New scientific discoveries pave the way for the development of new opioid analgesics

The picture shows a brain blooming with poppies, a great source of opioid analgesics. A frog on the poppy indicates that dervanins from the surface of the frog’s skin were used in this study. On the right side of the image are the structures of four activated opioid receptors embedded within the cell membrane. The opioid substances from the opium poppy mimic the function of opioid peptides in the human body, and all regulate physiological activities such as analgesia, perception, and reward in the human body by binding to opioid receptors. This study reports the binding of endogenous polypeptides across the entire opioid receptor family, as well as the three-dimensional structure of exogenous dermorphins on the skin surface of DOR and frogs.

The endogenous opioid system is composed of four opioid receptor members and a series of opioid peptides, which are widely distributed in the central nervous system, peripheral nervous system and immune system, regulating analgesia, euphoria, reward, cognition, stress and other signaling pathways, and are important targets for the treatment of pain, anxiety and other diseases.

There are four members of the opioid receptor family, namely μOR, δOR, κOR, and NOPR, all of which belong to G protein-coupled receptors and mainly function by conjugating downstream Gi proteins. Opioid receptors are activated not only by opioid peptides produced in the body, but also by exogenous opioids/non-opioids, including well-known drugs such as morphine, fentanyl, and more. In the journal Cell on November 10, 2022, the team of researcher Huaqiang Xu/Associate Professor Youwen Zhuang, together with researcher Xie Xin’s team and Professor Wang Mingwei’s team, reported for the first time the structure of morphine and fentanyl binding human μOR-Gi protein signaling complex, revealing the key differences between the two in binding to μ opioid receptors.

Small molecule opioids targeting μOR are widely used in clinical trials for moderate to severe acute pain, but they are accompanied by many serious side effects such as drug resistance, respiratory depression, and addiction while they are strong in analgesia. In order to develop analgesic drugs with lower toxic side effects, researchers have focused on agonists of other subtypes of opioid receptors, and successfully promoted the development of some analgesic drugs, including diazocine targeting κOR, pentazocine hydrochloride, nalbuphine sebacate, etazocine hydrobromide and other listed drugs, clinical candidates such as SRI-22138 and BMS986187 targeting δOR, PPL-138 targeting NOPR, LY-2940094 and other clinical drug candidates (derived from Yaodu data). Despite great progress, due to the poor selectivity and pharmacological activity of small molecule drugs, other side effects: hallucinogenicity, convulsions, epilepsy, etc., resulting in their clinical progress being hindered or even terminated.

Opioid peptides in the human body are not only a natural template for exogenous opioids such as powerful analgesic morphine, but also do not lead to side effects such as addiction and respiratory depression at the concentration secreted by the body, and endogenous opioid peptides have high affinity and agonistic activity for the four opioid receptors, and most importantly, they are highly selective, which gives them important research and development value. In recent years, with the widespread spread of the Internet saying “teenagers only know dopamine, middle-aged people understand endorphins”, inspirational activities that stimulate endorphin production have been gradually promoted. Endorphins are a polypeptide neurotransmitter among the many members of opioid peptides, which will be released when the body feels pain or stress, helping to reduce pain and relieve stress, and endorphins will also be released in massage, running, swimming and other exercise sports, through the body’s reward signal pathway to enhance people’s happiness. Opioids are enzymatically hydrolyzed and modified by a series of precursor proteins, including endorphins, dynorphins, enkephalins, and pain-sensitive peptides. In addition to endogenous opioid peptides, opioid receptors are also activated by exogenous polypeptides, including tyrosinins in food and dermorphins on the surface of frog skin.

With the development of X-ray crystallography technology and cryo-EM technology, in the past 20 years, more than 800 GPCR-related structures have been reported (derived from GPCRdb), opioid receptors as a pivotal class of drug targets, the inactivation and activation structures of this family have also been published, in previous studies, many scientific questions of much concern have been explained, including the binding mode of opioid receptors to small molecule antagonists/agonists, the activation mechanism mediated by sodium ion pockets, and the bias mechanism of μOR. However, the molecular mechanism of how endogenous opioid peptide ligands selectively recognize and activate opioid receptors of different subtypes is unclear, which hinders researchers’ understanding and rational design modification of opioid peptides.

On January 12, 2023, the team of Huaqiang Xu/Youwen Zhuang of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences and the team of Bryan L. Roth of the University of North Carolina at Chapel Hill published a research paper entitled “Structures of the entire human opioid receptor family” online in the top international journal Cell. This study systematically analyzed and clarified the signaling activity of endogenous opioid peptides on opioid receptors, and further analyzed the cryo-EM complex structures of four subtypes μOR, δOR, ΚOR, NOPR and their respective selective opioid peptides including endorphins, endorphins, dynorphins, dermorphins and pain-sensitive peptides, combined with a large number of functional experiments at the cellular level, revealed the binding patterns of peptide ligands and opioid receptors, and elaborated their selectivity and conservation mechanisms, as “messenger-mailbox-potency.” “Patterns provide structural and functional support.

