Structure-Function and Inhibition of Rnr1

描述（由申请人提供）：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来催化从头合成前体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标。最近，我们从酿酒酵母中首次获得了真核生物核糖核苷酸还原酶1的X射线结构。这12个结构揭示了两个新的结构域，即真核生物底物选择的结构基础和抗癌药物吉西他滨与核糖核苷酸还原酶基肽的结合模式；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。我们表达并纯化了人Rnr1用于结晶。在哺乳动物Rnr1结构被解决之前，酵母结构为设计靶向Rnr1的抑制剂提供了宝贵的起点。我们将解决Rnr1与靶向效应位点的抑制剂络合的x射线结构(例如；氯法拉滨)和催化位点（3NUDP），以及靶向效应位点和催化位点的双功能分子（dGTP-ADP共价连接），以及破坏RNR组装的肽拟物。这些结构将为基于知识的药物设计提供一个起点。作为一个原理证明，我们已经证明了一个基于小鼠Rnr2的抑制剂可以结合酵母Rnr1。这些化合物由我们的合作者Barry Cooperman博士（UPENN）， Vasha Ghandi博士（MD Anderson）和william Parker博士在南方研究所提供。我们还将研究SmM如何结合Rnr1使用交联，有限的蛋白水解，表面制图串联质谱。完整的Sml1-Rnr1和rnr1 - sml1肽复合物的结构将被确定。我们将使用位点导向突变来确认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通过我们的结构确定的赋予底物特异性的关键残基的作用。最后，我们将研究由Fred Hutchninson癌症中心的Julian Simon博士发现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重要的核糖核苷酸还原酶是如何调节的理解，以及基于结构的抑制剂设计对增殖性疾病（如癌症）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.