Structure-Function and Inhibition of Rnr1

描述(申请人提供)：核糖核苷酸还原酶(RNR)是一种多亚基酶，通过将核苷酸二磷酸转化为脱氧核苷酸二磷酸，催化从头合成前体DMA的限速步骤。RNR对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，我们首次解决了酿酒酵母核糖核苷酸还原酶1的X射线结构。解决的12个结构揭示了两个新的结构域，真核细胞中底物选择的结构基础和抗癌药物吉西他滨和基于核糖核苷酸还原酶的多肽的结合模式；后者复合体为设计破坏酶的多亚基组装的抗癌药物提供了一个框架。我们已经表达并纯化了人Rnr1，用于结晶。在哺乳动物的Rnr1结构被解决之前，酵母结构为设计靶向Rnr1的抑制剂提供了一个宝贵的起点。我们将解决Rnr1与靶向效应部位的抑制剂复合的X射线结构(例如。Clofarabine)和催化部位(3NUDP)，针对效应部位和催化部位的双功能分子(dGTP-ADP共价连接)，以及破坏RNR组装的肽类药物。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，我们已经证明了基于小鼠Rnr2的抑制剂与酵母Rnr1结合。这些化合物是由我们的合作者巴里·库珀曼博士(UPenn)、瓦莎·甘地博士(MD Anderson)和南方研究所的Willam Parker博士提供的。我们还将研究SMM如何通过交联法、有限蛋白水解法、表面图谱和质谱仪来结合Rnr1。将确定完整的Sml1-Rnr1和Rnr1-Sml1多肽复合体的结构。我们将使用定点突变来确认Sml1结合位点上的MS结果，确定新发现的插入结构域的功能以及由我们的结构确定的关键残基的作用，这些结构赋予底物特异性。最后，我们将调查由弗雷德·哈奇宁森癌症中心的朱利安·西蒙博士鉴定的合成致命错配修复突变体的结构基础。这项工作将进一步加深我们对核糖核苷酸还原酶这一重要酶的调控方式的理解，以及针对它的基于结构的抑制剂的设计对于癌症等增殖性疾病的治疗干预将是重要的。

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.