Chemistry and Pharmacology Branch (CP)

Research Highlight

Conformational dynamics of the µ-opioid receptor determine ligand intrinsic efficacy

Jiawei Zhao, Matthias Elgeti, Evan S. O’Brien, Cecília P. Sár, Amal EI Daibani, Jie Heng, Xiaoou Sun, Elizabeth White, Tao Che, Wayne L. Hubbell, Brian K. Kobilka, Chunlai Chen. Nature 629, 474-480 (2024).

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Structurally diverse ligands including alkaloids from the opium poppy and kratom leaf as well as synthetic compounds such as fentanyl bind and activate the μ opioid receptor (μOR). These agonists range in efficacy from full agonists (fentanyl) to strong partial agonists (morphine and mitragynine pseudoindoxyl) to weak partial agonists (buprenorphine). Despite the recent success in the determination of the active-state cryoEM structures of the µOR complexes, the structural and mechanistic basis for differences in ligand efficacies has remained elusive. In a recent effort, scientists used double electron-electron resonance (DEER) and single-molecule fluorescence resonance energy transfer (smFRET) to monitor conformational changes in transmembrane domain 6 (TM6) and the second intracellular loop (ICL2) in response to a panel of µOR ligands with different efficacy profiles. Results from this study showed that full agonists stabilized two distinct outward conformations in TM6, as well as conformational changes in ICL2. The G protein Gi appears to prefer the smaller outward movement of TM6 while arrestin prefers the larger conformational change. The binding of partial agonists such as morphine induce conformational changes in ICL2 without inducing any outward movement of TM6 until the µOR is coupled to Gi. Moreover, the binding of a full agonist induces a conformational change that dramatically lowers GDP affinity within the ternary complex. Findings from this study suggest targeting the sparsely populated conformational states of µOR as a potential approach for the design of therapeutic agents with fewer adverse effects.

Past Highlights

Structural insights into opposing actions of neurosteroids on GABAA receptors

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Structural insights into opposing actions of neurosteroids on GABAA receptors

Dagimhiwat H. Legesse, Chen Fan, Jinfeng Teng, Yuxuan Zhuang, Rebecca J. Howard, Colleen M. Noviello, Erik Lindahl & Ryan E. Hibbs. Nature Communications. 2023 14(1):5091.

Neurosteroids are endogenous modulators of neurotransmission and are emerging as valuable therapeutics for treating depression. In a recent effort, scientists determined how different classes of neurosteroids bind to the GABAA receptor that is the target of the primary inhibitory neurotransmitter of the brain, GABA. This receptor is part of a superfamily of pentameric ligand-gated ion channels. Cryo-electron microscopy revealed the binding site for the positive modulator, allopregnanolone, deep in the membrane, at an interface of two subunits in the receptor (see Figure). This neurosteroid is currently used to treat post-partum depression. The investigators further found that two sulfated steroids, DHEAS and pregnenolone sulfate, bind in the ion channel itself, and thereby inhibit channel activity. The findings reveal how chemically similar modulators act through distinct receptor sites to generate opposing activities. The details of allopregnanolone binding in particular may be helpful in developing improved antidepressants.

Structure-based design of bitopic ligands for the µ-opioid receptor

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Structure-based design of bitopic ligands for the µ-opioid receptor

Abdelfattah Faouzi, Haoqing Wang, Saheem A. Zaidi, Jeffrey F. DiBerto, Tao Che, Qianhui Qu, Michael J. Robertson, Manish K. Madasu, Amal El Daibani, Balazs R. Varga, Tiffany Zhang, Claudia Ruiz, Shan Liu, Jin Xu, Kevin Appourchaux, Samuel T. Slocum, Shainnel O. Eans, Michael D. Cameron, Ream Al-Hasani, Ying Xian Pan, Bryan L. Roth, Jay P. McLaughlin, Georgios Skiniotis, Vsevolod Katritch, Brian K. Kobilka & Susruta Majumdar. Nature. 2023, 613, 767-774.

