化學(xué)生物學(xué)導(dǎo)論-雷曉光.ppt

1、雷曉光 北京大學(xué) 化學(xué)學(xué)院 化學(xué)生物學(xué)系 北大-清華 生命科學(xué)聯(lián)合研究中心 北京生命科學(xué)研究所,化學(xué)生物學(xué)導(dǎo)論,一. 化學(xué)生物學(xué)概述二. 細(xì)胞死亡的化學(xué)生物學(xué)研究,個(gè)人教育與工作經(jīng)歷,教育背景： 20012006美國(guó)波士頓大學(xué)博士（有機(jī)化學(xué)與化學(xué)生物學(xué)） 導(dǎo)師：Prof. John Porco 19972001北京大學(xué)學(xué)士（化學(xué)） 導(dǎo)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(jí)研究員 2014現(xiàn)在 北京大學(xué) 化學(xué)學(xué)院 教授 20082014 北京生命科學(xué)研究所 研究員，高級(jí)研究員 20082014 天津大學(xué) 藥學(xué)院 副教授，教授 20062008美國(guó)哥倫比亞大學(xué)博士后（

2、生物有機(jī)化學(xué)） 合作導(dǎo)師：Prof. Samuel Danishefsky,一. 化學(xué)生物學(xué)概述二. 細(xì)胞死亡的化學(xué)生物學(xué)研究,生命,化學(xué),醫(yī)學(xué),化學(xué) 生物學(xué),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(xué)生物學(xué)是一門(mén)利用外源的化學(xué)物質(zhì)、化學(xué)方法或途徑，在分子層面上對(duì)生命體系進(jìn)行

6、精準(zhǔn)和動(dòng)態(tài)的修飾、調(diào)控和闡釋的科學(xué)。,8,化學(xué)生物學(xué)與生物化學(xué)的區(qū)別,核心思想：外源化學(xué) 核心工具：化學(xué)探針 核心任務(wù)：分子精度 核心目標(biāo)：揭示生命本質(zhì)并服務(wù)于化學(xué),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(xué)生物學(xué),主要研究方向,國(guó)際發(fā)展與資助情況

7、,我國(guó)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(xué)小分子 蛋白質(zhì)靶標(biāo),為生命科學(xué)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(guó),NIH Directors New Innovator Program重點(diǎn)資助的研究方向,18,生物醫(yī)學(xué)路線圖計(jì)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(kù)和成像計(jì)劃 (Molecular Libraries and Imaging, MLI),19,美國(guó)的化學(xué)生物學(xué)高水準(zhǔn)研究單位：,20,支持化學(xué)生物學(xué)研究的基金機(jī)構(gòu): EPSRC: Engineering and

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

10、學(xué)生物學(xué)（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(xué)生物學(xué)和生物化學(xué)的資助比例（A）參與這些研究的學(xué)生人數(shù)比例（B） EPSRC （2008）,22,德國(guó),合作研究中心（CRC） 目前獲DFG資助的多個(gè)CRC中心都是在化學(xué)與生物學(xué)的交叉界面上開(kāi)展研究工作，化學(xué)生物學(xué)是其主要的研究領(lǐng)域之一。,德國(guó)科學(xué)基金會(huì)（DFG）,23,日本,日本學(xué)術(shù)振興會(huì)（JSPS）,24,什么是化學(xué)生物學(xué),主要研究方向,國(guó)際發(fā)展與資助情況,我國(guó)發(fā)展與資助情況,25,2003年 化學(xué)二處設(shè)立了化學(xué)生物學(xué)學(xué)科 2005年 生物有機(jī)與化學(xué)生物學(xué)學(xué)科代碼 B020

11、7 2013年 化學(xué)生物學(xué)學(xué)科代碼B0212,學(xué)科建設(shè)情況（國(guó)家自然科學(xué)基金委員會(huì)）,26,基金資助引領(lǐng)化學(xué)生物學(xué)發(fā)展,80年代初 攀登計(jì)劃“生命過(guò)程中的化學(xué)問(wèn)題” 九五計(jì)劃 化學(xué)與生命科學(xué)交叉的重大項(xiàng)目5項(xiàng) 十五計(jì)劃 化學(xué)與生命科學(xué)交叉的重點(diǎn)項(xiàng)目32項(xiàng) 2003-2005 化學(xué)與生命科學(xué)交叉的重大國(guó)際合作 (8項(xiàng), 60-95萬(wàn)/項(xiàng)) 2004-2006 健康領(lǐng)域的交叉面上項(xiàng)目 (2000萬(wàn)/年) 2007-2015 重大研究計(jì)劃“基于化學(xué)小分子探針的 信號(hào)轉(zhuǎn)導(dǎo)過(guò)程研究”（20000萬(wàn)）,27,化學(xué)生物學(xué)研究機(jī)構(gòu)分布,28,什么是化學(xué)生物學(xué),主要研究方向,國(guó)際發(fā)展與資助情況,我國(guó)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(xué)探針”與“藥物”區(qū)別的總結(jié),32,（2） 生物分子的化學(xué)合成與標(biāo)記,33,（3） 生物相容反應(yīng)的發(fā)展及應(yīng)用,34,（4） 化學(xué)遺傳學(xué),35,（5） 生物合成化學(xué),36,（6） 藥物發(fā)現(xiàn)的化學(xué)生物學(xué),37,（7） 應(yīng)用化學(xué)生物學(xué),38,（8） 新方法、新理論及應(yīng)用,39,一. 化學(xué)生物學(xué)概述二. 細(xì)胞死亡的化學(xué)生物學(xué)研究,活性化學(xué)小分子探針,基礎(chǔ)生命科學(xué)研究,轉(zhuǎn)化醫(yī)學(xué) 創(chuàng)新藥物研究,發(fā)現(xiàn),制備,評(píng)價(jià),研究背景：利用活性小分子探索化學(xué)與生物醫(yī)學(xué)交叉,研究策略,細(xì)胞死亡的 小分子化學(xué)調(diào)控,高通量篩選 化學(xué)

13、遺傳學(xué),活性天然產(chǎn)物 導(dǎo)向的化學(xué) 生物學(xué),通過(guò)天然產(chǎn)合成化學(xué)、化學(xué)遺傳學(xué)、分子生物學(xué)等多學(xué)科交叉，利用活性小分子研究細(xì)胞死亡的生物作用機(jī)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(xì)胞死亡途徑（細(xì)胞凋亡和壞死）,43,細(xì)胞凋亡 (apoptosis),細(xì)胞壞死 (necrosis),細(xì)胞死亡的分子作用機(jī)制和調(diào)控方法 科學(xué)難點(diǎn)：調(diào)控基因致命性，信號(hào)轉(zhuǎn)導(dǎo)通路復(fù)雜 研究

15、現(xiàn)狀：經(jīng)典生物遺傳學(xué)無(wú)法闡明所有未知生物同路； 高效，可視，可控，定量化的化學(xué)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(xì)胞壞死分子作用機(jī)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. （