Cell Shanghai Drug reveals the structural basis of the mechanism of action of the potent analgesic fentanyl and morphine

On November 10, 2022, the team of Xu Huaqiang/Zhuang Youwen, Xie Xin and Wang Mingwei of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences cooperated to publish a research paper
entitled "Molecular recognition of morphine and fentanyl by the human μ-opioid receptor" online in the top international journal Cell 。 This study solved and reported the high-resolution three-dimensional structure of opioid analgesics such as fentanyl, morphine and olicuridine to activate μ opioid receptors (μOR), respectively, revealing for the first time the mechanism
of action of fentanyl and morphine to recognize and activate μOR 。 This study further combines a variety of cellular-level functional analysis and molecular dynamics simulation methods to clarify the structure-activity relationship between fentanyl series derivatives and the target μOR interaction, as well as the key structural basis of μOR-mediated inhibitory protein (Arrestin) signaling, systematically explore and deepen the understanding and understanding of the regulatory mechanism of μOR signaling, and point out the direction
for promoting the development of new opioid analgesics with high efficiency and low toxicity.

Pain, especially chronic pain, is a common neurological disorder
.
According to statistics, nearly 20% of adults worldwide suffer from chronic pain, and nearly 40%
in some economically backward countries.
Common chronic pains include low back pain, arthritis pain, migraine and cancer pain, which not only lead to weakened or lost behavioral capacity, but also bring about depression, sleep disorders and suicidal tendencies, which seriously affect people's physical and mental health and cause huge social and economic burdens
.

Figure 1.
Poppy (https://unsplash.
com/)

Opioids are currently the most widely used and highly effective analgesics
.
The use of opioids by humans dates back thousands of years to the use of the plant opium poppy for analgesic and sedative and recreational purposes (to produce pleasure and euphoria).

Subsequent studies found that the opioid morphine was the main substance
that exerted activity in the poppy.
Common opioids include natural opioid alkaloids such as morphine and cocaine, as well as synthetic opioids such as demeraldine and fentanyl, and synthetic opioids produce morphine-like effects
in the human body.
Opioids act on opioid receptors in the G protein-coupled receptor family, especially μ μ opioid receptors (μOR), mainly activating downstream inhibitory Gi/O proteins to exert pharmacological activities
such as analgesia.
The development of opioid receptor drugs has long been a hot spot in analgesic drug research, and most of the opioids that have been marketed are μOR agonists, and representative classic opioid analgesic drugs such as morphine and fentanyl have shown high selectivity
for μOR.
However, the use of opioid analgesics can lead to many toxic side effects, including addiction, respiratory depression and constipation, which greatly limits their clinical application and makes the development of safe and effective analgesic drugs targeting opioid receptors a major
difficulty.
Respiratory depression deaths caused by opioid addiction have also directly contributed to the widespread "opioid crisis", mainly concentrated in North America and Canada, causing more than 100,000 deaths per year, mainly due to the abuse of fentanyl and its derivatives
.
As the main factor of the "opioid crisis" and a powerful analgesic drug still in clinical use, the molecular mechanism of fentanyl and its receptor μOR interaction has been unknown for a long time, and elucidating the relevant molecular mechanism is of great
significance for us to rationally design safer and more efficient fentanyl-derived analgesics.

Figure 2.
Drug Addiction (www.
shutterstock.
com)

Previous studies have shown that the analgesic effects of opioids are mediated by the μOR G protein signaling pathway, while their side effects are caused by the Arrestin signaling pathway
.
However, recent studies have questioned this hypothesis, arguing that neurotoxic side effects such as respiratory depression are not associated with arrestin signaling1
.
Despite the doubts, a large amount of research has been invested in the development of G protein-biased μOR agonist drugs, aiming to discover highly effective and low-toxicity-targeted μOR analgesics2
.
In 2020, the US FDA approved the first and so far only μOR analgesic designed based on the concept of G protein bias, Olisteridine (TRV130), for the treatment of moderate to severe pain, which exhibits lower toxic side effects
than morphine.
Due to the lack of understanding of the molecular mechanism of G protein preference of μOR, the discovery of G protein biased agonists of μOR has been obtained through large-scale high-throughput blind screening for nearly 20 years since the above hypothesis was proposed, which greatly hindered the rational design and discovery
of similar innovative analgesic drugs.

