The antiseizure properties of ibogalogs, including ibogaminalog (DM506), ibogainalog (IBG), and nor-IBG, were assessed in rodents using the pentylenetetrazol (PTZ)-induced seizure test. The behavioral findings indicated that ibogalogs exhibited mild acute antiseizure effects in mice, with endpoint- and time window-dependent differences between the compounds. The antiseizure effect was suppressed by volinanserin and SB242084, consistent with the involvement of 5-HT2A and 5-HT2C receptors. The antiseizure activity after repeated administration (7 and 14 days) of subthreshold doses of nor-IBG (3 mg/kg) or DM506 (5 mg/kg) was higher than that after acute treatment, indicating augmented efficacy. Subthreshold doses of DM506 and nor-IBG restored the impact of PTZ on monoamine levels in hippocampal tissue following repeated administration, but not after a single dose. Additionally, the influence of ibogalogs was evaluated on epileptiform discharges induced by kainic acid (KA) in the CA3 region of the hippocampus. The results showed that nor-IBG and DM506 decreased epileptiform discharges in a concentration-dependent manner. Nor-IBG activity was inhibited by volinanserin, supporting a role for the 5-HT2AR. Functional studies have shown that ibogalogs are more potent agonists at 5-HT2A/2CRs than at 5-HT1A/1BRs, supporting the role of 5-HT2AR. In conclusion, repetitive treatment with ibogalogs induced antiseizure activity in mice through 5-HT2A/2CR activation, accompanied by normalization of PTZ-induced alterations in hippocampal monoamines. In the hippocampal CA3 subfield, ibogalogs reduced KA-induced epileptiform discharges, where nor-IBG activity was mediated by 5-HT2AR activation.

Abstract NBCn2 (SLC4A10), a member of the SLC4 solute carrier (SLC) family, is a sodium-dependent (bi)carbonate transporter that regulates acid extrusion in various brain regions. Mutations in NBCn2 cause severe neurodevelopmental disorders in humans, and knock out studies suggest that its role in regulating neuronal excitability could hold therapeutic potential for seizure disorders such as epilepsy. Despite its physiological importance, NBCn2’s molecular mechanisms remain largely unknown, and there is limited availability of tool compounds to further probe its role in health and disease. Combining cryoEM with computational docking and simulation studies, we herein elucidate NBCn2’s molecular architecture and substrate binding mechanisms on the atomic scale. Via structure-based drug discovery we further identify a compound series that inhibits NBCn2-mediated transport, and characterize its inhibitory mechanisms via cryoEM. Lastly, we showcase the potential of this compound series to template useful probes by demonstrating pharmacological activity both in primary culture as well as brain slices.

Abstract Positive allosteric modulators (PAMs) of the μ opioid receptor (MOR) offer a promising path toward safer opioid therapeutics, yet their mechanisms of action remain poorly understood. Here, we uncover the structural and mechanistic basis of BMS-986187, a chemically distinct MOR PAM with in vivo efficacy, using an integrated approach combining cryogenic electron microscopy (cryo-EM), molecular dynamics (MD) simulations, signaling assays, and site-directed mutagenesis. We identify a previously uncharacterized allosteric site for BMS-986187, a lipid-facing pocket formed by MOR transmembrane helices 2, 3, and 4, distinct from sites occupied by other known MOR PAMs or negative allosteric modulators. BMS-986187 engages both receptor residues and a neighboring cholesterol molecule, suggesting a cooperative ligand–lipid mechanism. Our studies pinpoint residues essential for allosteric modulation, while information-theory analysis of MD trajectories uncovers specific allosteric communication pathways linking the PAM site to both the orthosteric agonist DAMGO and the G protein interface. Together, these findings redefine the landscape of MOR allosteric modulation by revealing a previously unknown binding site, a potentially lipid-sensitive allosteric mechanism, and the molecular wiring of long-range communication within MOR. This work provides a molecular framework for the rational design of PAMs targeting opioid receptors with improved precision and possible therapeutic potential.

Activation of the serotonin receptor 5-HT1A has been shown to regulate mood and cognition, making 5-HT1A an important target in the treatment of anxiety, depression, and psychosis. Although the receptor signals through inhibitory G proteins, more work is necessary to understand differences in transducer coupling and its relation to functional activity. To develop a molecular understanding of the differences underlying transducer coupling and activation, we performed structure-activity relationship studies of 5-HT1A with distinct G proteins. Through a combination of in vitro assays, we identified a potent partial agonist that selectively engages a G protein subtype. We further investigated the differences in G protein engagement at 5-HT1A with cryo-electron microscopy, determining structures of 5-HT1A bound to distinct ligands and G protein subtypes. Combined with subsequent structure-guided mutagenesis and signaling assays, our studies uncover both orthosteric and allosteric determinants of agonist-specific stimulation of distinct transducers.

Psychedelics show tremendous promise for treating psychiatric disorders and other illnesses, including pain and migraine. Despite decades of research, there is uncertainty which signaling mechanisms are necessary for rapid-acting and durable therapeutic effects of psychedelics. Although activation of the serotonin 5-HT 2A receptor is critical for their psychopharmacological effects, the precise signaling pathways and receptor conformations responsible are still under investigation. This review summarizes progress in studying 5-HT 2A signaling mechanisms and recent developments in the discovery of biased agonist tool compounds to disentangle therapeutic from adverse effects. Moreover, we review insights from structural studies regarding the design of psychedelic-derived compounds with tailored pharmacology and briefly discuss other 5-HT receptors that may be important for shaping therapeutic effects. Finally, by drawing parallels between 5-HT 2A biased signaling and the opioid field, we conclude with lessons learned and discuss the need for more rigor and reproducibility to facilitate the development of novel psychedelic-based pharmacotherapies.

