Design & Development of Allosteric effectors of PK

设计

• 批准号: 6431259
• 负责人: DONALD J ABRAHAM
• 金额: $ 36.25万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6431259
• 关键词: X ray crystallography; allosteric site; clinical research; drug design /synthesis /production; enzyme inhibitors; enzyme mechanism; enzyme structure; isozymes; molecular cloning; protein structure function; pyruvate kinase

DESCRIPTION (provided by applicant): Our collaborators and we have successfully cloned and overexpressed human erythrocyte pyruvate kinase (R-PK). This reliable source of enzyme has allowed for the biological testing of new classes of molecules, and has subsequently enabled us to discover novel R-PK inhibitors that bind at micro molar (uM) concentrations. In conjunction with our collaborators, we have also successfully solved the X-ray crystal structure of human R-PK at a resolution of 2.7 A. This crystallization technique for the native structure will permit us to develop methods for co-crystallizing the newly discovered effectors with this enzyme, and thus facilitating structure-function studies (see preliminary results). Our approach to the generation of allosteric effectors of R-PK will integrate: Protein cloning and over-expression, X-ray crystallography, molecular modeling, organic synthesis, and biological studies.Therefore our specific aims include: (1) Comparison of human PK isozymes: cloning and structure determination: Ml-, M2-, and L-PK isozymes will be cloned for crystallographic structure solution and for comparison of allosteric binding sites between isozymes; (2) Structural determination of effector binding sites: we will co-crystallize newly identified inhibitors with R-PK to determine their binding sites and amino acid residue interactions. Refined Effector-Pyruvate Kinase (PK) crystal structures should also provide some information concerning which additional amino acids may be involved in regulating the allosteric transition of R-PK between the Tand R- States; (3) Structure based design of R-PK inhibitors: Initial studies will involve working with recently discovered lead compounds that are discussed in the proposal. Molecular modeling studies will employ fundamental drug design principles SAR, SAR, 3-D database searching, CoMFA, and HINT] to suggest modifications for improved binding of these compounds, as well as in the generation of novel allosteric effectors for synthesis. Development of new molecules will be directed by information from specific aims 1,2, and 4. An iterative process to improve compound binding and to increase potency will be developed as results are obtained and (4) Allosteric modulation of R-PK: Examination of R-PK- Effector kinetics to characterize the degree of allosteric function and effector/protein Structure activity relationships that arise. In summary, the goal of this proposal is to discover and employ new R-PK allosteric effectors that will also aid in unraveling the allosteric switch mechanism of this enzyme, and further the discovery of selective effector(s), which may translate into treatments for hypoxic diseases, including Alzheimer's disease. Another important aspect of this research may evolve from results obtained from specificity studies: the M2-PK isozyme is re-expressed in solid tumors, and any tested compounds that show specificity for this isozyme might lead to a new class of compounds for the treatment of cancer.

描述（由申请人提供）： 我们和我们的合作者已经成功克隆并过度表达了人类 红细胞丙酮酸激酶（R-PK）。这种可靠的酶来源使得 用于新类别分子的生物测试，并随后 使我们能够发现以微摩尔 (uM) 结合的新型 R-PK 抑制剂 浓度。我们还与我们的合作者一起 成功解析人R-PK的X射线晶体结构 2.7 A。这种天然结构的结晶技术将允许 我们开发新发现的效应器与 这种酶，从而促进结构功能研究（见初步 结果）。我们生成 R-PK 变构效应子的方法将 整合： 蛋白质克隆和过度表达、X 射线晶体学、分子建模、 有机合成和生物学研究。因此我们的具体目标包括： (1) 人PK同工酶的比较：克隆和结构测定：Ml-， M2-和L-PK同工酶将被克隆以获得晶体结构解决方案 以及同工酶之间变构结合位点的比较； (2) 结构 效应子结合位点的确定：我们将新共结晶 用 R-PK 鉴定抑制剂以确定其结合位点和氨基酸 残基相互作用。精制效应器丙酮酸激酶 (PK) 晶体结构 还应该提供一些关于哪些额外氨基酸的信息 可能参与调节 T 和 T 之间 R-PK 的变构转变 R- 状态； (3) R-PK抑制剂的基于结构的设计：初步研究 将涉及最近发现的先导化合物的研究 在提案中。分子建模研究将采用基础药物设计 SAR、SAR、3-D 数据库搜索、CoMFA 和 HINT 原则来建议 为改善这些化合物的结合而进行的修饰，以及 生成用于合成的新型变构效应子。开发新 分子将受到来自特定目标 1,2 和 4 的信息的指导。 改进化合物结合并增加效力的迭代过程将是 (4) R-PK 的变构调节： 检查 R-PK-效应动力学以表征变构程度 功能和效应器/蛋白质结构产生的活性关系。 总之，该提案的目标是发现并使用新的 R-PK 变构效应器也将有助于解开变构开关 这种酶的机制，并进一步发现选择性效应器， 这可能转化为缺氧疾病的治疗，包括阿尔茨海默病 疾病。这项研究的另一个重要方面可能源自结果 从特异性研究中获得：M2-PK 同工酶在固体中重新表达 肿瘤，以及任何对该同工酶表现出特异性的测试化合物都可能 产生一类用于治疗癌症的新化合物。