation and tachyphylaxis, and have an anti-psychotic-like behavioral profile. Overall, 5-HT2A receptor signaling can be fine-tuned to generate ligands with properties distinct from classical psychedelics.

Angiotensin AT1A receptor signal switching in Agouti-related peptide neurons mediates metabolic rate adaptation during obesity.

Resting metabolic rate (RMR) adaptation occurs during obesity and is hypothesized to contribute to failed weight management. Angiotensin II (Ang-II) type 1 (AT1A) receptors in Agouti-related peptide (AgRP) neurons contribute to the integrative control of RMR, and deletion of AT1A from AgRP neurons causes RMR adaptation. Extracellular patch-clamp recordings identify distinct cellular responses of individual AgRP neurons from lean mice to Ang-II: no response, inhibition via AT1A and Gαi, or stimulation via Ang-II type 2 (AT2) receptors and Gαq. Following diet-induced obesity, a subset of Ang-II/AT1A-inhibited AgRP neurons undergo a spontaneous G-protein "signal switch," whereby AT1A stop inhibiting the cell via Gαi and instead begin stimulating the cell via Gαq. DREADD-mediated activation of Gαi, but not Gαq, in AT1A-expressing AgRP cells stimulates RMR in lean and obese mice. Thus, loss of AT1A-Gαi coupling within the AT1A-expressing AgRP neuron subtype represents a molecular mechanism contributing to RMR adaptation.

Structure activity relationships of 5-HT2B and 5-HT2C serotonin receptor antagonists: N6, C2 and 5'-Modified (N)-methanocarba-adenosine derivatives.

(N)-Methanocarba adenosine derivatives were structurally modified to target 5-HT2B serotonin receptors as antagonists, predominantly containing branched N6-alkyl groups. N6-Dicycloalkyl-methyl groups, including their asymmetric variations, as well as 2-iodo, were found to generally favor 5-HT2Rs, while only N6-dicyclohexyl-methyl derivative 35 showed weak 5-HT2AR affinity (Ki 3.6 µM). The highest 5-HT2BR affinities were Ki 11-23 nM (N6-dicyclopropyl-methyl-2-iodo 11, 2-chloro-5'-deoxy-5'-methylthio 15 and N6-((R)-cyclobuty-cyclopropyl-methyl)-2-iodo 43), and Ki 73 nM at 5-HT2CR for 36. Direct comparison of adenine ribosides and their corresponding rigid (N)-methanocarba derivatives (cf. 51 and MRS8099 45) indicated a multifold affinity enhancement with the bicyclic ring system. Compounds 43, 45 and 48 were functional 5-HT2BR (KB 2-3 nM) and 5-HT2CR (KB 79-328 nM) antagonists in a Gq-mediated calcium flux assay, with 5-HT2BR functional selectivity ranging from 45- (48) to 113-fold (43). Substantial adenosine receptor (AR) affinity (Ki, A1AR < Ki, A3AR < Ki, A2AAR) was still present in this series, suggestive of dual acting compounds: 5-HT2B antagonist and A1AR agonist, potentially useful for treating chronic conditions (fibrosis; pain). Given its affinity (17 nM) and moderate 5-HT2BR binding selectivity (32-fold vs. 5-HT2CR, 4-fold vs. A1AR), 43 (MRS7925) could potentially be useful for anti-fibrotic therapy.

Mu-opioid receptor selective superagonists produce prolonged respiratory depression.

Synthetic opioids are increasingly challenging to combat the opioid epidemic and act primarily at opioid receptors, chiefly the G protein-coupled receptor (GPCR) µ-opioid receptor (MOR), which signals through G protein-dependent and ß-arrestin pathways. Using a bioluminescence resonance energy transfer (BRET) system, we investigate GPCR-signaling profiles by synthetic nitazenes, which are known to cause overdose and death due to respiratory depression. We show that isotonitazene and its metabolite, N-desethyl isotonitazene, are very potent MOR-selective superagonists, surpassing both DAMGO G protein and ß-arrestin recruitment activity, which are properties distinct from other conventional opioids. Both isotonitazene and N-desethyl isotonitazene show high potency in mouse analgesia tail-flick assays, but N-desethyl isotonitazene shows longer-lasting respiratory depression compared to fentanyl. Overall, our results suggest that potent MOR-selective superagonists may be a pharmacological property predictive of prolonged respiratory depression resulting in fatal consequences and should be examined for future opioid analgesics.

A non-hallucinogenic LSD analog with therapeutic potential for mood disorders.