Serotonin (5-hydroxytryptamine, 5-HT) acts via 13 different receptors in humans. Of these receptor subtypes, all but 5-HT1eR have confirmed roles in native tissue and are validated drug targets. Despite 5-HT1eR's therapeutic potential and plausible druggability, the mechanisms of its activation remain elusive. To illuminate 5-HT1eR's pharmacology in relation to the highly homologous 5-HT1FR, we screened a library of aminergic receptor ligands at both receptors and observe 5-HT1e/1FR agonism by multicyclic drugs described as pan-antagonists at 5-HT receptors. Potent agonism by tetracyclic antidepressants mianserin, setiptiline, and mirtazapine suggests a mechanism for their clinically observed anti-migraine properties. Using cryoEM and mutagenesis studies, we uncover and characterize unique agonist-like binding poses of mianserin and setiptiline at 5-HT1eR distinct from similar drug scaffolds in inactive-state 5-HTR structures. Together with computational studies, our data suggest that these binding poses alongside receptor-specific allosteric coupling in 5-HT1eR and 5-HT1FR contribute to the agonist activity of these antidepressants.

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Learn More CiteCitationCitation and abstractCitation and referencesMore citation options ShareShare onFacebookX (Twitter)WeChatLinkedInRedditEmailJump toExpandCollapse First ReactionsAugust 8, 2024Can Targeting the Sodium Site via Water Molecules Lead to the Development of Safer Opioids?Click to copy article linkArticle link copied!Targeting the Na+ site through water molecules has led to the development of a novel bitopic ligand, showing promise for future therapeutic applications.Daniel WackerDaniel WackerDepartment of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York 10029-6574, United StatesMore by Daniel Wackerhttps://orcid.org/0000-0003-4951-7230Marta Filizola*Marta FilizolaDepartment of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York 10029-6574, United States*Email: [email protected]More by Marta Filizolahttps://orcid.org/0000-0002-4382-8276Open PDFACS Central ScienceCite this: ACS Cent. Sci. 2024, XXXX, XXX, XXX-XXXClick to copy citationCitation copied!https://pubs.acs.org/doi/10.1021/acscentsci.4c01105https://doi.org/10.1021/acscentsci.4c01105Published August 8, 2024 Publication History Published online 8 August 2024newsPublished 2024 by American Chemical Society. This publication is licensed under CC-BY 4.0. License Summary*You are free to share (copy and redistribute) this article in any medium or format and to adapt (remix, transform, and build upon) the material for any purpose, even commercially within the parameters below:Creative Commons (CC): This is a Creative Commons license.Attribution (BY): Credit must be given to the creator.View full license*DisclaimerThis summary highlights only some of the key features and terms of the actual license. It is not a license and has no legal value. Carefully review the actual license before using these materials. This publication is licensed underCC-BY 4.0 . License Summary*You are free to share(copy and redistribute) this article in any medium or format and to adapt(remix, transform, and build upon) the material for any purpose, even commercially within the parameters below: Creative Commons (CC): This is a Creative Commons license. Attribution (BY): Credit must be given to the creator.View full license *DisclaimerThis summary highlights only some of the key features and terms of the actual license. It is not a license and has no legal value. Carefully review the actual license before using these materials. License Summary*You are free to share(copy and redistribute) this article in any medium or format and to adapt(remix, transform, and build upon) the material for any purpose, even commercially within the parameters below: Creative Commons (CC): This is a Creative Commons license. Attribution (BY): Credit must be given to the creator. View full license *DisclaimerThis summary highlights only some of the key features and terms of the actual license. It is not a license and has no legal value. Carefully review the actual license before using these materials. License Summary*You are free to share(copy and redistribute) this article in any medium or format and to adapt(remix, transform, and build upon) the material for any purpose, even commercially within the parameters below: Creative Commons (CC): This is a Creative Commons license. Attribution (BY): Credit must be given to the creator. View full license *DisclaimerThis summary highlights only some of the key features and terms of the actual license. It is not a license and has no legal value. Carefully review the actual license before using these materials. ACS PublicationsPublished 2024 by American Chemical SocietySubjectswhat are subjectsArticle subjects are automatically applied from the ACS Subject Taxonomy and describe the scientific concepts and themes of the article.LigandsMoleculesReceptorsScreening assaysTherapeuticsThe μ-opioid receptor (μOR), a member of the G protein-coupled receptor (GPCR) family, continues to be a key target for pain management and opioid use disorder (OUD), with several μOR agonists still considered the gold standard for effective analgesics and OUD treatments. (1) However, the numerous serious adverse effects associated with these drugs, including─but not limited to─the respiratory depression linked to the alarming statistics of the opioid epidemic, (2) underscore the urgent need for safer analgesics and for addressing the chronic and relapsing nature of OUD. To reduce the side effects associated with μOR activation while maintaining its beneficial effects, drug discovery has focused on modulating μOR activity to engage different pathways with distinct efficacies and potencies. However, considerable ambiguity remains about which pathways are linked to specific (patho)physiological outcomes, and even promising preclinical effects in mice have so far failed to translate into safer medications devoid of any liability. (3) Novel tools are therefore needed to complement existing chemical probes that bind at the orthosteric site, where endogenous opioid ligands bind. Of particular interest are tools that can leverage the potential of allosteric sites, such as the highly conserved allosteric binding site of sodium ions (Na+). In this issue of ACS Central Science, Majumdar, McLaughlin, Wang, Hüttenhain, and colleagues propose a new strategy to generate safer opioid-based analgesics. Specifically, the authors report on the development of novel fentanyl derivatives, best exemplified by RO76, which engage residues of the Na+ site within the μOR via a water-mediated interaction. (4) Along with their precursor lead analog, C6 guano, these fentanyl derivatives introduce a novel class of tools to the existing arsenal, as they achieve distinct pharmacological activities by extending to the allosteric Na+ site from their main μOR orthosteric binding site.The differential modulation of opioid agonists and antagonists by Na+ has been known for more than half a century. (5) The collapsed Na+ binding site and key protonated (neutral) residue D1142.50 in active μOR