Mu-opioid receptor agonists such as fentanyl have long been used for pain management, but are considered a major public health concern due to their adverse side effects, including lethal overdose. In a recent effort, scientists have designed novel bitopic ligands that simultaneously engage the orthosteric binding site and a conserved allosteric sodium ion-binding site within the µ-opioid receptor. Cryo-electron microscopy structure of the µ-opioid receptor bound to the bitopic ligand “C6guano” revealed that the positively charged guanidine group of the ligand engages the Asp2.50 residue in the sodium binding site. The bitopic ligand also shows a distinct G-protein signaling profile compared to fentanyl. In mice, C6guano produced potent antinociception with attenuated adverse effects including reduced respiratory depression. These findings reveal that both efficacy and functional selectivity can be modulated by the design of ligands that occupy the orthosteric and sodium binding sites in a bitopic manner, thus providing a novel strategy to develop safer opioid analgesics.

Insights into distinct signaling profiles of the µOR activated by diverse agonists

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Insights into distinct signaling profiles of the µOR activated by diverse agonists

Qianhui Qu, Weijiao Huang, Deniz Aydin, Joseph M. Paggi, Alpay B. Seven, Haoqing Wang, Soumen Chakraborty, Tao Che, Jeffrey F. DiBerto, Michael J. Robertson, Asuka Inoue, Carl-Mikael Suomivuori, Bryan L. Roth, Susruta Majumdar, Ron O. Dror, Brian K. Kobilka & Georgios Skiniotis. Nat Chem Biol. 2022 Nov 21. doi: 10.1038/s41589-022-01208-y. Epub ahead of print. PMID: 36411392.

Using cryoEM, scientists have now determined the structures of the µ-opioid receptor (MOR) in complex with mitragynine pseudoindoxyl (MP) and lofentanil (LFT). The structures show that the ligands occupy distinct sub-pockets in the same overall binding site within MOR. Atomistic simulations show that the ligands can stabilize different receptor conformations, potentially affecting the propensity of MOR to recruit signaling transducers. These results provide insights into the mechanisms that contribute to low propensity of MP in promoting G protein activation and arrestin recruitment compared to higher efficacy of LFT in signaling through both transducers. These findings reveal molecular mechanisms by which diverse ligands differentially engage distinct signaling pathways while binding to the same orthosteric site. The work was supported by NIDA grants R33DA045884 and R37DA036246.

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

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

Arman A. Sadybekov, Anastasiia V. Sadybekov, Yongfeng Liu, Christos Iliopoulos-Tsoutsouvas, Xi-Ping Huang, Julie Pickett, Blake Houser, Nilkanth Pate, Ngan K. Tran, Fei Tong, Nikolai Zvonok, Manish K. Jain, Olena Savych, Dmytro S. Radchenko, Spyros P. Nikas, Nicos A. Petasis, Yurii S. Moroz, Bryan L. Roth, Alexandros Makriyannis & Vsevolod Katritch; Nature. 2021 Dec 15. doi: 10.1038/s41586-021-04220-9.

Structure-based virtual ligand screening is emerging as a key paradigm for early drug discovery owing to the availability of high-resolution target structures and ultra-large libraries of virtual compounds. In a recent study, NIDA-funded scientists have introduced a conceptually new approach, called V-SYNTHES, that enables computational screening of billions of compounds to identify novel ligands to keep pace with the explosive growth of screening libraries. The method utilizes a hierarchical combinatorial approach that first identifies the best synthon-scaffold combinations as seeds suitable for further growth, and then iteratively elaborates these seeds to select complete molecules with the best docking scores. This method allows rapid detection of the best-scoring compounds in the Giga-scale chemical space while performing docking of only a small fraction (<0.1%) of the library compounds. In this study, the investigators applied the V-SYNTHES approach to screen REAL Space library of more than 11 billion compounds to identify new ligands of the cannabinoid receptors CB1 and CB2. Compared with the standard virtual screening, the V-SYNTHES methods demonstrated dramatically improved hit rate. Follow-up synthesis and evaluations led to the identification of several compounds with sub-micromolar potencies including one ligand with CB2 binding Ki of 0.9 nM that displayed 35 to 65-fold CB2/CB1 functional selectivity as an antagonist.

