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    Home > Biochemistry News > Biotechnology News > ANGEW Shanghai Institute of Pharmaceuticals collaborated to discover LC3 covalent small molecule probes targeting acetylation sites

    ANGEW Shanghai Institute of Pharmaceuticals collaborated to discover LC3 covalent small molecule probes targeting acetylation sites

    • Last Update: 2021-10-22
    • Source: Internet
    • Author: User
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    Autophagy is an important cell life activity for cells to regulate cell homeostasis and respond to stress conditions [1]
    .
    Many reports have shown that autophagy is importantly related to the occurrence and development of tumors, metabolic diseases, immune disorders, and neurodegenerative diseases [2-5]
    .
    However, most of the existing autophagy regulators directly target upstream regulatory proteins (such as mTOR, ULK1), or regulate lysosomal functions (such as chloroquine), and still lack small molecular probes that specifically target proteins in the autophagy process.
    [6]
    .
    LC3 protein, as a marker protein in the process of autophagy, not only mediates the recruitment of autophagy substrates into the process of lysosome degradation, but is also related to the formation and fusion of autophagosomes
    .
    However, the LC3 protein mainly functions through protein-protein interactions and lacks a relatively specific binding pocket
    .
    Therefore, the development of effective small molecule probes for the autophagy core protein LC3 has always been a problem in this field
    .
    In previous reports, there are various posttranslational modifications (PTM) (such as phosphorylation, acetylation) at the protein-protein interaction interface of the LC3 protein, and the posttranslational modification located at this interface destroys the LC3 protein and its interacting proteins The ability of interaction [7-9]
    .
    This puts forward a possible hypothesis for this study, that is, is it possible to target these post-translational modification sites to achieve their chemical probe discovery?
    In the preliminary research, Luo Cheng's research group from Shanghai Institute of Materia Medica, Chinese Academy of Sciences explored the establishment of research theory and method and application of disease intervention for the functional regulation and chemical intervention of post-translational modification sites: cooperation with the Dang Yongjun research group of Fudan University, etc.
    , For the phosphorylation sites of the Rho subfamily, a new allosteric regulatory pocket was discovered, and its allosteric regulatory small molecule compound DC-Rhoin (Covalent Inhibitors Allosterically Block the Activation of Rho Family Proteins and Suppress Cancer Cell Invasion.
    Adv Sci .
    2020 May 13;7(14):2000098.
    ); Cooperating with Zhao Kehao's research group at Yantai University, proposed targeted post-translational modification typing (PTM-isoforms), a possible strategy and method for drug discovery and disease typing (Drug design targeting active posttranslational modification protein isoforms.
    Med Res Rev.
    2021 May;41(3):1701-1750.
    ); such as cooperation with Soochow University Liang Zhongjie's research group to establish kinase family translation based on molecular dynamics simulation and elastic network Post-modification site and functional regulation database, to obtain the crystal structure of small molecules and their complexes targeting Src kinase phosphorylation sites (Dynamics of Post-Translational Modification Inspires Drug Design in the Kinase Family.
    J Med Chem.
    2021: DOI: 10.
    1021/acs.
    jmedchem.
    1c01076)
    .
    These preliminary researches on chemical probes based on post-translational modifications have laid a solid foundation for the discovery of small molecule probes targeting the autophagy core protein LC3
    .
    Therefore, in this study, Luo Cheng's group, together with the Fudan University/Chongqing Medical University Party Yongjun's group, the Shanghai Institute of Medicine Jiang Hualiang's group, and Zhou Bing's group, used the reactivity of lysine on the LC3 protein to find the first A chemical probe that can covalently bind to the acetylation modification site of LC3 protein
    .
    The probe can interfere with the function of LC3 and lay a foundation for the development of probes for autophagy regulation and the development of potential drug candidates for related diseases
    .
    The research paper was published online in Angewandte Chemie ("Inhibition of autophagy by a small molecule through covalent modification of LC3") on September 29, 2021
    .
      In this study, the joint team analyzed the presence of post-translationally modified lysine residues in the LC3B protein (the main functional protein in the LC3 family of proteins) at the protein-protein interaction interface, and found that the 49th lysine has a low pKa , There may be potential covalent reactivity
    .
    Subsequently, the team screened the small molecule library of in house covalent probes and found the sprout compound DC-LC3in, which can covalently bind to position K49 of LC3B, with an IC 50 of 3.
    06 μM
    .
    Figure 1.
    Covalent inhibitor discovery based on post-translational modification
    .
    (AB) The interaction interface of LC3B protein and the prediction of lysine pKa properties
    .
    (CE) Screening of covalent small molecule libraries and discovery of spontaneous compounds
    .
      Furthermore, in order to explore the interaction of LC3B-chemical probe molecules and provide clues for the optimization of medicinal chemistry, the team analyzed the crystal structure of the complex of LC3B and DC-LC3in derivatives (resolution of about 1.
    6 angstroms)
    .
    Finally, through optimization of medicinal chemistry, the team obtained a more active lead compound DC-LC3in-D5 (IC 50 of 0.
    2 μM)
    .
    In order to evaluate the potential off-target risk of covalent compounds in detail, the team used the click chemistry-protein mass spectrometry method, combined with in vivo and in vitro experimental verification, and proved that DC-LC3in-D5 is selective in whole-cell proteins, mainly binding to LC3 family proteins.
    LC3A/B protein has lower off-target properties
    .
    Figure 2.
    The lead compound DC-LC3in-D5 has good selectivity: (A) DC-LC3in-D5 and the negative molecule DC-LC3in-D9; (B) the compound has good kinetic properties
    .
    (CD) The compound binds to the K49 residue of LC3B, but not to the K49 residue on the synthetic LC3B peptide
    .
    (EG) Click on Chemistry-Mass Spectrometry and Mass Spectrometry Results Verification
    .
      Finally, cell experiments show that DC-LC3in-D5 can inhibit the lipid acylation process of LC3B protein from LC3B-I to LC3B-II
    .
    Moreover, it can alleviate the degradation of P62 protein under starvation conditions, reduce the number of autophagosomes, and finally exert the function of inhibiting autophagy
    .
    Figure 3.
    DC-LC3in-D5 inhibits autophagy
    .
    (AB) DC-LC3in-D5 inhibits the acylation of LC3B without affecting the acylation of GABARAP and reduces the degradation of P62
    .
    (CF) DC-LC3in-D5 inhibits autophagosome formation
    .
      In summary, this research work discovered the first small molecule inhibitor that covalently binds to LC3B K49 , which functions by disrupting protein-protein interactions
    .
    In addition, this study targets the sites of post-translational modification and finds its covalent inhibitors, which can provide a reference for the development of small molecules that are difficult to target proteins with conventional strategy chemical molecules
    .
      