and other GPCR structures likely contribute to a weaker binding affinity of Na+ ions. This allows Na+ to more easily translocate through the receptor and egress from the cytosol in an active receptor compared to an inactive one, as we demonstrated a few years ago using a combination of tens of microseconds of standard molecular dynamics (MD) and umbrella sampling simulations, Markov State Models, and machine learning tools. (6)In the pursuit of developing novel opioid tools, the authors not only generate new bitopic ligands that interact with both orthosteric and allosteric sites of μOR, but also employ an interesting strategy in which Na+ binding site residues are engaged indirectly through water molecules. Given the conservation of this site in other class A GPCRs, the implications of the design and pharmacological activities extend far beyond opioid receptors. Exploiting the Na+ binding site of other GPCRs, with or without water-mediated interactions, could in principle be applied to develop pathway-selective drugs for a variety of therapeutic targets, potentially revolutionizing the treatment of numerous conditions.The design of RO76 was motivated by the discovery that its precursor lead analog C6 guano exhibited μOR-dependent antinociception with reduced adverse effects compared to clinically used opioids but had limited therapeutic potential due to poor blood-brain barrier (BBB) penetration. Unlike RO76, which exhibits a partial agonism profile at all Gαi/o/z subtypes and low recruitment of both β-arrestin subtypes in vitro, C6 guano interacts directly with D1142.50 in the Na+ site and shows reduced activation of Gαo and Gαz compared to Gαi (7) (Figure 1). Encouragingly, RO76 is reported to show better BBB penetration while maintaining analgesic properties with reduced side effects compared to morphine in animal models. These pharmacological distinctions are rationalized as the likely result of the different interactions in the Na+ site. However, it remains unknown how the interactions of C6 guano and RO76 in the Na+ site result in these different activities. The authors propose that the indirect contact with D1142.50 through a single water molecule is responsible for RO76's distinct pharmacology. However, MD studies reveal that this indirect contact is only formed in ∼20% of cases, requiring further clarifying studies. As lower intrinsic activity has recently emerged as a key strategy to reduce μOR-related side effects, (8) and the precise G protein-mediated pathways governing therapeutic and adverse outcomes remain understudied, (9−12) RO76 is a valuable new tool to investigate the precise pathways and activation thresholds required to address opioid safety.Figure 1Figure 1. μOR-mediated Gα subtype selectivity profiles induced by C6 guano or RO76 using the Trupath assay. RO76 data were reproduced with permission from ref (4). Copyright 2024 American Chemical Society. C6 guano data were reproduced with permission from ref (7). Copyright 2024 Springer Nature.High Resolution ImageDownload MS PowerPoint SlideWhile RO76 shows favorable in vivo activity, its lower in vitro potency compared to other analogs indicates that further optimization is needed.Despite these promising findings, several challenges and opportunities lie ahead. First and foremost, what are the mechanisms by which different interactions in the Na+ site promote different signaling outputs, and could such insights lead to the rational design of pathway-selective probes via direct/indirect interaction with distinct Na+ site residues? While RO76 shows favorable in vivo activity, its lower in vitro potency compared to other analogs indicates that further optimization is needed. This optimization may or may not require G-protein selectivity for optimal therapeutic outcomes. This selectivity is lost with RO76, suggesting that water-mediated and direct interactions with residues of the Na+ binding site may lead to the activation of different G protein pathways. Another challenge is understanding the mechanisms underlying the reduced respiratory depression and physical dependence observed with RO76. Initial findings suggest that targeting the Na+ site indirectly through water molecules may modulate signaling pathways differently from traditional opioids. More research is needed to fully elucidate these mechanisms, including further investigating the role of proximal proteins like putative phosphatase Paladin 1 (PALD1) in μOR signaling, as highlighted by the authors. Additionally, since RO76 is derived from fentanyl, it will be important to compare its in vivo effects directly to those of fentanyl to better understand and contrast the underlying mechanisms. Future research could also explore the potential of targeting Na+ sites in other GPCRs. The rational design of ligands that exploit structural waters in the sodium binding pockets of these receptors could lead to the development of pathway-selective drugs with improved therapeutic profiles. This requires a multidisciplinary approach, combining medicinal chemistry, structural biology, and computer simulations to identify and optimize new ligands.The rational design of ligands that exploit structural waters in the sodium binding pockets of these receptors could lead to the development of pathway-selective drugs with improved therapeutic profiles.In summary, this paper represents a significant advancement in the field of GPCR research. By targeting the Na+ site through water molecules, the authors have developed a novel bitopic ligand with a potential toward the development of promising therapeutics. Although challenges remain, the insights gained from this study provide a strong foundation for future research aimed at developing safer and more effective opioid drugs. Moreover, this strategy can be adopted to target other GPCRs, providing complementary tool compound sets to study a wide variety of mechanisms in health and disease. The research community should build on these findings, embracing the innovative strategies and interdisciplinary collaboration needed to push the frontiers of drug discovery even further.Author InformationClick to copy section linkSection link copied!Corresponding AuthorMarta Filizola - Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York 10029-6574, United States; https://orcid.org/0000-0002-4382-8276; Email: [email protected]AuthorDaniel Wacker - Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York 10029-6574, United States; https://orcid.org/0000-0003-4951-7230NotesThe authors declare no competing financial interest.AcknowledgmentsClick to copy section linkSection link copied!The authors thank the National Institute on Drug Abuse for funding (DA045473 to M.F. and DA058681 to D.W.).ReferencesClick to copy section linkSection link copied! This article references 12 other publications. 