化學生物學導論-雷曉光.ppt

1、雷曉光 北京大學 化學學院 化學生物學系 北大-清華 生命科學聯(lián)合研究中心 北京生命科學研究所,化學生物學導論,一. 化學生物學概述二. 細胞死亡的化學生物學研究,個人教育與工作經(jīng)歷,教育背景： 20012006美國波士頓大學博士（有機化學與化學生物學） 導師：Prof. John Porco 19972001北京大學學士（化學） 導師：王劍波 教授 工作經(jīng)歷： 2014現(xiàn)在 北大-清華生命中心 高級研究員 2014現(xiàn)在 北京大學 化學學院 教授 20082014 北京生命科學研究所 研究員，高級研究員 20082014 天津大學 藥學院 副教授，教授 20062008美國哥倫比亞大學博士后（

2、生物有機化學） 合作導師：Prof. Samuel Danishefsky,一. 化學生物學概述二. 細胞死亡的化學生物學研究,生命,化學,醫(yī)學,化學 生物學,5,Nat. Chem. Biol. 2005, 1, 3.,“In our view, chemical biology focuses on understanding biological systems at the molecular level and using these mechanistic insights to expand chemistry and biology in new directions”.,Na

3、t. Chem. Biol. 2005, 1, 64.,Stuart Schreiber “Chemical Biology provides the missing small-molecule piece of the central dogma. Chemical Biologists make both small and large “small molecules”, and they use them to illuminate the principles that underlie life.”,6,ChemBioChem 2009, 10, 16-29,“Chemical

4、biologyas the name suggests-employs the methods and techniques of chemistry for the study of biological phenomena.,Herbert Waldmann: “Chemical biologyas the name suggests-employs the methods and techniques of chemistry for the study of biological phenomena.,7,J. Biol. Chem. 2010, 285, 11031-11032.,“

5、Chemical biology is a scientific discipline spanning the fields of chemistry and biology that involves the application of chemical techniques and tools, often compounds produced through synthetic chemistry, to the study and manipulation of biological systems.”,化學生物學是一門利用外源的化學物質(zhì)、化學方法或途徑，在分子層面上對生命體系進行

6、精準和動態(tài)的修飾、調(diào)控和闡釋的科學。,8,化學生物學與生物化學的區(qū)別,核心思想：外源化學 核心工具：化學探針 核心任務(wù)：分子精度 核心目標：揭示生命本質(zhì)并服務(wù)于化學,9,(1996),(1996),10,NO特殊生理活性發(fā)現(xiàn) （ 1774年, Joseph Priestly ）,1994年創(chuàng)刊,1997年創(chuàng)刊,2001年創(chuàng)刊,2008年創(chuàng)刊,(Elsevier),(Springer),(cell),11,2004*年創(chuàng)刊,2005年創(chuàng)刊,12,2006年創(chuàng)刊,2004年創(chuàng)刊,2005年創(chuàng)刊,(Royal Society),2005年創(chuàng)刊,13,什么是化學生物學,主要研究方向,國際發(fā)展與資助情況

7、,我國發(fā)展與資助情況,14,Stuart Schreiber,Carolyn Bertozzi,Peter Schultz,利用外源物質(zhì)（小分子探針）研究生命體系,化學小分子 蛋白質(zhì)靶標,為生命科學研究提供全新的方法和手段,15,15,M. Bucci, C. Goodman, T. L. Sheppard,“A decade of chemical biology” Nat. Chem. Biol. 2010, 6 (12), 847-854.,16,Methods of the Year 2010-2013, Nature Method,17,NSF: National Science F

8、oundation NIH: National Institute of Health DOE: Department of Energy,美國,NIH Directors New Innovator Program重點資助的研究方向,18,生物醫(yī)學路線圖計劃 (NIH Roadmap for Medical Research ),NIH Roadmap資助的小分子篩選中心網(wǎng)絡(luò),分子庫和成像計劃 (Molecular Libraries and Imaging, MLI),19,美國的化學生物學高水準研究單位：,20,支持化學生物學研究的基金機構(gòu): EPSRC: Engineering and

