Structure-Function and Inhibition of Rnr1

Rnr1 的结构-功能和抑制

基本信息

批准号：
7909255
负责人：
Chris G Dealwis
金额：
$ 40.46万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-01 至 2011-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7909255
关键词：
Antineoplastic Agents Antiviral Agents Binding Binding Sites Biological Assay C-terminal Cancer Center Catalytic Domain Cells Chemicals Clofarabine Complex Crystallization Digestion Diphosphates Disease Drug Design Enzymes Equilibrium Eukaryota Fluorescence Fred Hutchinson Cancer Research Center Human Link Malignant Neoplasms Maps Mass Spectrum Analysis Mismatch Repair Mus Mutation Nucleotides Peptides Production Proliferating Proteins Proteolysis Research Institute Ribonucleotide Reductase Roentgen Rays Role Saccharomyces cerevisiae Site Site-Directed Mutagenesis Specificity Structure Substrate Specificity Suppressor Mutations Surface Techniques Testing Therapeutic Intervention Viral Work Yeast Model System Yeasts base chemotherapeutic agent crosslink deoxyguanosine triphosphate design gemcitabine inhibitor/antagonist knowledge base mutant novel peptidomimetics protein complex research study small molecule three dimensional structure

项目摘要

DESCRIPTION (provided by applicant): Ribonucleotide Reductase, (RNR) is a multi-subunit enzyme that catalyzes the rate-limiting step of de novo precursor DMA synthesis by converting nucleotide diphosphates to deoxynucleotide diphosphates. Crucial for rapidly proliferating cells, RNR is a target for anti-cancer and anti-viral therapy. Recently, we solved the first X -ray structures of eukaryotic ribonucleotide reductase 1 from Saccharomyces cerevisiae. The twelve structures solved reveal two new domains, the structural basis for substrate selection in eukaryotes and the mode of binding of the anti-cancer drug Gemcitabine and ribonucleotide reductase based peptides; the latter complex provides a framework for designing anti-cancer drugs that disrupt the enzyme's multi-subunit assembly. We have expressed and purified the human Rnr1 for crystallization. Until the mammalian Rnr1 structure is solved, the yeast structures provide an invaluable starting point for designing inhibitors that target Rnr1. We will solve the X-ray structures of Rnr1 complexed with inhibitors that target the effector sites (Eg. Clofarabine) and catalytic site (3NUDP) as well as bifunctional molecules that target both effector and catalytic sites (dGTP-ADP linked covalently), and peptidomimetics that disrupt RNR assembly. These structures will provide a starting point for knowledge based drug design. As a proof of principle we have shown that a mouse Rnr2 based inhibitor binds yeast Rnr1. The compounds have been provided by our collaborators Dr. Barry Cooperman (UPENN), Dr. Vasha Ghandi (MD Anderson), and Dr. Willam Parker at the Southern Research Institute. We will also study how SmM binds Rnr1 using cross-linking, limited proteolysis, surface mapping in tandem with mass spectrometry. The structures of intact Sml1-Rnr1 and Rnr1-Sml1peptide complexes will be determined. We will use site-directed mutagenesis to confirm the MS results on the Sml1 binding site, identify the function of the newly identified insert domains and the role of crucial residues identified by our structures that confer substrate specificity. Finally, we will investigate the structural basis of the synthetically lethal mismatch repair mutants identified by Dr. Julian Simon at the Fred Hutchninson Cancer Center. The work proposed will further our understanding on how the vital enzyme ribonucleotide reductase is regulated and the structure based design of inhibitors against it will be important for the therapeutic intervention of proliferative diseases such as cancer.

描述（由申请人提供）：核糖核苷酸还原酶（RNR）是一种多亚基酶，其通过将核苷酸二磷酸转化为脱氧核苷酸二磷酸来催化从头前体DMA合成的限速步骤。 RNR 对于快速增殖的细胞至关重要，是抗癌和抗病毒治疗的靶点。最近，我们首次解析了酿酒酵母真核核糖核苷酸还原酶 1 的 X 射线结构。解决的十二个结构揭示了两个新的结构域，真核生物中底物选择的结构基础以及抗癌药物吉西他滨和基于核糖核苷酸还原酶的肽的结合模式；后一种复合物为设计破坏酶的多亚基组装的抗癌药物提供了框架。我们已经表达并纯化了人 Rnr1 用于结晶。在哺乳动物 Rnr1 结构得到解决之前，酵母结构为设计针对 Rnr1 的抑制剂提供了宝贵的起点。我们将解析 Rnr1 与针对效应位点（例如氯法拉滨）和催化位点（3NUDP）的抑制剂以及针对效应位点和催化位点（共价连接的 dGTP-ADP）的双功能分子复合的 X 射线结构，以及破坏 RNR 组装的肽模拟物。这些结构将为基于知识的药物设计提供起点。作为原理证明，我们已经证明基于小鼠 Rnr2 的抑制剂可以结合酵母 Rnr1。这些化合物由我们的合作者南方研究所的 Barry Cooperman 博士 (UPENN)、Vasha Ghandi 博士 (MD Anderson) 和 Willam Parker 博士提供。我们还将研究 SmM 如何使用交联、有限蛋白水解、表面图谱与质谱结合来结合 Rnr1。将确定完整的 Sml1-Rnr1 和 Rnr1-Sml1 肽复合物的结构。我们将使用定点诱变来确认 Sml1 结合位点的 MS 结果，识别新识别的插入结构域的功能以及由我们的结构识别的赋予底物特异性的关键残基的作用。最后，我们将研究 Fred Hutchninson 癌症中心的 Julian Simon 博士鉴定的合成致死错配修复突变体的结构基础。所提出的工作将进一步加深我们对重要酶核糖核苷酸还原酶如何调节的理解，并且基于结构的抑制剂设计对于癌症等增殖性疾病的治疗干预非常重要。