1Valentino, R. J.; Volkow, N. D. Untangling the complexity of opioid receptor function. Neuropsychopharmacology 2018, 43 (13), 2514– 2520, DOI: 10.1038/s41386-018-0225-3 Google Scholar1Untangling the complexity of opioid receptor functionValentino Rita J; Volkow Nora DNeuropsychopharmacology : official publication of the American College of Neuropsychopharmacology (2018), 43 (13), 2514-2520 ISSN:. Mu opioid receptor agonists are among the most powerful analgesic medications but also among the most addictive. The current opioid crisis has energized a quest to develop opioid analgesics that are devoid of untoward effects. Since their discovery in the 1970's, there have been major advances in our understanding of the endogenous opioid systems that these drugs target. Yet many questions remain and the development of non-addictive opioid analgesics has not been achieved. However, access to new molecular, genetic and computational tools have begun to elucidate the structural dynamics of opioid receptors, the scaffolding that links them to intracellular signaling cascades, their cellular trafficking and the distinct ways that various opioid drugs modify them. This mini-review highlights some of the chemical and pharmacological findings and new perspectives that have arisen from studies using these tools. They reveal multiple layers of complexity of opioid receptor function, including a spatiotemporal specificity in opioid receptor-induced cellular signaling, ligand-directed biased signaling, allosteric modulation of ligand interactions, heterodimerization of different opioid receptors, and the existence of slice variants with different ligand specificity. By untangling these layers, basic research into the chemistry and pharmacology of opioid receptors is guiding the way towards deciphering the mysteries of tolerance and physical dependence that have plagued the field and is providing a platform for the development of more effective and safer opioids. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3czitlShsA%253D%253D&md5=5053c0bd385d822a8274d0f9ae5476a12 VSRR Provisional Drug Overdose Death Counts; Center for Disease Control and Prevention, 2023, https://data.cdc.gov/d/xkb8-kh2a.Google ScholarThere is no corresponding record for this reference.3Niu, J.; Hu, W.; Lu, Y.; Tang, H. Efficacy and safety of oliceridine treatment in patients with postoperative pain: a systematic review and meta-analysis of randomized controlled trials. Expert Rev. Clin Pharmacol 2023, 16 (6), 589– 599, DOI: 10.1080/17512433.2023.2213889 Google ScholarThere is no corresponding record for this reference.4Ople, R. S.; Ramos-Gonzalez, N.; Li, Q.; Sobecks, B. L.; Aydin, D.; Powers, A. S.; Faouzi, A.; Polacco, B. J.; Bernhard, S. M.; Appourchaux, K.; Sribhashyam, S.; Eans, S. O.; Tsai, B. A.; Dror, R. O.; Varga, B. R.; Wang, H.; Huttenhain, R.; McLaughlin, J. P.; Majumdar, S. Signaling modulation mediated by ligand water interactions with sodium site at μOR. ACS Central Sci. 2024, DOI: 10.1021/acscentsci.4c00525 .Google ScholarThere is no corresponding record for this reference.5Pert, C. B.; Pasternak, G.; Snyder, S. H. Opiate agonists and antagonists discriminated by receptor binding in brain. Science 1973, 182 (4119), 1359– 1361, DOI: 10.1126/science.182.4119.1359 Google Scholar5Opiate agonists and antagonists discriminated by receptor binding in brainPert, Candace B.; Pasternak, Gavril; Snyder, Solomon H.Science (Washington, DC, United States) (1973), 182 (4119), 1359-61CODEN: SCIEAS; ISSN:0036-8075. Administration of either opiate agonists or antagonists rapidly increased stereospecific 3H-labeled dihydromorphine [466-99-9] binding to mouse brain exts. by 40-100%, but antagonists were 10-1000 times more potent than agonists. Naloxone-HCl (I-HCl) [357-08-4] (0.02 mg/kg) significantly increased opiate receptor binding. Na [7440-23-5] enhanced antagonist binding in vitro but decreased agonist binding, a qual. difference that may be relevant to the divergent pharmacol. properties of opiate agonists and antagonists. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE2cXnvVGjsQ%253D%253D&md5=4887755e094db5205617a58e1d872e0f6Hu, X.; Wang, Y.; Hunkele, A.; Provasi, D.; Pasternak, G. W.; Filizola, M. Kinetic and thermodynamic insights into sodium ion translocation through the mu-opioid receptor from molecular dynamics and machine learning analysis. PLoS Comput. Biol. 2019, 15 (1), e1006689 DOI: 10.1371/journal.pcbi.1006689 Google ScholarThere is no corresponding record for this reference.7Faouzi, A.; Wang, H.; Zaidi, S. A.; DiBerto, J. F.; Che, T.; Qu, Q.; Robertson, M. J.; Madasu, M. K.; El Daibani, A.; Varga, B. R. Structure-based design of bitopic ligands for the μ-opioid receptor. Nature 2023, 613 (7945), 767– 774, DOI: 10.1038/s41586-022-05588-y Google ScholarThere is no corresponding record for this reference.8Gillis, A.; Gondin, A. B.; Kliewer, A.; Sanchez, J.; Lim, H. D.; Alamein, C.; Manandhar, P.; Santiago, M.; Fritzwanker, S.; Schmiedel, F.; Low intrinsic efficacy for G protein activation can explain the improved side effect profiles of new opioid agonists. Sci. Signal 2020, 13 (625). DOI: 10.1126/scisignal.aaz3140 .Google ScholarThere is no corresponding record for this reference.9Lamberts, J. T.; Jutkiewicz, E. M.; Mortensen, R. M.; Traynor, J. R. μ-Opioid receptor coupling to Galpha(o) plays an important role in opioid antinociception. Neuropsychopharmacology 2011, 36 (10), 2041– 2053, DOI: 10.1038/npp.2011.91 Google ScholarThere is no corresponding record for this reference.10Leck, K. J.; Bartlett, S. E.; Smith, M. T.; Megirian, D.; Holgate, J.; Powell, K. L.; Matthaei, K. I.; Hendry, I. A. Deletion of guanine nucleotide binding protein alpha z subunit in mice induces a gene dose dependent tolerance to morphine. Neuropharmacology 2004, 46 (6), 836– 846, DOI: 10.1016/j.neuropharm.2003.11.024 Google ScholarThere is no corresponding record for this reference.11Raffa, R. B.; Martinez, R. P.; Connelly, C. D. G-protein antisense oligodeoxyribonucleotides and μ-opioid supraspinal antinociception. Eur. J. Pharmacol. 1994, 258 (1–2), R5– 7, DOI: 10.1016/0014-2999(94)90073-6 Google ScholarThere is no corresponding record for this reference.12Sakloth, F.; Sanchez-Reyes, O. B.; Ruiz, A.; Nicolais, A.; Serafini, R. A.; Pryce, K. D.; Bertherat, F.; Torres-Berrio, A.; Gomes, I.; Devi, L. A. A Regional and Projection-Specific Role of RGSz1 in the Ventrolateral Periaqueductal Grey in the Modulation of Morphine Reward. Mol. Pharmacol. 2023, 103 (1), 1– 8, DOI: 10.1124/molpharm.122.000528 Google ScholarThere is no corresponding record for this reference.Cited By Click to copy section linkSection link copied!This article has not yet been cited by other publications.Download PDFFiguresReferencesOpen PDF Get e-AlertsGet e-AlertsACS Central ScienceCite this: ACS Cent. Sci. 2024, XXXX, XXX, XXX-XXXClick to copy citationCitation copied!https://doi.org/10.1021/acscentsci.4c01105Published August 8, 2024 Publication History Published online 8 August 2024Published 2024 by American Chemical Society. This publication is licensed under CC-BY 4.0. License Summary*You are free to share (copy and redistribute) this article in any medium or format and to adapt (remix, transform, and build upon) the material for any purpose, even commercially within the parameters below:Creative Commons (CC): This is a Creative Commons license.Attribution (BY): Credit must be given to the creator.View full license*DisclaimerThis summary highlights only some of the key features and terms of the actual license. It is not a license and has no legal value. Carefully review the actual license before using these materials. Article Views366Altmetric-Citations-Learn about these metrics closeArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated.Recommended Articles FiguresReferencesAbstractHigh Resolution ImageDownload MS PowerPoint SlideFigure 1Figure 1. μOR-mediated Gα subtype selectivity profiles induced by C6 guano or RO76 using the Trupath assay. RO76 data were reproduced with permission from ref (4). Copyright 2024 American Chemical Society. C6 guano data were reproduced with permission from ref (7). Copyright 2024 Springer Nature.High Resolution ImageDownload MS PowerPoint SlideReferences This article references 12 other publications. 