In this study, the researchers first analyzed the three-dimensional structure of human μOR binding to balanced agonists such as fentanyl, morphine and DAMGO (showing bidirectional signaling activity of G protein and Arrestin) and G protein-biased agonists such as TRV130, SR17018 and PZM21 by cryo-EM, and further characterized the signaling characteristics of μOR under the activation of different signaling active agonists through molecular dynamics simulation and cellular-level functional analysis
。 The study found that fentanyl occupies an additional binding pocket at the TM2 to TM3 proximal outer end of μOR compared to morphine, and in addition, fentanyl's aniline ring side chain forms a direct π-π hydrophobic interaction with amino acid residues W295 and Y328, which confers it with receptor-activating activity
up to 50-100 times higher than morphine 。 Through molecular docking and point-mutation function verification of different fentanyl derivatives, the researchers further explored the structure-activity relationship between fentanyl and its derivatives and μOR, and found that different degrees of interaction between drug molecules and amino acid residues such as D149, Y150, W135 and W320 play a key role
in determining the different activities of fentanyl and its derivatives (carfentanil, sufentanil and oxymetrentanyl, etc.
).
。 The analysis of the analyzed series of structures and molecular dynamics simulation showed that G protein biased agonist PZM21 and others tended to bind to the TM2/3 side of the μOR ligand binding pocket, while the equilibrium agonist fentanyl showed a broader and more balanced interaction with the μOR transmembrane region, and made the intracellular domain of μOR more compacted, which was conducive to the binding of μOR to Arrestin, thus explaining the molecular mechanism of equilibrium agonist manifestation of Arrestin activity
。 Based on these findings, the researchers also designed novel G protein-biased fentanyl derivatives FBD1 and FBD3
with different activities based on the fentanyl molecular backbone.

Figure 3.
Structures
of opioids with different chemical structures bound to human μOR.
Top left: Different binding patterns of fentanyl and morphine; Bottom left: Structure-activity analysis of fentanyl and its derivatives interacting with μOR; Top right: In molecular dynamics simulations, the equilibrium agonist of μOR mediates a more compact intracellular lumen conformation than the G protein biased agonist; Bottom right: Novel G protein-biased fentanyl derivatives FBD1 and FBD3
with different activities based on structural design.
Intermediate: TM6/7 interaction of attenuated ligand and μOR causes μOR signal bias
.

This study was completed by the team of Xu Huaqiang/Zhuang Youwen, Xie Xin and Wang Mingwei of Shanghai Institute of Materia Medica
.
Youwen Zhuang, associate researcher at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, and Yue Wang, Bingqing He and Xinheng He, doctoral students, are co-first authors
of the paper.
Professors Xu Huaqiang, Xie Xin and Zhuang Youwen of Shanghai Institute of Materia Medica, and Professor Wang Mingwei, Department of Pharmacology, School of Basic Medical Sciences, Fudan University, are co-corresponding authors
of the paper.
Also participating in this study are Researcher Cheng Xi, Researcher Yang Dehua, Researcher Jiang Yi, Researcher Jiang Xiangrui, Dr.
Guo Shimeng of Shanghai Institute of Materia Medica, Rao Qidi, a graduate student jointly trained by Shanghai Institute of Materia Medica, Fudan University and ShanghaiTech University, Zhou Qingtong, researcher of the School of Basic Medical Sciences of Fudan University, and Professor Karsten Melcher and Dr.
X.
Edward Zhou of the Wen Anluo Research Institute
.
At the same time, the research work has received strong support and help
from Academician Jiang Hualiang and researcher Shen Jingshan of Shanghai Institute of Materia Medica.
Shanghai Yuansi Biotech provided compound samples
for this study.
The work was supported by the cryo-EM platform of Shanghai Institute of Materia Medica, the peak electron microscopy platform of Shanghai Institute of Materia Medica, and was funded by the National Natural Science Foundation of China, the Key R&D Program of the Ministry of Science and Technology, the Pilot Project of the Chinese Academy of Sciences, the National Science and Technology Major Project (Key Project of New Drug Creation), the Shanghai Science and Technology Major Project and the Special Assistant Research Project of the Chinese Academy of
Sciences.

