Structure-Function and Inhibition of Rnr1

Rnr1 的结构-功能和抑制

• 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
• 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

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）是一种多生核苷酸酶，通过将核苷酸二磷酸二磷酸二磷酸二磷酸二磷酸二磷酸核苷酸DMA合成的速率限制步骤催化。对于快速增殖的细胞至关重要，RNR是抗癌和抗病毒疗法的靶标。最近，我们解决了酿酒酵母的真核核糖核苷酸还原酶1的第一个X射线结构。解决的十二个结构揭示了两个新领域，这是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肽的结合方式；后一种复合物为设计抗癌药物的框架提供了破坏酶多支出组件的抗癌药物。我们已经表达并纯化了人类RNR1进行结晶。在溶解哺乳动物RNR1结构之前，酵母结构为设计靶向RNR1的抑制剂提供了宝贵的起点。 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.这些结构将为基于知识的药物设计提供一个起点。作为原理的证明，我们表明基于RNR2的抑制剂结合酵母RNR1。这些化合物由我们的合作者Barry Cooperman博士（Upenn），Vasha Ghandi博士（MD Anderson）和南方研究所的Willam Parker博士提供。我们还将研究SMM如何使用交联，有限的蛋白水解，与质谱串联的表面映射结合RNR1。将确定完整的SML1-RNR1和RNR1-SML1肽配合物的结构。我们将使用位置定向的诱变来确认SML1结合位点上的MS结果，确定新鉴定的插入域的功能以及由赋予底物特异性的结构确定的关键残基的作用。最后，我们将研究朱利安·西蒙（Julian Simon）博士在弗雷德·哈奇尼森（Fred Hutchninson）癌症中心确定的合成性不匹配修复突变体的结构基础。提出的工作将进一步了解我们如何调节重要酶核糖核苷酸还原酶，并且针对其抑制剂的结构设计对于癌症等增殖性疾病的治疗干预至关重要。