Design & Development of Allosteric effectors of PK

设计

• 批准号: 6782522
• 负责人: DONALD J ABRAHAM
• 金额: $ 33.75万
• 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 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6782522
• 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)： 我们的合作者和我们成功地克隆并过度表达了人 红细胞丙酮酸激酶(R-PK)。这种可靠的酶来源使 用于新类别分子的生物测试，并随后 使我们能够发现结合在微摩尔(Um)上的新型R-PK抑制剂 浓度。与我们的合作者一起，我们还 成功地解决了人R-PK的X-射线晶体结构 2.7A，这种本征结构的晶化技术将允许 美国将开发方法使新发现的效应器与 这种酶，从而促进结构-功能研究(见初步 结果)。我们对R-PK变构效应产生的方法将 集成： 蛋白质克隆和过度表达，X射线结晶学，分子模拟， 有机合成和生物研究。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同工酶将被克隆用于晶体结构溶液 用于比较同工酶之间的变构结合部位；(2)结构 效应器结合部位的测定：我们将重新结晶 用R-PK鉴定抑制剂的结合部位和氨基酸 残基相互作用。精制效应丙酮酸激酶(PK)的晶体结构 也应该提供一些关于哪些额外的氨基酸的信息 可能参与调节T和T之间R-PK的变构转换 R-态；(3)基于结构的R-PK抑制剂设计：初步研究 将涉及到使用最近发现的先导化合物，这些化合物将被讨论 在提案中。分子建模研究将采用基础药物设计 原理SAR、SAR、3-D数据库搜索、CoMFA和提示]建议 用于改善这些化合物的结合的修饰，以及在 用于合成的新型变构效应器的生成。新技术的发展 分子将由来自特定目标1、2和4的信息引导。 改进化合物结合和提高效力的迭代过程将是 得到的结果和(4)R-PK的变构调制： 表征变构程度的R-PK效应动力学研究 产生的功能和效应器/蛋白质结构活性关系。 总之，该提案的目标是发现和使用新的R-PK 变构效应器也将有助于解开变构开关 该酶的作用机制，以及选择性效应子的进一步发现(S)， 这可能会转化为治疗包括阿尔茨海默氏症在内的缺氧性疾病 疾病。这项研究的另一个重要方面可能是从结果演变而来的 来自特异性研究：M2-PK同工酶在固体中重新表达 肿瘤，以及任何对这种同工酶表现出特异性的测试化合物可能 导致了一类新的治疗癌症的化合物。