Structural Determination and Design of Drug Interactions with Ribonucleotide Reductase

项目总结/摘要 脱氧核糖核苷三磷酸（dNTPs）是DNA的结构单元，其平衡对于DNA的合成至关重要。 维护人类健康。核糖核苷酸还原酶（RNR）被称为dNTP生物合成的调节剂， 是在所有生物体中发现的必需酶，其催化核糖核苷酸的还原， 脱氧核糖核苷酸是DNA复制和修复的基本反应。细胞无法维持 适当的dNTP浓度可导致增加的诱变和不受控制的增殖， 促进癌症发展的特征。RNR抑制已经涉及几种类型的 癌症，并且是药物设计的目标。虽然目前临床使用的药物有效，但我们的理解是， 抑制机制是不完整的。具体地，核苷类似物用作α-抑制剂，并且具有 已显示在加入RNR α亚基后引起明显的构象变化。一旦加入 核苷类似物，形成α-六聚体环。α-六聚化已被观察到， 三磷酸化核苷类似物，氯法拉滨，克拉屈滨和氟达拉滨;然而，没有近- 原子分辨率结构可用。本提案中所述的工作旨在获得高分辨率 研究了每种α-抑制剂与人RNR的结构，并设计和评估了新的RNR α-抑制剂。冷冻- 电子显微镜将用于检查添加三磷酸化后α-六聚体的结构 克拉屈滨、氯法拉滨和氟达拉滨，以确定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-六聚化 防止RNR活动。此外，将设计新的核苷类似物，其目标是增加 结合亲和力的核苷类似物，以研究电子性质对α- 六聚体总之，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 可诱导α-六聚化的核苷类似物。

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.