Article link: https://doi.
org/10.
1016/j.
cell.
2022.
09.
041

References:

　　1.
zevedo Neto, J.
et al.
Biased versus Partial Agonism in the Search for Safer Opioid Analgesics.
Molecules 25, 3870, doi:10.
3390/molecules25173870 (2020).

　　2.
Che, T.
, Dwivedi-Agnihotri, H.
, Shukla, A.
K.
& Roth, B.
L.
Biased ligands at opioid receptors: Current status and future directions.
Sci Signal 14, doi:10.
1126/scisignal.
aav0320 (2021).

Expert Reviews:

Zhang Xu (Academician, Chinese Academy of Sciences)

According to the 2020 China Pain Medicine Development Report, nearly 10%-20% of the population in China is plagued by chronic pain including cancer pain, postoperative pain, nerve damage and muscle fiber pain, and the group of chronic pain patients has exceeded 300 million and is still growing
.
Pain has developed into the third major health problem in the current society after cancer and cardiovascular and cerebrovascular diseases, which has caused a huge impact and burden on people's lives and social and economic development, and the development of efficient and safe analgesics is a major demand
for current medical and health care.

Opioids are currently the most widely used and effective analgesic drugs
.
The earliest human use of opioids was reflected in the discovery
of the value of the plant opium poppy.
As an ancient medicinal plant, the poppy was mentioned as early as the Sumerian text around 4000 BC and was called Hul Gil (the delightful plant).

Opium is a class of alkaloid active substances extracted from the opium poppy, and its application in human history dates back thousands of years, mainly for pain relief treatment and recreational use
.
The core active substance in opium is morphine, named after the Greek god of dreams, Morpheus, first isolated from opium in 1805 by the German pharmacist Friedrich Sertürner
.
Morphine has a potent analgesic effect, but it is also accompanied by toxic side effects
such as high addiction and respiratory depression.
In the pursuit of powerful, low-addiction analgesics, a series of opioids with different structures producing morphine-like physiological effects have been synthesized, including heroin discovered in 1874 and fentanyl synthesized in 1959
.
Although these synthetic opioids exhibit stronger analgesic effects than natural opioid alkaloid morphine, they are also associated with more intense toxic side effects
.
The design and development of new opioid analgesics and even non-opioid analgesics that are highly effective in analgesia and avoid neurotoxic side effects has always been the unremitting pursuit
of scientists.

Opioids exert analgesic effects
by mimicking the antinociceptive physiological effects of endogenous opioid peptides, such as endorphins and dynorphins, and activating opioid receptors in the G protein-coupled receptor family.
Among them, morphine and fentanyl, as representatives of classical opioid analgesics, mainly play a role by activating μ opioid receptor (μ μOR), and are still used as clinical analgesics
.
For a long time, a large amount of research has focused on the scientific question of how the two can be combined with μOR, in order to design safer opioid analgesics based on structural information, especially after
the first μOR (inactive) structure was resolved in 2012.
However, agonist binding models based on molecular docking and kinetic simulations are complex and cannot reflect the binding patterns
of real agonists.
This study is the first to resolve a series of near-atomic-resolution structures of agonist morphine and fentanyl binding μOR, clarifies the understanding of the confusion of fentanyl binding patterns, gives us the first understanding of how it interacts with μOR, and provides a precise template
for the design of future painkillers.
The μOR-mediated arrestin signaling pathway is considered to be an important factor in the occurrence of opioid toxicity, which also led to the discovery of the G protein-biased ligand TRV130 and its approval
by the FDA in 2020 。 This study is also based on structural and multiple pharmacological function experiments and found that weakening the interaction of opioid molecules with the sixth and seventh transmembrane regions of μOR can weaken or even eliminate arrestin signaling, thereby triggering G protein biased signal transduction, which provides new ideas for subsequent design and discovery of opioids with pathway bias, which will promote the discovery
of highly effective and low-addictive analgesics.