Hallucinations limit widespread therapeutic use of psychedelics as rapidly acting antidepressants. Here we profiled the non-hallucinogenic lysergic acid diethylamide (LSD) analog 2-bromo-LSD (2-Br-LSD) at more than 33 aminergic G protein-coupled receptors (GPCRs). 2-Br-LSD shows partial agonism at several aminergic GPCRs, including 5-HT2A, and does not induce the head-twitch response (HTR) in mice, supporting its classification as a non-hallucinogenic 5-HT2A partial agonist. Unlike LSD, 2-Br-LSD lacks 5-HT2B agonism, an effect linked to cardiac valvulopathy. Additionally, 2-Br-LSD produces weak 5-HT2A ß-arrestin recruitment and internalization in vitro and does not induce tolerance in vivo after repeated administration. 2-Br-LSD induces dendritogenesis and spinogenesis in cultured rat cortical neurons and increases active coping behavior in mice, an effect blocked by the 5-HT2A-selective antagonist volinanserin (M100907). 2-Br-LSD also reverses the behavioral effects of chronic stress. Overall, 2-Br-LSD has an improved pharmacological profile compared with LSD and may have profound therapeutic value for mood disorders and other indications.

Psychedelics promote neuroplasticity through the activation of intracellular 5-HT2A receptors.

Decreased dendritic spine density in the cortex is a hallmark of several neuropsychiatric diseases, and the ability to promote cortical neuron growth has been hypothesized to underlie the rapid and sustained therapeutic effects of psychedelics. Activation of 5-hydroxytryptamine (serotonin) 2A receptors (5-HT2ARs) is essential for psychedelic-induced cortical plasticity, but it is currently unclear why some 5-HT2AR agonists promote neuroplasticity, whereas others do not. We used molecular and genetic tools to demonstrate that intracellular 5-HT2ARs mediate the plasticity-promoting properties of psychedelics; these results explain why serotonin does not engage similar plasticity mechanisms. This work emphasizes the role of location bias in 5-HT2AR signaling, identifies intracellular 5-HT2ARs as a therapeutic target, and raises the intriguing possibility that serotonin might not be the endogenous ligand for intracellular 5-HT2ARs in the cortex.

DPP4-Truncated CXCL12 Alters CXCR4/ACKR3 Signaling, Osteogenic Cell Differentiation, Migration, and Senescence.

Bone marrow skeletal stem cells (SSCs) secrete many cytokines including stromal derived factor-1 or CXCL12, which influences cell proliferation, migration, and differentiation. All CXCL12 splice variants are rapidly truncated on their N-terminus by dipeptidyl peptidase 4 (DPP4). This includes the common variant CXCL12 alpha (1-68) releasing a much less studied metabolite CXCL12(3-68). Here, we found that CXCL12(3-68) significantly inhibited SSC osteogenic differentiation and RAW-264.7 cell osteoclastogenic differentiation and induced a senescent phenotype in SSCs. Importantly, pre-incubation of SSCs with CXCL12(3-68) significantly diminished their ability to migrate toward CXCL12(1-68) in transwell migration assays. Using a high-throughput G-protein-coupled receptor (GPCR) screen (GPCRome) and bioluminescent resonance energy transfer molecular interaction assays, we revealed that CXCL12(3-68) acts via the atypical cytokine receptor 3-mediated ß-arrestin recruitment and as a competitive antagonist to CXCR4-mediated signaling. Finally, a reverse phase protein array assay revealed that DPP4-cleaved CXCL12 possesses a different downstream signaling profile from that of intact CXCL12 or controls. The data presented herein provides insights into regulation of CXCL12 signaling. Importantly, it demonstrates that DPP4 proteolysis of CXCL12 generates a metabolite with significantly different and previously overlooked bioactivity that helps explain discrepancies in the literature. This also contributes to an understanding of the molecular mechanisms of osteoporosis and bone fracture repair and could potentially significantly affect the interpretation of experimental outcomes with clinical consequences in other fields where CXCL12 is vital, including cancer biology, immunology, cardiovascular biology, neurobiology, and associated pathologies.

Molecular and cellular basis of praziquantel action in the cardiovascular system.

