毒蕈碱乙酰胆碱受体M3以及配体结构与功能研究
发布时间：2023-06-04 23:33
G蛋白偶联受体(GPCRs),是人体中最大膜蛋白受体家族,拥有约800个家族成员,负责将细胞外或者环境中的信号分子或刺激通过下游G蛋白偶联或者arrestin蛋白及第二信使分子的级联扩大,将信息传递到细胞内,引起细胞内生理反应。GPCRs也是非常重要的药物靶点,全球治疗药物市场中大约有1/3的药物分子是靶向GPCRs的。毒蕈碱乙酰胆碱受体家族包含5个家族成员M1,M2,M3,M4和M5,调控身体不同的生理功能,并且其分布非常广泛。其中,M3受体主要分布于血管以及肺部呼吸系统中的平滑肌,M3受体被神经递质乙酰胆碱激活,活化下游Gq蛋白并且偶联介导细胞内钙离子水平上升,导致平滑肌收缩。本研究是基于已经解析的M2和M3受体的结构,根据配体结合口袋的差异,设计并合成新的小分子,通过生化实验筛选对M3有选择性的配体。本课题的设计是在M受体原有配体的骨架上添加一个新的基团,使其嵌入M3的配体结合口袋并面向口袋中的亮氨酸残基(Leu225),由于M2相同位置是较大的苯丙氨酸(Phe181)从而弱化配体的结合,这样就可以设计出对M3有选择性的配体。首先,合作者通过计算机分子对接(...
【文章页数】：167 页
【学位级别】：博士
【文章目录】：
摘要
abstract
Chapter1.Introduction
1.1 G protein coupled receptors(GPCRs)
1.1.1 Classification
1.1.2 Physiological Function
1.1.3 Significance
1.1.4 Signaling of GPCRs
1.1.5 GPCRs ligands
1.1.6 GPCR Structure
1.1.7 GPCRs dynamics
1.2 Muscarinic acetylcholine receptors
1.2.1 Classification and function
1.2.2 Signal transduction
1.2.3 Muscarinic receptor structures
1.2.4 Significance and diseases model for muscarinic M3 receptor
1.2.5 Ligands of muscarinic receptors
1.2.6 Design of the project in the thesis
Chapter2.Materials and methods
2.1 Materials
2.1.1 General reagents and kits
2.1.2 Cell culture medium and reagents
2.1.3 Ligands
2.1.4 Radio-ligands
2.1.5 General buffer
2.2 Methods
2.2.1 Cloning
2.2.2 Transfection and Sf9 cell culture
2.2.3 Expression and purification
2.2.4 Crystallization
2.2.5 Binding assay
2.2.6 Data collection and processing
2.2.7 NMR spectrum
Chapter3.Structure insight into M3R with selective antagonists
3.1 M3R purification and crystallization with tiotropium
3.1.1 Constructs
3.1.2 Solubilization test
3.1.3 M3R purification and crystallization with tiotropium
3.1.4 M3R crystallization with compound6b(ABH423)
3.2 Optimization of M3R construct and ligands for crystallography
3.2.1 Analog of compound6b(ABH423)
3.2.2 Covalent M3R selective antagonists
3.2.3 M3R thermostabilizing constructs
3.3 Selective antagonist optimization
3.4 Crystallization of M3 receptor with compound6o(BS46)
3.4.1 M3R with6o(BS46)expression test and binding assay
3.4.2 M3R-6o(BS46)crystallization
3.4.3 Data collection and process of M3R bound compound6o(BS46)
3.4.4 M3R-6o(BS46)crystals optimization
3.4.5 Data collection and process of M3R-6o(BS46)
3.5 M3mT4L bound compound6o(BS46)crystal structure determination
3.5.1 M3mT4L bound compound6o(BS46)crystals packing
3.5.2 Overall structure and conformation of M3mT4L crystals
3.5.3 Orthosteric binding site of M3R
3.6 M3 receptor with selective antagonist Darifenacin(DAR)
3.6.1 Introduction of darifenacin
3.6.2 M3R-DAR purification
3.6.3 M3R-DAR crystallization
3.7 Summary
Chapter4.M3 receptor dynamics with NMR spectrometry
4.1 Design and optimization of M3R constructs for NMR spectrometry
4.1.1 M3R constructs for Lys labeling
4.1.2 M3R constructs for Met labeling
4.1.3 Stability of M3?i
4.2 NMR sample preparation for Lys labeling
4.2.1 M3?i3 purification and13C-Lys label
4.2.2 M3?i3 NMR spectrum with different ligands
4.3 NMR sample preparation for methionine labeling
4.4 Summary