The Shanghai Institute of Chinese Academy of Sciences has a long history in the mechanism of action of analgesic drugs and the discovery of new drugs, and has made a series of important discoveries and contributions
.
Mr.
Zhao Chengga and Mr.
Jin Guozhang, the first directors of the Institute of Medicine, respectively carried out purification and systematic pharmacological studies on the analgesic tetrahydrobarmatine in traditional Chinese medicine fumaso
.
Mr.
Zou Gang of the Institute of Medicine has conducted in-depth and meticulous research on the mechanism of morphine action and neuropeptide pharmacology, and confirmed that the effective site of morphine analgesia is the third ventricle and the central gray matter around the brain aqueduct, which is known as a "milestone" in the study of the mechanism of morphine action
.
Mr.
Chi Chi of the Institute of Pharmaceuticals has long been engaged in research on the neuropharmacology and analgesics of opioid receptors, and led the discovery of the highly potent analgesic oxymetfentanyl
.
This research is also a good inheritance
of the research direction of opioid receptor analgesics where the drug is located.

Pei Gang (Academician, Chinese Academy of Sciences)

Opioid receptor is the most important target molecule of clinical analgesic drugs, and its related research is very necessary
.
Recently, researchers Xu Huaqiang, Xie Xin, Wang Mingwei and Zhuang Youwen of Shanghai Institute of Materia Medica, together with multiple teams, have made important progress in the study of the mechanism of μ opioid receptor recognition of fentanyl, morphine and other drug molecules, and related work has been published in Cell
.
After the opioid receptor is activated by the ligand, it mainly mediates two downstream signaling pathways, namely G protein and β-arrestin signaling pathway, to achieve analgesic effect, but it is also accompanied by side effects
such as addiction, respiratory depression, and constipation.
At present, many studies in this field suggest that the analgesic effect is mainly mediated by the G protein signaling pathway, while the side effects are mediated by the β-arrestin pathway
.
Although there are many experiments that do not support the above observations, many scientists are still committed to the research of G protein pathway preference drugs, of which the G protein pathway preference ligand TRV130 was approved by the US FDA in 2020 for the treatment of moderate to severe acute pain in adults, but it still emphasizes its remaining side effects in the form of a "black box warning", which is "known, not known"
.

In this paper, we systematically elucidate the mode of opioid receptor binding to preferred and non-preferred drugs, and breakthrough has found the key binding characteristics
that mediate the two signaling pathways.
Both ligands have stronger interactions with the anterior transmembrane helix, while preference ligands interact weakly
with the posterior transmembrane helix compared to non-preference ligands such as fentanyl and morphine.
These results have not only been verified at the cellular level and molecular dynamics simulations, but the authors have also accurately modified fentanyl based on the mechanism provided by structural biology to synthesize FBD1 and FBD3, two derivatives with G protein pathway preference, further confirming the concept
of preference key sites.
These structures allow us to "know and why"
the pathway selection mechanisms of opioid receptors.
But the biological laws of nature are very complex and diverse, and I look forward to further validation
of this mechanism of preference and the efficacy of potential opioid receptor modulators at the animal level.

With the development of structural biology, Chinese researchers not only analyze the structure of biological macromolecules through multidisciplinary means such as biochemistry combined with computer, but also further explore and solve major problems and urgent needs
in the frontier field of life science and biomedicine from these structures.
The work, published in Cell, is a good example of translational research that not only advances the understanding of the molecular mechanisms of opioids, but also lays the foundation
for structure-based drug design and development.