The anthelmintic drug praziquantel (PZQ) causes contraction of parasitic schistosomes as well as constriction of blood vessels within the mesenteric vasculature of the host where the adult blood flukes reside. The contractile action of PZQ on the vasculature is mediated by the activation of host serotonergic 5-HT2B receptors (5-HT2BRs). However, the molecular basis for PZQ interaction with these targets and the location of these 5-HT2B receptors in the vessel wall have not been experimentally defined. Evaluation of a PZQ docking pose within the 5-HT2BR orthosteric site, using both Ca2+ reporter and bioluminescence resonance energy transfer (BRET) assays, identified residues F3406.51 and F3416.52 (transmembrane helix 6, TM6) as well as L209EL2 (extracellular loop 2) as critical for PZQ-mediated agonist activity. A key determinant of PZQ selectivity for the 5-HT2B receptor over the 5-HT2A/2C receptors was determined by M2185.39 in transmembrane helix 5 (TM5) of the orthosteric site. Mutation of this residue to valine (M218V), as found in 5-HT2A and 5-HT2C, decreased PZQ agonist activity, whereas the reciprocal mutation (V215M) in 5-HT2C increased PZQ activity. Two-photon imaging in intact mesenteric arterial strips visualized PZQ-evoked Ca2+ transients within the smooth muscle cells of the vessel wall. PZQ also triggered cytoplasmic Ca2+ signals in arterial smooth muscle cells in primary culture that were isolated from mesenteric blood vessels. These data define the molecular basis for PZQ action on 5-HT2B receptors localized in vascular smooth muscle.

Pharmacological Mechanism of the Non-hallucinogenic 5-HT2A Agonist Ariadne and Analogs.

Ariadne is a non-hallucinogenic analog in the phenylalkylamine chemical class of psychedelics that is closely related to an established synthetic hallucinogen, 2,5-dimethoxy-4-methyl-amphetamine (DOM), differing only by one methylene group in the α-position to the amine. Ariadne has been tested in humans including clinical trials at Bristol-Myers Company that indicate a lack of hallucinogenic effects and remarkable therapeutic effects, such as rapid remission of psychotic symptoms in schizophrenics, relaxation in catatonics, complete remission of symptoms in Parkinson's disease (PD), and improved cognition in geriatric subjects. Despite these provocative clinical results, the compound has been abandoned as a drug candidate and its molecular pharmacology remained unknown. Here, we report a detailed examination of the in vitro and in vivo pharmacology of Ariadne and its analogs, and propose a molecular hypothesis for the lack of hallucinogenic effects and the therapeutic potential of this compound class. We also provide a summary of previous clinical and preclinical results to contextualize the molecular signaling data. Our results show that Ariadne is a serotonin 5-HT2 receptor agonist, exhibits modest selectivity over 5-HT1 receptors, has no relevant activity at 5-HT4,5,7 and other aminergic receptors, and no substantial affinity at plasma membrane monoamine transporters. Compared to DOM, Ariadne shows lower signaling potency and efficacy in multiple signaling pathways examined (Gq, G11, and ß-arrestin2) coupled to 5-HT2A receptors. We confirmed the shift in signaling for an α-propyl analog and provide a molecular docking rationale for the progressive decrease in signaling potency with the growing length of the α-substituent. Ariadne versus DOM exhibits no apparent change in the relative preference between Gq/11 activation and ß-arrestin2 recruitment; instead, there is a small but consistent drop in efficacy in these signaling channels. Ariadne acts as a 5-HT2A agonist in vivo in mice and shows markedly attenuated head twitch response (HTR) in comparison to its hallucinogenic analogs, consistent with previous studies in rabbits, cats, and dogs. Hence, we propose the lower 5-HT2A receptor signaling efficacy of this compound class as an explanatory model for the lack of hallucinogenic effects of Ariadne in humans and the dramatically attenuated hallucinosis-like effects in animals (5-HT2A signaling efficacy hypothesis). In terms of reverse translation of the noted clinical therapeutic effects, we used an auxilin knockout model of Parkinson's disease where Ariadne rescued severe motor deficits in this mouse line, on par with the effects of l-DOPA, a notable finding considering Ariadne's lack of activity at dopamine receptors and transporters. Ariadne emerges as a prototype of a new drug class, non-hallucinogenic 5-HT2A agonists, with considerable therapeutic potential across psychiatric and neurological indications.

Bespoke library docking for 5-HT2A receptor agonists with antidepressant activity.

