Computational and NMR Methods to define Conformations of Protein-Ligand Complexes

定义蛋白质-配体复合物构象的计算和核磁共振方法

• 批准号: 7265740
• 负责人: GERHARD WAGNER
• 金额: $ 35.01万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7265740
• 关键词: Address; Affinity; Algorithms; Amino Acids; Apoptosis; Apoptotic; Area; BH3 Domain; Binding; Binding Sites; Biological Assay; Boston; Budgets; Cancer cell line; Categories; Cell Line; Chemical Structure; Chemicals; Cloning; Collaborations; Complex; Computer Simulation; Computer software; Computers; Computing Methodologies; Condition; Data; Databases; Development; Diffusion; Direct Costs; Dissociation; Docking; Electrostatics; Enzymes; Equilibrium; Equipment; Exhibits; Face; Facilities and Administrative Costs; Fluorescence Polarization; Glucose; Growth; Human; Human Resources; Individual; Inpatients; Instruction; Isotope Labeling; Label; Last Name; Libraries; Ligand Binding; Ligands; Maintenance; Maps; Measures; Membrane Proteins; Memory; Methods; Modeling; Molecular Conformation; NMR Spectroscopy; Names; Nucleic Acids; Nucleotides; Numbers; Oncogenes; Outpatients; Patient Care; Peptide Initiation Factors; Peptides; Pharmaceutical Preparations; Pliability; Polymerase Chain Reaction; Positioning Attribute; Principal Investigator; Protein Conformation; Proteins; Protons; RNA Cap-Binding Proteins; RNA Helicase; Range; Rate; Recruitment Activity; Registries; Relative (related person); Relaxation; Reporting; Research; Research Personnel; Resolution; Role; Rotation; Scaffolding Protein; Screening procedure; Side; Signal Transduction; Simulate; Site; Solubility; Speed; Structure; Subgroup; Sum; Surface; Testing; Time; Translations; Travel; Trees; Tube; Universities; Untranslated Regions; Vertebral column; Wages; Work; bak protein; base; cost; high throughput screening; human embryonic stem cell; inhibitor/antagonist; macromolecule; medical schools; member; novel; nuclear Overhauser enhancement; particle; performance tests; programs; protein protein interaction; protein structure; receptor; reconstruction; repaired; research study; scaffold; small molecule; small molecule libraries; symposium; tumor

The general objective of the proposed research is to combine a new computational approach with complementary NMR methods for characterizing binding of small ligands to proteins. This will address a wide need for defining interactions of proteins with natural ligands, such as peptides, non-peptidic biomolecules, or with non-natural ligands, including drug leads or other chemicals. While this Component 2 is on interactions of small ligands with proteins, this approach can readily be applied to protein-protein or protein-nucleic acid interactions. This connects this research to Components 1 and 4. Thus, both aspects address the problem of characterizing weak but specific iinteractions of proteins with small molecules and other macromolecules in situations where experimental constraints are sparse. We proposeto develop an approach that will build on the recently