Structural Determination and Design of Drug Interactions with Ribonucleotide Reductase

项目摘要/摘要 脱氧核糖核苷三磷酸(DNTPs)是DNA的组成成分，它的平衡对 维护人类健康。被称为dNTP生物合成调节剂的核糖核苷酸还原酶(RNRs) 是在所有生物体中发现的重要酶，它们催化核糖核苷酸还原为 脱氧核糖核苷酸，DNA复制和修复的基本反应。单元格维护失败 适当的dNTP浓度会导致突变增加和不受控制的增殖， 促进癌症发展的特征。RnR抑制与几种类型的 癌症，是药物设计的目标。虽然目前临床使用的药物是有效的，但我们对 抑制机制的研究还不完善。具体地说，核苷类似物被用作α抑制剂，并具有 被证明在加入RnRα-亚基后会引起明显的构象变化。在添加 形成了核苷类似物、α-六聚体环。α-六聚体已经观察到有三个 三磷酸化核苷类似物，氯法拉滨、克拉立滨和氟达拉滨；然而，没有近- 可用的原子分辨率结构。本提案中描述的工作旨在获得高分辨率 每种α-抑制剂与人RNR结合的结构，并设计和评价新的RNR-α抑制剂。冷藏- 电子显微镜将被用来检查加入三磷酸化后的α-六聚体的结构 克拉立滨、氯法拉滨和氟达拉滨用于确定α抑制剂结合位置，可能的构象 可以解释α-六聚体稳定性的变化和非共价相互作用，以及α-六聚体如何 阻止RNR活动。此外，将设计新的核苷类似物，目标是增加 核苷类似物结合亲和力研究电子性质对α-稳定性的影响 六角人。总之，这项工作旨在加深我们对RNR抑制机制的理解 了解α-六聚体的形成并利用基于结构的药物设计来扩大文库 核苷类似物，可诱导α-六聚体。

PROJECT SUMMARY/ABSTRACT The equilibrium of deoxyribonucleoside triphosphates (dNTPs), the building blocks of DNA, is critical for maintaining human health. Known as the regulator of dNTP biosynthesis, ribonucleotide reductases (RNRs) are essential enzymes found in all organisms that catalyze the reduction of ribonucleotides to deoxyribonucleotides, an essential reaction for DNA replication and repair. Failure of cells to maintain appropriate dNTP concentrations can lead to increased mutagenesis and uncontrolled proliferation, characteristics that promote cancer development. RNR inhibition has been implicated in several types of cancers and is a target for drug design. Although current drugs in clinical use are effective, our understanding of the inhibition mechanism is incomplete. Specifically, nucleoside analogs are used as α-inhibitors and have been shown to cause a distinct conformational change upon addition to the RNR α-subunit. Upon addition of nucleoside analogs, α-hexamer rings are formed. α-hexamerization has been observed with three triphosphorylated nucleoside analogs, clofarabine, cladribine and fludarabine; however, there are no near- atomic resolution structures available. The work described in this proposal aims to obtain high resolution structures of each α-inhibitor with Human RNR and to design and evaluate new RNR α-inhibitors. Cryo- electron microscopy will be used to examine the structures of α-hexamers after addition of triphosphorylated cladribine, clofarabine, and fludarabine to determine α-inhibitor binding locations, possible conformational changes and noncovalent interactions that could explain α-hexamer stability, and how α-hexamerization prevents RNR activity. Furthermore, new nucleoside analogs will be designed with the goal of increasing binding affinity of the nucleoside analogs to study the effects of electronic properties on the stability of α- hexamers. Together, this work aims to deepen our understanding of the mechanism of RNR inhibition by understanding the formation of α-hexamers and utilize structure-based drug design to expand the library of nucleoside analogs that can induce α-hexamerization.