Chapter5 Discussion and expectation
5.1 M3R crystallization with selective antagonists
5.1.1 M3R-6o(BS46)crystal structure
5.1.2 M3R crystallization with darifenacin
5.2 M3R dynamics with different ligands
Reference
致谢
个人简历、在学期间发表的学术论文与研究成果
本文编号：3831241
【文章页数】：167 页
【学位级别】：博士
【文章目录】：
摘要
abstract
Chapter1.Introduction
1.1 G protein coupled receptors(GPCRs)
1.1.1 Classification
1.1.2 Physiological Function
1.1.3 Significance
1.1.4 Signaling of GPCRs
1.1.5 GPCRs ligands
1.1.6 GPCR Structure
1.1.7 GPCRs dynamics
1.2 Muscarinic acetylcholine receptors
1.2.1 Classification and function
1.2.2 Signal transduction
1.2.3 Muscarinic receptor structures
1.2.4 Significance and diseases model for muscarinic M3 receptor
1.2.5 Ligands of muscarinic receptors
1.2.6 Design of the project in the thesis
Chapter2.Materials and methods
2.1 Materials
2.1.1 General reagents and kits
2.1.2 Cell culture medium and reagents
2.1.3 Ligands
2.1.4 Radio-ligands
2.1.5 General buffer
2.2 Methods
2.2.1 Cloning
2.2.2 Transfection and Sf9 cell culture
2.2.3 Expression and purification
2.2.4 Crystallization
2.2.5 Binding assay
2.2.6 Data collection and processing
2.2.7 NMR spectrum
Chapter3.Structure insight into M3R with selective antagonists
3.1 M3R purification and crystallization with tiotropium
3.1.1 Constructs
3.1.2 Solubilization test
3.1.3 M3R purification and crystallization with tiotropium
3.1.4 M3R crystallization with compound6b(ABH423)
3.2 Optimization of M3R construct and ligands for crystallography
3.2.1 Analog of compound6b(ABH423)
3.2.2 Covalent M3R selective antagonists
3.2.3 M3R thermostabilizing constructs
3.3 Selective antagonist optimization
3.4 Crystallization of M3 receptor with compound6o(BS46)
3.4.1 M3R with6o(BS46)expression test and binding assay
3.4.2 M3R-6o(BS46)crystallization
3.4.3 Data collection and process of M3R bound compound6o(BS46)
3.4.4 M3R-6o(BS46)crystals optimization
3.4.5 Data collection and process of M3R-6o(BS46)
3.5 M3mT4L bound compound6o(BS46)crystal structure determination
3.5.1 M3mT4L bound compound6o(BS46)crystals packing
3.5.2 Overall structure and conformation of M3mT4L crystals
3.5.3 Orthosteric binding site of M3R
3.6 M3 receptor with selective antagonist Darifenacin(DAR)
3.6.1 Introduction of darifenacin
3.6.2 M3R-DAR purification
3.6.3 M3R-DAR crystallization
3.7 Summary
Chapter4.M3 receptor dynamics with NMR spectrometry
4.1 Design and optimization of M3R constructs for NMR spectrometry
4.1.1 M3R constructs for Lys labeling
4.1.2 M3R constructs for Met labeling
4.1.3 Stability of M3?i
4.2 NMR sample preparation for Lys labeling
4.2.1 M3?i3 purification and13C-Lys label
4.2.2 M3?i3 NMR spectrum with different ligands
4.3 NMR sample preparation for methionine labeling
4.4 Summary
Chapter5 Discussion and expectation
5.1 M3R crystallization with selective antagonists
5.1.1 M3R-6o(BS46)crystal structure
5.1.2 M3R crystallization with darifenacin
5.2 M3R dynamics with different ligands
Reference
致谢
个人简历、在学期间发表的学术论文与研究成果
本文编号：3831241
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