developed TreeDock algorithm and will combine it with information from NMR spectroscopy on small-molecule inhibitors of protein-protein interactions obtained from high-throughput library screening. To define structures of weak protein-inhibitor complexes, TreeDock can search the docking space very fast by eliminating up front all configurations in which the components to be docked have no contact or penetrate each other. This is achieved with kd search trees. The algorithm uses complete enumeration of the search space at high resolution and can handle ligand mobility by querying a database of docked rigid subgroups. TreeDock can be ideally interfaced with sparse data from NMR experiments. We propose NMR methods that can provide few specific protein-ligand contacts even under difficult experimental conditions, such as intermediate exchange or poor solubility. The combination of the computational and experimental NMR methods will also be capable of dealing with structural changes of receptor proteins upon ligand binding. The research will be pursued four Specific Aims: 1: Development of a flexible docking algorithm (TreeDock II) that is capable of screening compound libraries efficiently, docking peptides and DNA/RNA to proteins. 2: Development of a compiler (parser, classifier) of chemical compound libraries. 3. Experimental methods for measuring NOEs in the intermediate exchange regime and defining anchors for TreeDock - Application to inhibitors of Bcl-2 type proteins. 4. Define complexes of small-molecule inhibitors with translation initiation factors 4E and 4A. Harvard Medical School, Boston, MA PHS 398 (Rev. 04/06) Page 126 Form Page 2 Principal Investigator/Program Director (Last, First, Middle): Wagner, Gerhard - Component 2 KEY PERSONNEL. See instructions. Use continuationpages as needed to provide the required information in the format shown below. Start with Principal Investigator(s). List all other key personnel in alphabetical order, last name first. Name eRA Commons User Name Organization Role on Project Wagner, Gerhard GWAGNER Harvard Medical School PI Fahmy, Amr AMRFAHMY Harvard Medical School coPI OTHER SIGNIFICANT CONTRIBUTORS Name Organization Role on Project Human Embryonic Stem Cells E] No CH Yes If the proposed project Involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the following list: http://stemcells.nih.qov/registrv/index.asp. Use continuation pages as needed. If a specific line cannot be referencedat this time, include a statement that one from the Registry will be used. Cell Line PHS 398(Rev. 04/06) Page 127 Form Pa9e 2-continued Number the following pages consecutively throughout the application: Do not use suffixes such as 4a, 4b. Principal Investigator/Program Director (Last, First, Middle): Wagner, Gerhard - Component 2 DETAILED BUDGET FOR INITIAL BUDGET PERIOD DIRECT COSTS ONLY PERSONNEL (Applicant organization only) Months Devoted to Project ROLE ON Cal. Acad. Sum. INST.BASE NAME PROJECT Mnths Mnths Mnths SALARY Principal Gerhard