G蛋白偶聯(lián)受體(GPCRs),是人體中最大膜蛋白受體家族,擁有約800個家族成員,負責(zé)將細胞外或者環(huán)境中的信號分子或刺激通過下游G蛋白偶聯(lián)或者arrestin蛋白及第二信使分子的級聯(lián)擴大,將信息傳遞到細胞內(nèi),引起細胞內(nèi)生理反應(yīng)。GPCRs也是非常重要的藥物靶點,全球治療藥物市場中大約有1/3的藥物分子是靶向GPCRs的。毒蕈堿乙酰膽堿受體家族包含5個家族成員M1,M2,M3,M4和M5,調(diào)控身體不同的生理功能,并且其分布非常廣泛。其中,M3受體主要分布于血管以及肺部呼吸系統(tǒng)中的平滑肌,M3受體被神經(jīng)遞質(zhì)乙酰膽堿激活,活化下游G_q蛋白并且偶聯(lián)介導(dǎo)細胞內(nèi)鈣離子水平上升,導(dǎo)致平滑肌收縮。本研究是基于已經(jīng)解析的M2和M3受體的結(jié)構(gòu),根據(jù)配體結(jié)合口袋的差異,設(shè)計并合成新的小分子,通過生化實驗篩選對M3有選擇性的配體。本課題的設(shè)計是在M受體原有配體的骨架上添加一個新的基團,使其嵌入M3的配體結(jié)合口袋并面向口袋中的亮氨酸殘基(Leu225),由于M2相同位置是較大的苯丙氨酸(Phe181)從而弱化配體的結(jié)合,這樣就可以設(shè)計出對M3有選擇性的配體。首先,合作者通過計算機分子對接(...

【文章頁數(shù)】：167 頁

【學(xué)位級別】：博士

【文章目錄】：
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 M3_mT4L bound compound6o(BS46)crystal structure determination
        3.5.1 M3_mT4L bound compound6o(BS46)crystals packing
        3.5.2 Overall structure and conformation of M3_mT4L 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
致謝
個人簡歷、在學(xué)期間發(fā)表的學(xué)術(shù)論文與研究成果



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