There is considerable interest in screening ultralarge chemical libraries for ligand discovery, both empirically and computationally1-4. Efforts have focused on readily synthesizable molecules, inevitably leaving many chemotypes unexplored. Here we investigate structure-based docking of a bespoke virtual library of tetrahydropyridines-a scaffold that is poorly sampled by a general billion-molecule virtual library but is well suited to many aminergic G-protein-coupled receptors. Using three inputs, each with diverse available derivatives, a one pot C-H alkenylation, electrocyclization and reduction provides the tetrahydropyridine core with up to six sites of derivatization5-7. Docking a virtual library of 75 million tetrahydropyridines against a model of the serotonin 5-HT2A receptor (5-HT2AR) led to the synthesis and testing of 17 initial molecules. Four of these molecules had low-micromolar activities against either the 5-HT2A or the 5-HT2B receptors. Structure-based optimization led to the 5-HT2AR agonists (R)-69 and (R)-70, with half-maximal effective concentration values of 41 nM and 110 nM, respectively, and unusual signalling kinetics that differ from psychedelic 5-HT2AR agonists. Cryo-electron microscopy structural analysis confirmed the predicted binding mode to 5-HT2AR. The favourable physical properties of these new agonists conferred high brain permeability, enabling mouse behavioural assays. Notably, neither had psychedelic activity, in contrast to classic 5-HT2AR agonists, whereas both had potent antidepressant activity in mouse models and had the same efficacy as antidepressants such as fluoxetine at as low as 1/40th of the dose. Prospects for using bespoke virtual libraries to sample pharmacologically relevant chemical space will be considered.

Characterization of Dual-Acting A3 Adenosine Receptor Positive Allosteric Modulators That Preferentially Enhance Adenosine-Induced Gαi3 and GαoA Isoprotein Activation.

The A3 adenosine receptor (A3AR) is a promising therapeutic target for inflammatory diseases, cancer, and chronic neuropathic pain, with agonists already in advanced clinical trials. Here we report an in-depth comparison of the pharmacological properties and structure-activity relationships of existing and expanded compound libraries of 2-substituted 1H-imidazo[4,5-c]quinolin-4-amine and 4-amino-substituted quinoline derivatives that function as A3AR positive allosteric modulators (PAMs). We also show that our lead compound from each series enhances adenosine-induced A3AR signaling preferentially toward activation of Gαi3 and GαoA isoproteins, which are coexpressed with the A3AR in immune cells and spinal cord neurons. Finally, utilizing an extracellular/intracellular chimeric A3AR approach composed of sequences from a responding (human) and a nonresponding (mouse) species, we provide evidence in support of the idea that the imidazoquinolin-4-amine class of PAMs variably interacts dually with the orthosteric ligand binding site as well as with a separate allosteric site located within the inner/intracellular regions of the receptor. This study has advanced both structural and pharmacological understanding of these two classes of A3AR PAMs, which includes leads for future pharmaceutical development.

Conformational selection guides ß-arrestin recruitment at a biased G protein-coupled receptor.

G protein-coupled receptors (GPCRs) recruit ß-arrestins to coordinate diverse cellular processes, but the structural dynamics driving this process are poorly understood. Atypical chemokine receptors (ACKRs) are intrinsically biased GPCRs that engage ß-arrestins but not G proteins, making them a model system for investigating the structural basis of ß-arrestin recruitment. Here, we performed nuclear magnetic resonance (NMR) experiments on 13CH3-ε-methionine-labeled ACKR3, revealing that ß-arrestin recruitment is associated with conformational exchange at key regions of the extracellular ligand-binding pocket and intracellular ß-arrestin-coupling region. NMR studies of ACKR3 mutants defective in ß-arrestin recruitment identified an allosteric hub in the receptor core that coordinates transitions among heterogeneously populated and selected conformational states. Our data suggest that conformational selection guides ß-arrestin recruitment by tuning receptor dynamics at intracellular and extracellular regions.

Discovery of Highly Potent Serotonin 5-HT2 Receptor Agonists Inspired by Heteroyohimbine Natural Products.

The serotonin 5-HT2 receptors are important pharmaceutical targets involved in signaling pathways underlying various neurological, psychiatric, and cardiac functions and dysfunctions. As such, numerous ligands for the investigation of these receptors' activity and downstream effects have been developed synthetically or discovered in nature. For example, the heteroyohimbine natural product alstonine exhibits antispychotic activity mediated by 5-HT2A/2C agonism. In this work, we identified a heteroyohimbine metabolite containing a serotonin pharmacophore and truncated the scaffold, leading to the discovery of potent agonist activity of substituted tetrahydro-ß-carbolines across the 5-HT2 receptor family. Extensive SAR development resulted in compound 106 with EC50 values of 1.7, 0.58, and 0.50 nM at 5-HT2A, 5-HT2B, and 5-HT2C, respectively. Docking studies suggest a π-stacking interaction between the tetrahydro-ß-carboline core and conserved residue Trp6.48 as the structural basis for this activity. This work lays a foundation for future investigation of these compounds in neurological and psychiatric disorders.