1Valentino, R. J.; Volkow, N. D. Untangling the complexity of opioid receptor function. Neuropsychopharmacology 2018, 43 (13), 2514– 2520, DOI: 10.1038/s41386-018-0225-3 1Untangling the complexity of opioid receptor functionValentino Rita J; Volkow Nora DNeuropsychopharmacology : official publication of the American College of Neuropsychopharmacology (2018), 43 (13), 2514-2520 ISSN:. Mu opioid receptor agonists are among the most powerful analgesic medications but also among the most addictive. The current opioid crisis has energized a quest to develop opioid analgesics that are devoid of untoward effects. Since their discovery in the 1970's, there have been major advances in our understanding of the endogenous opioid systems that these drugs target. Yet many questions remain and the development of non-addictive opioid analgesics has not been achieved. However, access to new molecular, genetic and computational tools have begun to elucidate the structural dynamics of opioid receptors, the scaffolding that links them to intracellular signaling cascades, their cellular trafficking and the distinct ways that various opioid drugs modify them. This mini-review highlights some of the chemical and pharmacological findings and new perspectives that have arisen from studies using these tools. They reveal multiple layers of complexity of opioid receptor function, including a spatiotemporal specificity in opioid receptor-induced cellular signaling, ligand-directed biased signaling, allosteric modulation of ligand interactions, heterodimerization of different opioid receptors, and the existence of slice variants with different ligand specificity. By untangling these layers, basic research into the chemistry and pharmacology of opioid receptors is guiding the way towards deciphering the mysteries of tolerance and physical dependence that have plagued the field and is providing a platform for the development of more effective and safer opioids. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3czitlShsA%253D%253D&md5=5053c0bd385d822a8274d0f9ae5476a12 VSRR Provisional Drug Overdose Death Counts; Center for Disease Control and Prevention, 2023, https://data.cdc.gov/d/xkb8-kh2a.There is no corresponding record for this reference.3Niu, J.; Hu, W.; Lu, Y.; Tang, H. Efficacy and safety of oliceridine treatment in patients with postoperative pain: a systematic review and meta-analysis of randomized controlled trials. Expert Rev. Clin Pharmacol 2023, 16 (6), 589– 599, DOI: 10.1080/17512433.2023.2213889 There is no corresponding record for this reference.4Ople, R. S.; Ramos-Gonzalez, N.; Li, Q.; Sobecks, B. L.; Aydin, D.; Powers, A. S.; Faouzi, A.; Polacco, B. J.; Bernhard, S. M.; Appourchaux, K.; Sribhashyam, S.; Eans, S. O.; Tsai, B. A.; Dror, R. O.; Varga, B. R.; Wang, H.; Huttenhain, R.; McLaughlin, J. P.; Majumdar, S. Signaling modulation mediated by ligand water interactions with sodium site at μOR. ACS Central Sci. 2024, DOI: 10.1021/acscentsci.4c00525 .There is no corresponding record for this reference.5Pert, C. B.; Pasternak, G.; Snyder, S. H. Opiate agonists and antagonists discriminated by receptor binding in brain. Science 1973, 182 (4119), 1359– 1361, DOI: 10.1126/science.182.4119.1359 5Opiate agonists and antagonists discriminated by receptor binding in brainPert, Candace B.; Pasternak, Gavril; Snyder, Solomon H.Science (Washington, DC, United States) (1973), 182 (4119), 1359-61CODEN: SCIEAS; ISSN:0036-8075. Administration of either opiate agonists or antagonists rapidly increased stereospecific 3H-labeled dihydromorphine [466-99-9] binding to mouse brain exts. by 40-100%, but antagonists were 10-1000 times more potent than agonists. Naloxone-HCl (I-HCl) [357-08-4] (0.02 mg/kg) significantly increased opiate receptor binding. Na [7440-23-5] enhanced antagonist binding in vitro but decreased agonist binding, a qual. difference that may be relevant to the divergent pharmacol. properties of opiate agonists and antagonists. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE2cXnvVGjsQ%253D%253D&md5=4887755e094db5205617a58e1d872e0f6Hu, X.; Wang, Y.; Hunkele, A.; Provasi, D.; Pasternak, G. W.; Filizola, M. Kinetic and thermodynamic insights into sodium ion translocation through the mu-opioid receptor from molecular dynamics and machine learning analysis. PLoS Comput. Biol. 2019, 15 (1), e1006689 DOI: 10.1371/journal.pcbi.1006689 There is no corresponding record for this reference.7Faouzi, A.; Wang, H.; Zaidi, S. A.; DiBerto, J. F.; Che, T.; Qu, Q.; Robertson, M. J.; Madasu, M. K.; El Daibani, A.; Varga, B. R. Structure-based design of bitopic ligands for the μ-opioid receptor. Nature 2023, 613 (7945), 767– 774, DOI: 10.1038/s41586-022-05588-y There is no corresponding record for this reference.8Gillis, A.; Gondin, A. B.; Kliewer, A.; Sanchez, J.; Lim, H. D.; Alamein, C.; Manandhar, P.; Santiago, M.; Fritzwanker, S.; Schmiedel, F.; Low intrinsic efficacy for G protein activation can explain the improved side effect profiles of new opioid agonists. Sci. Signal 2020, 13 (625). DOI: 10.1126/scisignal.aaz3140 .There is no corresponding record for this reference.9Lamberts, J. T.; Jutkiewicz, E. M.; Mortensen, R. M.; Traynor, J. R. μ-Opioid receptor coupling to Galpha(o) plays an important role in opioid antinociception. Neuropsychopharmacology 2011, 36 (10), 2041– 2053, DOI: 10.1038/npp.2011.91 There is no corresponding record for this reference.10Leck, K. J.; Bartlett, S. E.; Smith, M. T.; Megirian, D.; Holgate, J.; Powell, K. L.; Matthaei, K. I.; Hendry, I. A. Deletion of guanine nucleotide binding protein alpha z subunit in mice induces a gene dose dependent tolerance to morphine. Neuropharmacology 2004, 46 (6), 836– 846, DOI: 10.1016/j.neuropharm.2003.11.024 There is no corresponding record for this reference.11Raffa, R. B.; Martinez, R. P.; Connelly, C. D. G-protein antisense oligodeoxyribonucleotides and μ-opioid supraspinal antinociception. Eur. J. Pharmacol. 1994, 258 (1–2), R5– 7, DOI: 10.1016/0014-2999(94)90073-6 There is no corresponding record for this reference.12Sakloth, F.; Sanchez-Reyes, O. B.; Ruiz, A.; Nicolais, A.; Serafini, R. A.; Pryce, K. D.; Bertherat, F.; Torres-Berrio, A.; Gomes, I.; Devi, L. A. A Regional and Projection-Specific Role of RGSz1 in the Ventrolateral Periaqueductal Grey in the Modulation of Morphine Reward. Mol. Pharmacol. 2023, 103 (1), 1– 8, DOI: 10.1124/molpharm.122.000528 There is no corresponding record for this reference.

