Note: at the end of the paper, there is a brief introduction of the research team and an analysis of the research ideas in this paper
The central nervous system (CNS) is the most important part of the human nervous system. It receives the incoming information from all parts of the body, and after it is integrated and processed, it becomes the coordinated motor outgoing, or stored in it to become the neural basis of learning and memory. Human thinking is the function of CNS. The pathological changes of CNS will seriously affect the normal work and life of patients and their families in a long period of time, and bring heavy burden to the society. Glioma and Parkinson's disease are two kinds of diseases with completely different pathogenetic mechanism, but they belong to the category of major diseases. The outline of "healthy China 2030" clearly points out that people's healthy development should be given priority, and health needs to be integrated into all policies. Among them, prevention, diagnosis and treatment of major diseases are the most important. In this context, researchers focus on the study of CNS disease pre diagnosis and treatment, and focus on the detection of biochemical markers and functional regulation. Monoamine oxidases (Maos) are a kind of functional protease expressed on the outer membrane of mitochondria. They oxidize and metabolize monoamines in organism, mainly including neurotransmitters and exogenous ammonia. Their abnormal function is closely related to CNS diseases. Based on the different metabolic substrates, Maos can be divided into two subtypes (MAO-A and MAO-B). The similarity rate of amino acid sequence between them is more than 70%. It is a challenging and biologically significant work to use fluorescent probes to distinguish the two. Because of the important functions of Maos, researchers have developed many fluorescent probes of Maos, among which two-photon fluorescent probes have great scientific significance and practical application value due to their deeper tissue penetration.
Recently, a new mao-a-specific two-photon fluorescence probe was designed and synthesized based on the concept of "spatial configuration conversion" by Huang Wei and Li Lin, academician of Nanjing University of technology, and Professor Yao Shao Q., National University of Singapore. The application of the probe in the detection of MAO-A activity in human glioma tissue was reported.
Based on the previous study of MAO-B specific probes (NAT. Commun., 2014, 5, 3276), the author found that the binding mode of probe U1 to MAO-A and MAO-B was completely different. Only when U1 was combined with MAO-B, warhead turned to the active site of Mao, coenzyme fad, but it was opposite to mao-a. Therefore, we hope to design a new MAO-A specific two-photon fluorescence probe based on the structure of U1. The new probe F1 selects tetrahydropyridine in the molecular structure of MPTP, the known Maos substrate, as the detection group, and produces the change of fluorescence signal through the mechanism of "construction of instantaneous conjugated structure", aiming to detect the activity of MAO-A by the way of fluorescence signal enhancement. According to the results of docking, only when F1 is combined with MAO-A, the nitrogen atom on tetrahydropyridine will point to the coenzyme fad, thus completing the enzyme catalytic oxidation, obtaining the product FD1 with fluorescence, generating fluorescence signal.
Firstly, the selectivity of F1 to MAO-A was successfully verified on a variety of mammalian cell lines and a cell model regulating Mao expression by CRISPR / cas9. Based on the above results, the author further explored the application of F1 in the tissue, and imaged the fresh brain tissue of mice, SH-SY5Y tumor tissue, human glioma tissue (tumor adjacent tissue section as reference), with the imaging depth of 220 μ M. In addition, they found that the addition of clorgyline (CL) can significantly inhibit the enhancement of fluorescence signal, and the activity of MAO-A in paracancerous tissue is far lower than that of other tissue samples, which is consistent with the previously reported low expression of MAO-A in paracancerous tissue. This work provides new ideas and tools for the detection and research of Maos.
This achievement was recently published on Angewandte Chemie International Edition in the form of hot paper. The first author of this article is Fang tsunami, a doctoral candidate from Advanced Materials Research Institute of Nanjing University of technology.
Original text (scan or long press QR code and go to the original page after identification): rational design of two photon fluorescent probe for visualizing monoamine oxidase A activity in human glioma tissues Fang Haixiao, Zhang hang, Li Lin *, Ni Yun, Shi RI, Li Zheng, Yang Xuekang, Ma Bo, Zhang Chengwu, Wu Qing, Yu Changmin, Yang naidi, Yao Shao Qin *, Huang Wei*Angew. Chem. Int. Ed., 2020, DOI: 10.1002/anie.202000059
About Professor Li Lin
Professor Li Lin's research group focuses on Biomedical Photonics Research of "mitochondrial optical detection and regulation", covering photonics / organic chemistry / Biomedical Engineering, focusing on the application of new methods of mitochondrial specific pathological biological system development in early diagnosis and treatment and the development of new drugs. The research results reveal the role of small molecule fluorescent probes in the detection and regulation of protein activity in vivo. Meanwhile, at the gene level, the great application prospects of this kind of light functional small molecule biological function regulators in biomedical fields such as disease diagnosis, signal transduction and drug development are described. So far, in nature communications, accounts of chemical research, Journal of the American Chemical Society, Angewandte Chemistry International Edition, Advanced materials and other journals published more than 80 papers included in SCI as the first / corresponding author, applied for / authorized 39 / 5 patents, participated in the compilation of a monograph molecular imaging and accurate diagnosis, presided over 7 national / provincial and ministerial scientific research projects, and participated in a project entrusted by enterprises. He was successively elected as the spokesperson of element MC of Chinese young chemist element periodic table, the young member of Biomedical Photonics branch of China biomedical engineering society, the young member of active oxygen biological effect Professional Committee of China Environmental Mutagen Society, the Editorial Committee of material guide, Chinese chemical Young editorial board member of letters, young editorial board member of Chinese clinical pharmacology and therapeutics, visiting researcher of NNI.