9、 Physical Sciences Research Council (英國工程和自然科學研究委員會) BBSRC: Biotechnology and Biological Sciences Research (英國生物技術(shù)與生物科學研究理事會) MRC: Medical Research Council (英國醫(yī)學研究理事會) Royal Society (Fellowships) (英國皇家學會). Wellcome Trust, Cancer Research UK, EU funding, Leverhulme Trust,英國,21,英國倫敦皇家學院、腫瘤研究所等單位聯(lián)合組建了化

10、學生物學（http:/www.chemicalbiology.ac.uk）研究中心，從事生物膜的結(jié)構(gòu)與功能研究。,化學生物學和生物化學的資助比例（A）參與這些研究的學生人數(shù)比例（B） EPSRC （2008）,22,德國,合作研究中心（CRC） 目前獲DFG資助的多個CRC中心都是在化學與生物學的交叉界面上開展研究工作，化學生物學是其主要的研究領(lǐng)域之一。,德國科學基金會（DFG）,23,日本,日本學術(shù)振興會（JSPS）,24,什么是化學生物學,主要研究方向,國際發(fā)展與資助情況,我國發(fā)展與資助情況,25,2003年 化學二處設(shè)立了化學生物學學科 2005年 生物有機與化學生物學學科代碼 B020

11、7 2013年 化學生物學學科代碼B0212,學科建設(shè)情況（國家自然科學基金委員會）,26,基金資助引領(lǐng)化學生物學發(fā)展,80年代初 攀登計劃“生命過程中的化學問題” 九五計劃 化學與生命科學交叉的重大項目5項 十五計劃 化學與生命科學交叉的重點項目32項 2003-2005 化學與生命科學交叉的重大國際合作 (8項, 60-95萬/項) 2004-2006 健康領(lǐng)域的交叉面上項目 (2000萬/年) 2007-2015 重大研究計劃“基于化學小分子探針的 信號轉(zhuǎn)導過程研究”（20000萬）,27,化學生物學研究機構(gòu)分布,28,什么是化學生物學,主要研究方向,國際發(fā)展與資助情況,我國發(fā)展與資助情

12、況,29,30,（1） 分子探針,31,Nature Chemical Biology關(guān)于“化學探針”與“藥物”區(qū)別的總結(jié),32,（2） 生物分子的化學合成與標記,33,（3） 生物相容反應(yīng)的發(fā)展及應(yīng)用,34,（4） 化學遺傳學,35,（5） 生物合成化學,36,（6） 藥物發(fā)現(xiàn)的化學生物學,37,（7） 應(yīng)用化學生物學,38,（8） 新方法、新理論及應(yīng)用,39,一. 化學生物學概述二. 細胞死亡的化學生物學研究,活性化學小分子探針,基礎(chǔ)生命科學研究,轉(zhuǎn)化醫(yī)學 創(chuàng)新藥物研究,發(fā)現(xiàn),制備,評價,研究背景：利用活性小分子探索化學與生物醫(yī)學交叉,研究策略,細胞死亡的 小分子化學調(diào)控,高通量篩選 化學

13、遺傳學,活性天然產(chǎn)物 導向的化學 生物學,通過天然產(chǎn)合成化學、化學遺傳學、分子生物學等多學科交叉，利用活性小分子研究細胞死亡的生物作用機制和調(diào)控方法,Nat. Chem. Biol. 2015, under review Nat. Chem. Biol. 2013, 9, 84-89 Cell 2012, 148, 213-227 Cell Res. 2010, 20, 1289-1305,Nat. Commun. 2015, 6, 6522 Angew. Chem. Int. Ed. 2015, 54, 1011-1015 Nat. Commun. 2014, 5, 4614 Angew.

14、Chem. Int. Ed. 2014, 53, 12111-12115 Angew. Chem. Int. Ed. 2014, 53, 9257-9261 J. Am. Chem. Soc. 2013, 135, 4996-4999 J. Am. Chem. Soc. 2012, 134, 12414-12417 Angew. Chem. Int. Ed. 2012, 51, 491-495,研究背景：經(jīng)典的細胞死亡途徑（細胞凋亡和壞死）,43,細胞凋亡 (apoptosis),細胞壞死 (necrosis),細胞死亡的分子作用機制和調(diào)控方法 科學難點：調(diào)控基因致命性，信號轉(zhuǎn)導通路復雜 研究