    Figure 4.
    Schematic diagram of the mode of action of LC3 covalent small molecules
      Researcher Luo Cheng from Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Professor Yongjun Dang from Fudan University/Chongqing Medical University, Researcher Zhou Bing from Shanghai Institute of Materia Medica, and Academician Jiang Hualiang are the co-corresponding authors of the paper; Ph.
    D candidates Fan Shijie and Dr.
    Le Liyan from Shanghai Institute of Materia Medica, Dr.
    Wan Wei, associate researcher Zhang Yuanyuan, and Dr.
    Zhang Bidong are the co-first authors of the paper
    .
    The research was also supported by Professor Takanori Otomo of Scripps Research Institute, Academician Chen Kaixian of Shanghai Institute of Medicine, Researcher Tan Minjia, and Researcher Zhang Naixia
    .
    The research was funded by the National Natural Science Foundation of China, the Major Scientific Program of Biomacromolecule Dynamic Modification, the Outstanding Youth Fund and the Shanghai Science and Technology Commission
    .
    The related patents of this project have been transferred to the enterprise to carry out comprehensive pre-clinical research research
    .
      Full text link: https://onlinelibrary.
    wiley.
    com/doi/10.
    1002/ange.
    202109464?af=R
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    e7.
      (Contribution department: Luo Cheng's research group)
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