Only lose fat but not meat? For obesity, help develop next-generation dual agonist drugs for amylin and calcitonin receptors

  Some people say that three points of exercise and seven points of eating. A few days ago, the social media shared “how to eat rice in different ways”, which mentioned the combination of “seven-color brown rice”: black rice, red rice, corn glutinous rice, oat kernels, brown rice, highland barley, and wheat kernels. In addition to being healthy, these whole grains are also more satiety.
  If you’ve ever tried to lose weight, you’ve probably heard of the practice of drinking water before meals to increase satiety and avoid consuming more energy. So, where does the human body’s sense of fullness come from?
  This starts with the discovery of amylin and its receptor protein. In 1986, scientists discovered the existence of amylin, whereby it was discovered that when we eat, the pancreatic islets begin to secrete insulin and amylin. The former helps to quickly control blood sugar, while amylin binds to amylin receptors distributed in the brain to produce satiety signals. When we receive the signal, we stop eating, thereby avoiding the metabolic diseases caused by overeating.
  Taking advantage of this natural mechanism of the body’s own energy balance, in 2005, amylin-derived polypeptide, pramlintide, was successfully launched as a blood sugar control drug for diabetic patients and is still in use today. Subsequent studies have revealed that the amylin signaling pathway has great potential for the development of weight loss therapy. As a result, the study of amylin and its receptors has attracted a number of drug companies, who hope to develop new treatments in the field of weight loss and obesity treatment.
How to achieve an excellent weight loss effect that only loses fat without losing muscle?

  From 1998 to 1999, the unique composition of the amylin receptor was first elucidated, which itself is a heterodimer formed by the binding of a calcitonin receptor and a receptor activity-modulating protein. The calcitonin receptor itself does not affect human metabolism. When activated by calcitonin polypeptide, it will produce a physiological signal that regulates the concentration of calcium in the blood, and promotes the deposition of blood calcium into the bones.
  However, once it binds to the receptor active protein, it no longer responds to the calcitonin polypeptide, but to the amylin polypeptide, thereby forming the corresponding amylin receptor. The latest research shows that if a polypeptide is artificially engineered to activate the calcitonin receptor and the amylin receptor at the same time, it can better stimulate basal metabolism, increase energy consumption, and achieve fat loss without muscle loss. Excellent weight loss effect.
  However, since these two receptors do not have protein structure information, related drug development can only focus on mutating natural polypeptides. However, calcitonin polypeptides and amylin polypeptides differ in their native sequences, even in lengths. If you want to artificially create a hybrid peptide that can stimulate two receptors at the same time, the question that must be faced is: which natural peptide is used as the base template?
  In previous studies, it was found that the difference between salmon calcitonin and human calcitonin is that salmon calcitonin can activate both the calcitonin receptor and the amylin receptor in the human body. Therefore, the earliest studies for more than ten years have used salmon calcitonin as a template for mutation transformation, which has a length of 32 amino acids. These studies have achieved effective results in both pharmacological and animal models, and have entered the clinical trial stage.
  However, when human experiments were carried out, the actual results were not ideal. In recent years, some drug companies have tried to start from the amylin peptide template. Among them, canaglitide developed by Novo Nordisk, an international biopharmaceutical company, has achieved surprising clinical trial results. However, in actual drug development, there is still an unavoidable difficulty. When establishing a pharmacological analysis experiment, due to the particularity of the composition of amylin receptors, the calcitonin receptor will exist in the system at the same time, and the signal generated by the calcitonin receptor cannot be excluded, which makes the corresponding drug characterization very difficult. difficulty. In this case, it is of great significance to drug development if the visualized protein structure information is grasped. However, in terms of structural biology, these two receptors have long been difficult to characterize their structures.

  The two receptors themselves belong to G protein-coupled receptors, which are an important target in the field of drug research and development. 30% of the existing drugs on the market act on G protein-coupled receptors. However, the structural study of G protein-coupled receptors has been in a difficult state for many years. The fundamental reason is that the protein molecular weight of the G protein-coupled receptor itself is very small, but it is very dynamic. This makes it difficult for traditional protein crystallography to achieve efficient crystal growth unless the receptor is genetically modified and a large number of thermodynamically stable modifications are introduced that would distort the natural structure.
  This dilemma was not resolved until 2017; with the rapid development of cryo-TEM technology, Cao Jianjun, a doctoral student at the Institute of Pharmaceutical Sciences at Monash University, Australia, and his Wootten/Sexton research group realized the first cryo-TEM analysis of G protein coupling. receptor structure. Thanks to this technological breakthrough, Cao Jianjun’s research project during his Ph.D. study was carried out. Since traditional pharmacological tools cannot fully distinguish the signaling of calcitonin receptors and amylin receptors, the emerging cryo-electron microscopy technology can completely characterize the structural characteristics of the two types of receptors, thereby filling this information gap.
  At the same time, he said that there are three subtypes of the receptor activity regulator protein family that constitute the amylin receptor, and they bind to the calcitonin receptor respectively to form three subtypes of amylin receptors. The three amylin receptors have some differences in the response to different peptides, and this difference is difficult to explain by traditional pharmacological experiments.
  Cao Jianjun recently published a paper “Structural Basis of Amylin Receptor Phenotype”, which is particularly rich in structural data. There are a total of ten structures in the paper: four receptors (calcitonin receptor and three subtype of amylin receptor) and three natural polypeptides (human calcitonin, salmon calcitonin, mouse amylin) binding mode.
  By analyzing the binding modes of this different cross-combination, a large amount of structural information can be presented, which can explain the different mechanisms of the binding of the two polypeptides to the corresponding receptors and the non-corresponding receptors, respectively.
  He said, “This is the first time that people have intuitively recognized this difference, and this realization has not been achieved by other means before. The reviewers feel that our study is very systematic and can provide great insights for future drug development. detailed information.”
Structural Analysis to and from Tokyo-Melbourne

  Although in 2017, the research group achieved the first breakthrough in the structural analysis of G protein-coupled receptors by cryo-electron microscopy. However, due to the special heterodimer composition of the amylin receptor, the process of protein expression and purification is more difficult and complicated. This problem was solved and refined in 2018 by the team’s Dr. Yi-Lynn Liang in the study of the homologous analog of the amylin receptor, the CGRP receptor. In 2018, Cao Jianjun joined the research group and started doctoral study and research.