In this study, the researchers first systematically analyzed the signaling properties of opioid peptides on wild-type opioid receptors through functional experiments at the cellular level. Since the degree of activation of endorphins and enkephalins on μOR and δOR is similar, in order to systematically elucidate the selectivity differences, the researchers finally locked the endorphins and the more selective endorphin, dynorphin, pain-sensitive peptide and exogenous dervin as the research object, respectively, analyzed the three-dimensional structure of endorphin-μOR, endorphin-μOR, dynorkin-κOR, pain-sensitive peptide-NOPR, and dervin-δOR in high-resolution cryo-EM, and used the inhibition test of the second messenger cAMP. G protein and arrestin protein recruitment experiments to analyze the characteristics of mutants.

Figure 1. Functional experiments and cryo-EM structures for opioid receptor binding to opioid peptides. A: Affinity of opioid peptide-induced opioid receptor activation, B:cAMP inhibition curve results, curve results of C:Gi recruitment experiment, cryo-EM structure and atomic model of D-H: opioid receptor binding to opioid peptides.

The study found that all opioid peptides entered the receptor in the form of N-terminal insertion, and the N-terminal motif YGGF (pain peptide is FGGF) of the opioid peptide “messenger” bound to the very conserved positive binding pocket chamber of the opioid receptor, the first tyrosine/ The amino groups on the phenylalanine backbone all form a polar network with the D3.32Q2.60Y7.43 motif and are essential for ligand recognition and pharmacological activity, and the synthesized YGGF tetrapeptide can activate the four receptors (EC50 about 10 μM) through a weaker action. Structural analysis found that the extracellular end of opioid receptors, mainly ECL2/3 and TM2/6/7, are different in sequence and chargeability, such as ECL2 of κOR and NOPR is rich in negatively charged amino acids, and the corresponding dynorphins and pain sensitive peptides are rich in positively charged amino acids at ECL2. The results of large-scale functional mutation experiments showed that the extracellular sequence conservatism and charge difference of opioid receptors ECL2/3 and TM2/6/7 jointly determined the selective binding of opioid receptors to opioid peptides.

NOPR is the last receptor discovered in the opioid receptor family, not named with the Greek alphabet, the structure of pain peptide binding to NOPR resolved in this study is also the structure of the first activated form NOPR, through structural alignment analysis, it is not surprising that it follows the common activation mechanism of opioid receptors. In addition, opioid receptors have high selectivity for downstream Gi proteins, and this study found that the ICL3 of opioid receptors will form a short α helix to form a hydrophobic effect with Gi, while conserved arginine forms an electrostatic interaction with Gi. This may also partly explain why the Gi-bound GPCR structure contains intact ICL3, while the electron cloud density of ICL3 is often missing in the GPCR structure bound to other subtypes of G proteins (Gs, Gq).

In summary, the research team systematically elucidated the binding pattern of peptide ligands by resolving the opioid receptor structure bound by opioid peptides, revealed the conserved binding pocket in opioid receptors and the unique mechanism of action of ECL2/3 co-mediating opioid peptides, and elucidated the general activation mechanism of opioid receptors and the selective characteristics of downstream Gi proteins. These works provide a solid structural basis for understanding the biology of the opioid system, paving the way for the design of novel and highly effective opioid peptide analgesics.

Figure 2. Selection of opioid receptors and opioid peptides and the structural basis of the “messenger-letterbox” concept.

The cryo-EM data of this study were collected at the peak EM center of Shanghai Institute of Materia Medica, and the cryo-EM platform of Shanghai Institute of Materia Medica. Yue Wang, a doctoral student at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Youwen Zhuang, an associate researcher, and Jeffery F. DiBerto at the University of North Carolina at Chapel Hill are co-first authors of the paper. Huaqiang Xu, Associate Professor Youwen Zhuang of the Shanghai Institute of Materia Medica, and Professor Bryan L. Roth of the University of North Carolina at Chapel Hill are co-corresponding authors of the paper. Also participating in the study were Weiyi Liu, a master’s graduate of Shanghai Institute of Materia Medica, Qingning Yuan, Executive Director of Summit Electron Microscopy Center, Professor Karsten Melcher of the Winanluo Institute and X. Dr. Edward Zhou et al. The work was funded by the National Natural Science Foundation of China, the Key R&D Program of the Ministry of Science and Technology, the Shanghai Science and Technology Major Project, the Special Assistant Research Program of the Chinese Academy of Sciences, and the National Institute of Mental Health and the National Institutes of Health. (Source: Shanghai Institute of Materia Medica, Chinese Academy of Sciences)

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