The human trace amine-associated receptor 1 (hTAAR1, hTA1) is a key regulator of monoaminergic neurotransmission and the actions of psychostimulants. Despite preclinical research demonstrating its tractability as a drug target, its molecular mechanisms of activation remain unclear. Moreover, poorly understood pharmacological differences between rodent and human TA1 complicate the translation of findings from preclinical disease models into novel pharmacotherapies. To elucidate hTA1's mechanisms on the molecular scale and investigate the underpinnings of its divergent pharmacology from rodent orthologs, we herein report the structure of the human TA1 receptor in complex with a Gαs heterotrimer. Our structure reveals shared structural elements with other TAARs, as well as with its closest monoaminergic ortholog, the serotonin receptor 5-HT4R. We further find that a single mutation dramatically shifts the selectivity of hTA1 towards that of its rodent orthologs, and report on the effects of substituting residues to those found in serotonin and dopamine receptors. Strikingly, we also discover that the atypical antipsychotic medication and pan-monoaminergic antagonist asenapine potently and efficaciously activates hTA1. Together our studies provide detailed insight into hTA1 structure and function, contrast its molecular pharmacology with that of related receptors, and uncover off-target activities of monoaminergic drugs at hTA1.

Psychedelic substances such as lysergic acid diethylamide (LSD) and psilocybin show potential for the treatment of various neuropsychiatric disorders1-3. These compounds are thought to mediate their hallucinogenic and therapeutic effects through the serotonin (5-hydroxytryptamine (5-HT)) receptor 5-HT2A (ref. 4). However, 5-HT1A also plays a part in the behavioural effects of tryptamine hallucinogens5, particularly 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT), a psychedelic found in the toxin of Colorado River toads6. Although 5-HT1A is a validated therapeutic target7,8, little is known about how psychedelics engage 5-HT1A and which effects are mediated by this receptor. Here we map the molecular underpinnings of 5-MeO-DMT pharmacology through five cryogenic electron microscopy (cryo-EM) structures of 5-HT1A, systematic medicinal chemistry, receptor mutagenesis and mouse behaviour. Structure-activity relationship analyses of 5-methoxytryptamines at both 5-HT1A and 5-HT2A enable the characterization of molecular determinants of 5-HT1A signalling potency, efficacy and selectivity. Moreover, we contrast the structural interactions and in vitro pharmacology of 5-MeO-DMT and analogues to the pan-serotonergic agonist LSD and clinically used 5-HT1A agonists. We show that a 5-HT1A-selective 5-MeO-DMT analogue is devoid of hallucinogenic-like effects while retaining anxiolytic-like and antidepressant-like activity in socially defeated animals. Our studies uncover molecular aspects of 5-HT1A-targeted psychedelics and therapeutics, which may facilitate the future development of new medications for neuropsychiatric disorders.