  After he enrolled, the above topics were officially established. The subsequent outbreak of the global new crown epidemic has brought difficulties to scientific research and practice. In the early stage of the project, the electron microscope equipment in the group has not been optimized yet. Therefore, they sent the samples to Tokyo, and the long-term collaborator, Professor Radostin Danev of the Graduate School of Medicine of the University of Tokyo, performed cryo-electron microscopy imaging, and then returned the data to Melbourne for structural analysis and comparison. Finally, the structural characteristics and Pharmacological linkages are described. Through such an international long-distance round-trip operation, the six structures of the research subject have been analyzed.

  Cao Jianjun said: “Structural analysis has been supported by the Supercomputing Center of Monash University. During the search process, we found that more structural information must be incorporated into the system to make the system more complete and extended. In the late stage of the project, the Melbourne-based Bio21’s ARC Membrane Protein Cryo-EM Center has been completed. At that time, the equipment conditions were relatively complete, we carried out supplementary photography of four structures on the spot, and finally realized the complete analysis of ten structures.”
Achieving adipose tissue-specific weight loss

  Relevant clinical application scenarios are imminent. Although drug companies do not have structural information, they are also exploring based on peptide sequences. In 2020, the canaglitide developed by Novo Nordisk was realized based on the 37 amino acid sequence template of the amylin polypeptide.
  Based on clinical phase I and clinical phase II studies, the relevant team has published two papers in The Lancet. These studies have demonstrated that amylin polypeptide-derived artificial polypeptides can activate both calcitonin receptors and amylin at the same time. receptors to achieve effective weight loss in obese patients. Plus, this weight loss specifically targets adipose tissue, so it doesn’t lose muscle mass.
  Cao Jianjun estimates that the above-mentioned polypeptides should already be in human trials in Phase III clinical trials, and these are just some preliminary attempts made by drug companies without obtaining structural information. His research group and his ARC Membrane Protein Cryo-EM Center have reached long-term cooperation with more than ten drug R&D companies.
  He said: “Based on our structure and previous explorations by pharmaceutical companies, they are very excited by the final results. The paper we are publishing now, although only endogenous natural peptides, also provide a lot of structural information for pharmaceutical companies. Reference. If it can be further optimized based on the structure, it will soon be able to enter the clinical trial stage.”

  At the same time, if these drug development companies lack structural information, they will not know how to optimize the peptide sequence, and will not be able to achieve better therapeutic effects, and they will not be able to continue to convince potential investors. With the structural information of Cao Jianjun’s research group, these companies have been further optimized. It is expected that more artificial polypeptides based on amylin receptor templates will appear in the future, and these artificial polypeptides can activate both receptor signals and achieve weight loss function. It is expected that similar drugs will continue to enter clinical trials until they are finally approved for use.
After working as a laboratory assistant at Sun Yat-Sen University for one year, he came to Australia to study for a Ph.D.

  According to reports, Cao Jianjun’s two main mentors are the laboratories chaired by Professor Dennis Wooten and Professor Patrick Sexton from the Institute of Pharmaceutical Sciences of Monash University. Research.
  During his studies in the laboratory, Dr. Matthew Belusov was very proficient in protein crystallography and cryo-electron microscopy. He taught Cao Jianjun to use cryo-transmission electron microscopy to analyze protein structures. Dr. Yilin Lian directly taught him related protein expression and purification methods. In 2017, with the announcement of the results of the research group at that time, she also became the world’s first analyzer of the cryo-electron microscope structure of G protein at that time.

Cao Jianjun

  Cao Jianjun said: “Dr. Lian Yilin was a very important technical mentor of mine and gave me a lot of help before the epidemic. Later, she transferred to the industry, but she also taught me all the corresponding methods and technologies. At the end of 2019 , the outbreak of the new crown epidemic, and the border control of various countries. I was in China at the time, and Patrick and Denise got the news and immediately funded me to quarantine in Thailand for 14 days. Finally, I successfully entered Australia and completed the project. Otherwise, I might have I have to face the situation of dropping out of school. During the long-term home closure process in the early stage of the epidemic, Matt and I held online meetings every day, remotely teaching me how to process and parse data. Without these four experts in their respective fields It is impossible for me to complete such research work alone. I often joke that I have a mentor ‘dream team’.”
  According to reports, Cao Jianjun was born in Ma’anshan, Anhui Province in 1991. Undergraduate studied chemistry at Sun Yat-sen University, and then joined the laboratory of Professor Mao Zongwan, School of Chemistry, Sun Yat-sen University. Associate Professor Tan Caiping. During the master’s degree, Jianjun Cao is mainly engaged in the research of transition metal complexes in the direction of anti-tumor therapy. After graduation, he worked as a laboratory assistant in Mao Zongwan’s laboratory for one year. During his master’s degree, he published a total of 11 papers, which also extended Cao Jianjun’s research interests from chemistry to biomedicine.