Wagner 1.2 183,500 Investigator Co- Amr Fahmy 6 92,920 Investigator Research Mikhail Reibarkh 12 48,542 Fellow SUBTOTALS hwi CONSULTANT COSTS EQUIPMENT (Itemize) FROM THROUGH 5/1/07 4/30/08 DOLLAR AMOUNT REQUESTED (omit cents) SALARY FRINGE REQUESTED BENEFITS TOTAL 18,350 4,597 22,947 46,460 11,638 58,098 48,542 11,626 60,168 113,352 27,861 141,2131 . SUPPLIES (Itemize by category) NMR tubes, Shigemi tubes 1,000 Labeled amino acids 2,000 Chemicals and disposables 2,000 Precursors for ILV,LV or L labeling 4,000 Enzymes for cloning 3,000 15NH3 , 13C glucose & D2O 1 5,000 PCR primers 3,000 30,000 TRAVEL Costs for 2 researchers to attend a relevant conference 3,000 PATIENT CARE COSTS INPATIENT - OUTPATIENT - ALTERATIONS AND RENOVATIONS (Itemize by category) _ OTHER EXPENSES (Itemize by category) NMR Spectrometer maintenance, repair & operating costs (detailed in Justification) $23,000 23,000 CONSORTIUM/CONTRACTUAL COSTS DIRECT COSTS SUBTOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD (Item 7a, Face Page) $ 197,213 CONSORTIUM/CONTRACTUAL COSTS FACILITIES AND ADMINISTRATIVE COSTS TOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD $ 197,213 PHS 398 (Rev. 04/06) Page 128 Form Page 4

拟议研究的总体目标是将联合收割机与 用于表征小配体与蛋白质的结合的互补NMR方法。这将解决 广泛需要定义蛋白质与天然配体如肽、非肽 生物分子，或与非天然配体，包括药物先导物或其它化学品。 虽然该组件2是关于小配体与蛋白质的相互作用，但该方法可以容易地被理解为是一种新的方法。 应用于蛋白质-蛋白质或蛋白质-核酸相互作用。这将本研究与组件1联系起来 和4.因此，这两个方面都解决了表征蛋白质弱但特异性相互作用的问题 小分子和其他大分子在实验约束条件稀疏的情况下。 我们建议开发一种方法，该方法将建立在最近开发的TreeDock算法上， 将联合收割机与核磁共振波谱学中有关蛋白质-蛋白质小分子抑制剂的信息相结合 从高通量文库筛选获得的相互作用。确定弱蛋白抑制剂的结构 复杂，TreeDock可以搜索对接空间非常快，消除了前面的所有配置， 其中待对接的部件彼此不接触或穿透。这是通过kd实现的 搜索树该算法在高分辨率下使用搜索空间的完全枚举， 通过查询对接的刚性亚组的数据库来处理配体迁移率。 TreeDock可以理想地与来自NMR实验的稀疏数据连接。我们提出了核磁共振方法 即使在困难的实验条件下， 中间交换或溶解性差。计算和实验NMR的结合 这些方法也能够处理配体结合后受体蛋白的结构变化。 该研究将追求四个具体目标： 1：开发能够筛选化合物文库的灵活对接算法（TreeDock II） 有效地将肽和DNA/RNA对接到蛋白质上。 2：开发化合物库的编译器（解析器、分类器）。 3.测量中间交换状态下NOE和确定NOE锚点的实验方法 TreeDock -应用于Bcl-2型蛋白的抑制剂。 4.定义小分子抑制剂与翻译起始因子4 E和4A的复合物。 哈佛医学院，马萨诸塞州波士顿 PHS 398（04/06修订版）第126页表格第2页 主要研究者/项目负责人（最后一名、第一名、中间名）：瓦格纳、Gerhard -组件2 关键人员。参见说明。根据需要使用续页，以下列格式提供所需信息。 从主要研究者开始。按字母顺序列出所有其他关键人员，姓在前。 名称eRA Commons用户名组织在项目中的角色 瓦格纳，格哈德GWAGNER哈佛医学院PI 哈佛医学院教授 其他重要贡献者 名称组织在项目中的角色 人胚胎干细胞（英文） 如果拟开展的项目涉及人类胚胎干细胞，请在下面列出以下列表中特定细胞系的注册号： http://stemcells.nih.qov/registrv/index.asp.根据需要使用续页。 如果此时无法引用特定的行，请包括一个声明，说明将使用注册表中的一行。 细胞系 PHS 398（2006年4月修订版）第127页表格Pa 9 e 2-续 将下列各页依次编号 应用：不要使用后缀，如4a，4 b。 主要研究者/项目负责人（最后一名、第一名、中间名）：瓦格纳、Gerhard -组件2 初步预算期的详细预算 仅直接费用 人员（仅限申请组织）投入项目的月份 加州大学的作用总计。INST.BASE 名称项目月工资 主要 格哈德瓦格纳1.2 183，500 研究者 共- Amr Fahmy 6 研究者 研究 Mikhail Reibarkh 12 48，542 家伙 小计hwi 咨询人费用 设备（逐项列出） 来自通 2007年1月5日2008年4月30日 申请金额（省略美分） 薪酬福利 要求的福利共计 18，350 4，597 22，947 46，460 11，638 58，098 48，542 11，626 60，168 113，352 27，861 . 用品（按类别逐项列出） 核磁共振管、重见管1，000标记氨基酸2，000 化学品和一次性用品2，000 ILV、LV或L标签前体4，000 用于克隆的酶3，000 15 NH3，13 C葡萄糖和D2 O 1 5，000 PCR引物3，000 30，000 旅行 2名研究人员出席相关会议的费用 住院病人护理费用- 门诊- 改建和翻修（按类别逐项列出） _ 其他费用（按类别分列） NMR光谱仪维护、维修和运营成本（详见理由）23，000美元 23,000 联合体/采购费用直接费用 初步预算期间直接费用小计（项目7a，正面） 联合体/行政费用、设施和行政费用 初步预算期间直接费用共计197 213美元 PHS 398（04/06修订版）第128页表格第4页