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 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.

Opioid analgesics exert their therapeutic and adverse effects by activating μ opioid receptors (MOPR); however, functional responses to MOPR activation are modulated by distinct signal transduction complexes within the brain. The ventrolateral periaqueductal gray (vlPAG) plays a critical role in modulation of nociception and analgesia, but the exact intracellular pathways associated with opioid responses in this region are not fully understood. We previously showed that knockout of the signal transduction modulator Regulator of G protein Signaling z1 (RGSz1) enhanced analgesic responses to opioids, whereas it decreased the rewarding efficacy of morphine. Here, we applied viral mediated gene transfer methodology and delivered adeno-associated virus (AAV) expressing Cre recombinase to the vlPAG of RGSz1fl\fl mice to demonstrate that downregulation of RGSz1 in this region decreases sensitivity to morphine in the place preference paradigm, under pain-free as well as neuropathic pain states. We also used retrograde viral vectors along with flippase-dependent Cre vectors to conditionally downregulate RGSz1 in vlPAG projections to the ventral tegmental area (VTA) and show that downregulation of RGSz1 prevents the development of place conditioning to low morphine doses. Consistent with the role for RGSz1 as a negative modulator of MOPR activity, RGSz1KO enhances opioid-induced cAMP inhibition in periaqueductal gray (PAG) membranes. Furthermore, using a new generation of bioluminescence resonance energy transfer (BRET) sensors, we demonstrate that RGSz1 modulates Gαz but not other Gαi family subunits and selectively impedes MOPR-mediated Gαz signaling events invoked by morphine and other opioids. Our work highlights a regional and circuit-specific role of the G protein–signaling modulator RGSz1 in morphine reward, providing insights on midbrain intracellular pathways that control addiction-related behaviors. SIGNIFICANCE STATEMENT This study used advanced genetic mouse models to highlight the role of the signal transduction modulator named RGSz1 in responses to clinically used opioid analgesics. We show that RGSz1 controls the rewarding efficacy of opioids by actions in ventrolateral periaqueductal gray projections to the ventral tegmental area, a key component of the midbrain dopamine pathway. These studies highlight novel mechanisms by which pain-modulating structures control the rewarding efficacy of opioids.