High-throughput screening and validation of antibodies against synaptic proteins to explore opioid signaling dynamics

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High-throughput screening and validation of antibodies against synaptic proteins to explore opioid signaling dynamics

Mariana Lemos Duarte, Nikita A. Trimbake, Achla Gupta, Christine Tumanut, Xiaomin Fan, Catherine Woods, Akila Ram, Ivone Gomes, Erin N. Bobeck, Deborah Schechtman, Lakshmi A. Devi; Commun Biol. 2021 Feb 22;4(1):238. doi: 10.1038/s42003-021-01744-8

Antibodies are powerful tools to explore signal transduction pathways. In a recent effort, NIDA-funded researchers have developed an integrated approach that combines a strategy of antibody development, a time- and cost-effective method of antibody validation, and a high-throughput microscopy/machine learning pipeline to identify antibodies. Using yeast display antibody libraries from the B cells of immunized rabbits and high-throughput functional screening they identified and validated 137 recombinant high affinity antibodies against understudied synaptic proteins. These antibodies could serve as potential tools to shed light on signaling in the central nervous system.

Using a subset of antibodies targeting the opioid receptors they examined the effect of morphine and fentanyl on mu opioid receptor phosphorylation and signaling. Their studies revealed differences in the rate, extent, and site of opioid receptor phosphorylation induced by morphine and fentanyl, thus highlighting the complexity of opioid signaling and illuminating ligand-dependant differential activation and desensitization of mu opioid receptors. These molecular differences may be relevant to the acceleration in opioid overdose deaths that have been attributed to increased use of fentanyl.

Structure of a Hallucinogen-Activated Gq-Coupled 5-HT_2A Serotonin Receptor

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Structure of a Hallucinogen-Activated Gq-Coupled 5-HT_2A Serotonin Receptor

Kuglae Kim, Tao Che, Ouiliana Panova, Jeffrey F. DiBerto, Jiankun Lyu, Brian E. Krumm, Daniel Wacker, Michael Robertson, Alpay B. Seven, David E. Nichols, Brian K. Shoichet, Georgios Skiniotis, Bryan Roth. Cell. 2020 Sep 17;182(6):1574-1588.e19. doi: 10.1016/j.cell.2020.08.024

Hallucinogens like lysergic acid diethylamide (LSD), psilocybin, and substituted N-benzyl phenylalkylamines are widely used for recreational purposes. In addition to their recreational use, substances such as psilocybin are being considered as therapeutics for many neuropsychiatric disorders including depression, anxiety, and substance abuse. Although the precise mechanisms of action of hallucinogens remain unclear, agonist activity at the 5-HT_2A serotonin (5-hydroxytryptamine [5-HT]) receptor is known to play an essential role in their psychedelic effects in humans.

In a landmark study, NIDA-funded scientists have solved active state structure of a prototypical hallucinogen 25-CN-NBOH bound to 5-HT_2A receptor in complex with an engineered αq heterotrimer by cryoelectron microscopy (cryo-EM). They also obtained X-ray crystal structures of the arrestin-biased agonist LSD and the inverse agonist methiothepin bound to 5-HT_2A receptor. These structures provide insights into the molecular details of both ligand recognition and coupling to the intracellular effectors that are key to gaining a molecular understanding of hallucinogen actions. Moreover, these studies provide a framework for a structure-guided search to identify more selective and efficacious HT_2A receptor agonists as potential therapeutic agents for neuropsychiatric disorders.