15、現(xiàn)狀：經(jīng)典生物遺傳學無法闡明所有未知生物同路； 高效，可視，可控，定量化的化學方法亟待發(fā)展,Hallmarks of Cancers,Hanahan and Weinberg Cell 2011, 144, 646,Amyotrophic lateral sclerosis (ALS)-肌萎縮側(cè)索硬化癥,Extrinsic Pathways for Programmed Cell Death,Our aims: using small molecules to dissect and modulate cell death!,necroptosis,Forward chemical-genet

16、ics includes screening exogenous ligands with a cell-based assay, selecting a ligand that shows a phenotype of interest, and identifying the protein target(s) of this ligand. Reverse chemical-genetics includes overexpressing a protein of interest, screening for a chemical ligand for the protein, and

17、 using the ligand to determine the phenotypic consequences of altering the function of this protein in a cellular context.,Chemical Genetics Using Small Molecules,Advantages of Chemical Genetics,Chemical genetic tools are more effective for studying essential genes or transient processes and in most

18、 part, feasible in the system most relevant to human health. Chemical genetic approach using small molecules is complementary to the classic genetic methods, because these small molecules act conditionally and rapidly, and can directly target the genes protein product. They can also hit on several p

19、roteins simultaneously, and can be used in a combination for multiple pathways in cultured human cells. In addition, if a study relates to human disease, a small molecule may also serve as a lead compound for drug development.,Beckman FX, ORCA Optimized Robot, Microplate Carousel and Paradigm Detect

20、ion Platform,Development of a Robust Bioassay for High-throughput Screening (HTS),Taking advantage of the robustness of necrosis induced by the combina-tional treatment of TNF-a, Smac mimetic, and z-VAD-fmk, We screened a chemical library of 200,000 for compounds that block necrosis,Cell-based assay

21、 for screening inhibitors of necrosis. HT-29 cell was pre-treated with DMSO or positive control necrostatin-1 (Nec-1) for 1 hour, followed by the treatment of DMSO alone or TNFa/Smac mimetic/z-VAD for additional 72 hours. Cell viability was determined by measuring ATP level using cell-titer glo assa

22、y (Promega),Yuan et al, Nat. Chem. Biol. 2008, 313,Identification of Hit 14 from HTS,Synthetic Plan for Structural Modifications of Hit,Substitution at R1by methyl is preferred, by propargyl, benzyl or hydrogen reduce the activity.,Substitution at R2 by H is preferred , by Br or alkyl little reduce

23、the activity.,Double bond is critical for the activity,Replaced by pyrimidine little reduces the activity.,2-NO2 thiophene is indispensable, 3-NO2 thiophene reduces activity ， 2-Br thiophene is inactive,Furan ring reduces activity, and increased toxicity, other nitro-substituted aromatic ring also r

24、educes activity,SAR Studies of the Hit Compound,Necrosulfonamide (NSA) and Its Negative Analogs,The Effects of Necrostatin-1 and Necrosulfonamide (NSA) on RIP3,The Effect of Necrosulfonamide (NSA) on the RIP1/RIP3 Interaction,Identification of the Mixed Lineage Kinase Domain-like Protein (MLKL) as a

25、 Necrosome Component,Evaluation of MLKL as a Valid Target for Necrosis,1. Is MLKL critical for necrotic pathway? 2. Is MLKL the cellular target for necrosulfonamide (NSA)?,We plan to use NSA-based chemical probes to address this question!,Insertion of a long, rigid polyproline helix between a small-

26、molecule bait and a biotin tag boosts the capacity of affinity purification and thereby allows for the isolation of low-abundance or low-affinity proteins.,J. Am. Chem. Soc. 2007, 129, 873-880,Design of the Chemical Probe,Synthesis of the Key Intermediates,Synthesis of the Chemical Probes,Identifica

27、tion of the Target Protein through Pull-down Experiment,Identification of MLKL as the Cellular Target of Necrosulfonamide (NSA),Identification of Cys86 of MLKL as the Necrosulfonamide-binding Residue,利用活性小分子發(fā)現(xiàn)全新的細胞壞死分子作用機制,Sun, L.; Wang, H.; Wang, Z.; He, S.; Chen, S.; Liao, D.; Wang, L.; Yan, J.; Liu, W.; Lei, X.*; Wang, X.* Cell 2012, 148, 213-227. （