https://www.x-mol.com/university/faculty/27721
Analysis of scientific research ideas
Q: What was the initial purpose of the study? Or how do ideas come about? A: As mentioned above, Maos have important biological functions and are closely related to human mental diseases and neurodegenerative diseases. Our research group has been concerned about the relationship between the functional differences of different subtypes of Maos and central nervous system diseases, and has published several targeted papers (nature communication, 2014, 5, 3276; angelw. Chem. Int. ed., 2015, 54, 10821; angelw. Chem. Int. ed., 2019, 58, 7657; chemochem, 2019, 20, 1487). In 2014, after solving the structural design problem of mao-b-specific two-photon fluorescence probe for the first time, we have been trying to find a solution to the structural design problem of mao-a-specific two-photon fluorescence probe for the purpose of perfectly realizing the classification and detection of homologous protein subtypes. We have tried a variety of design ideas successively, and many graduate students have made contributions in this. Until 2017, we finally found a way to verify the specificity and sensitivity of probes in various biological systems. Finally, this article (angel. Chem. Int. ed., 2020, DOI: 10.1002/anie. 202000059) has formed a perfect closed-loop on the function verification of two homologous subtypes of Maos. So far, we are also trying to summarize / revise the "design concept of fluorescence probe for spatial configuration conversion", hoping to provide a reference for the design of small molecule fluorescence enzyme contact probe in the future.
Q: What are the challenges in the research process? A: There are two main challenges in the research process: one is how to distinguish homologous proteins rationally, so as to design corresponding fluorescent probes based on this. The similarity of amino acid sequence between MAO-A and MAO-B is as high as 70%, and they have the same oxidation mechanism. How to make the designed molecular probe specific is one of the challenges. Based on the previous work and experience, we use molecular docking as a tool to change different molecular structures to connect with different protein crystals. The results are used as a reference for probe design. For a more accurate selection mechanism, we are still in the research, hoping to have a more regular summary as soon as possible, to provide guidance for the later design of fluorescent probes, and to have a greater breakthrough. Another challenge is the validation of different levels of biological systems. We are an interdisciplinary research group with the background of Photonics and chemistry. We have accumulated too little in biological mechanism and disease model, and have gone a lot of detours. Whether it's the use of artificial recombinant protein, or the establishment of representative immortalized cell lines (we specially took the time to find the company's help to establish CRISPR / cas9 edited cell lines), to mouse tumor, to human glioma samples, with the help of too many predecessors and peers, we can finally complete the verification of the whole system, providing a satisfactory answer for all partners Volume. We have learned a lot and accumulated a lot in this process. Now we are working hard in the new marker system, but this time, we are more confident to complete the task. At the same time, after several years of study, the research group has also been tempered. Now many colleagues give us the label of "photonics + chemistry + biomedicine" multi interdisciplinary subject attribute research group.
Q: What are the important applications of the research results? What areas of business or research institutions are likely to benefit from the results? A： At present, we expect that this version of MAO-A probe will play an important role in the study of glioma signaling molecular pathway. It is of great significance to understand the role of MAO-A in the occurrence and development of gliomas. On a deeper level, our study provides a small two-photon fluorescence probe for both subtypes of Maos homology, which is a step further than the method that can only be used to distinguish antibodies originally, and will provide a very powerful research tool for the correlation research of glioma and Parkinson's disease, two CNS diseases with completely different mechanisms. Health disease is an eternal topic for human beings. Diagnostic reagent companies and drug development companies are constantly striving to develop new preparations and tools to help human beings solve the problems we are facing. We feel that as young scientific and technological workers, it is the responsibility and responsibility of our generation to contribute their own strength and wisdom. We will continue to work hard in the field of small molecule fluorescent enzyme contact probes, and strive for greater breakthroughs.
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