5-hydroxytryptamine (serotonin) 1E and 1F receptors (5-HT1E and 5-HT1F, respectively) are highly expressed human G protein-coupled receptors with high sequence identity among themselves and other 5-HT1 receptor subtypes. Although the physiological role of 5-HT1E and 5-HT1F is not fully understood, they are molecular targets of drugs that are effective to treat migraine, depression, and schizophrenia, albeit not without also producing adverse effects. Notably, no selective or high-affinity drugs have been reported for the 5-HT1E receptor to date. Thus, there is widespread interest in understanding at an atomic level of detail how small molecules bind and activate these receptors for the ultimate purpose of designing improved therapeutics. Through computational studies including docking calculations, metadynamics rescoring, and molecular dynamics simulations of cryo-electron microscopy structures, we predict the binding mode and mechanism of action of tricyclic small molecules that are capable of simultaneously activating the 5-HT1E and 5-HT1F receptors. Our results provide testable hypotheses of ligand-receptor interactions and ligand-induced allosteric modulation that may be used to improve the efficacy of these small molecules towards specific biological endpoints.

Of the 13 serotonin (5-hydroxytryptamine, 5-HT) receptors in humans, the 5-HT1E and 5-HT1F subtypes (1E and 1F, respectively), are understudied G protein-coupled receptors with high sequence identity among themselves, and to a lesser degree, the other 5-HT1 receptor subtypes. While 1F is a validated target for anti-migraine drugs, the physiological function of 1E remains unknown due to a lack of rodent orthologs and selective compounds. To expand the repertoire of potential chemical scaffolds that could be used to generate 1E- and 1F-selective compounds, we screened a small library of medications and research compounds with known activities at various aminergic receptors. This screen led us to discover that a series of clinically-used drugs featuring a tetracyclic scaffold are high-affinity agonists at 1E and 1F, while they have generally been reported as pan-aminergic antagonists. Seeking to elucidate their mechanisms, we determined structures of 5-HT1E complexed with Gi1 and bound by two of these drugs at 3.31 and 3.28 Å. These structures reveal unambiguous binding poses that demonstrate key interactions with binding pocket residues that confer high affinity in a ligand-specific fashion, and lay the stage for the generation of 1E-specific agonists for physiological studies. Additionally, our functional work offers a putative mechanism of action for the reported anti-migraine activities of the medications characterized in this study.

Anion exchanger 1 (AE1), a member of the solute carrier (SLC) family, is the primary bicarbonate transporter in erythrocytes, regulating pH levels and CO2 transport between lungs and tissues. Previous studies characterized its role in erythrocyte structure and provided insight into transport regulation. However, key questions remain regarding substrate binding and transport, mechanisms of drug inhibition and modulation by membrane components. Here we present seven cryo-EM structures in apo, bicarbonate-bound and inhibitor-bound states. These, combined with uptake and computational studies, reveal important molecular features of substrate recognition and transport, and illuminate sterol binding sites, to elucidate distinct inhibitory mechanisms of research chemicals and prescription drugs. We further probe the substrate binding site via structure-based ligand screening, identifying an AE1 inhibitor. Together, our findings provide insight into mechanisms of solute carrier transport and inhibition.

Anion Exchanger 1 (AE1, SLC4A1) is the primary bicarbonate (HCO3-) transporter expressed in erythrocyte membranes where it mediates transport of CO2 between lungs and other tissues via import/export of bicarbonate. It is also a key regulator of erythrocyte structure and antigenic recognition. Previous biochemical studies, and a low-resolution crystal structure of the transmembrane domain have provided initial insight into AE1 structure and function. However, key questions remain regarding substrate binding and transport as well as the mechanism of inhibition. The orientation of the intracellular domain as well as the localization of lipid and sterol binding sites also remain enigmatic. We herein present seven novel high resolution cryo-EM structures of the full length human transporter in the apo, bicarbonate-bound, and several inhibitor-bound states combined with uptake- and computational studies. To our knowledge, these studies represent the first full length human, and substrate bound, SLC4 transporter structure. Our results reveal important molecular details about substrate binding and transport, as well as the diverse mechanisms of AE1 inhibition by both research chemicals and prescription drugs. We also provide novel insights into the full-length transporter architecture, identify the conformational space of the Diego blood antigen system and elucidate multiple lipid and sterol binding sites.

The cover image is based on the Review Article Artificial intelligence and machine learning-aided drug discovery in central nervous system diseases: State-of-the-arts and future directions by Sezen Vatansever et al., https://doi.org/10.1002/med.21764.