Combined Substrate Polymerase Inhibitors

描述（由申请人提供）：核苷酸是天然产物，对于涉及基因复制、维持、表达和蛋白质合成（在转录因子循环中）的所有过程都是必需的，更不用说它们在能量储存、基团转移反应、化学偶联和信号转导（例如，G蛋白）。我们对修饰核苷酸研究的主要目标是：1）设计、合成和动力学分析细菌和病毒DNA聚合酶和病毒逆转录酶的组合底物，也称为模板竞争性抑制剂。我们建议在本申请中将我们的研究扩展到克隆病毒RNA依赖性RNA聚合酶（RDRP）的抑制剂。这些研究的目标是通过修饰核苷酸、糖（对于本申请是新的）、侧链部分和磷酸酯2）确定这些抑制剂对一系列克隆的细菌和病毒靶标的活性和选择性;第三章扩大核苷酸类似物库，以研究修饰核苷酸作为肌苷单磷酸的组合底物抑制剂的结合和抑制脱氢酶（IMPDH），一种适用于组织排斥、癌症、细菌和寄生虫治疗的靶标（本申请的新技术）;和4）开发用于快速表达和纯化这些研究所需靶标的克隆方法（本申请的新技术）。这些研究为本科生培训提供了许多机会，包括使用标准和组合方法进行修饰核苷、NMP和NTP合成和纯化;开发用于分析聚合酶和IMPDH活性的连续动力学方法，并分析抑制模式;用于cDNA产生、克隆、转化和选择、细菌细胞生长、收获和破碎的PCR，以及酶纯化，包括亲和色谱和离子交换色谱。这些方法涵盖了实验设计，执行，数据收集和分析中的各种技术，可以使化学和生物学本科生在生物医学科学领域寻求学士后教育。组合底物抑制剂概念是一种药物设计，用于寻找可能用于治疗癌症和细菌，寄生虫和病毒感染的新实体。这些化合物特异性靶向这些细胞或颗粒类型复制中的关键蛋白质。这些靶点包括DNA聚合酶、逆转录酶、病毒RNA依赖性DNA聚合酶和肌苷一磷酸脱氢酶（GMP细胞合成中的控制酶）。

DESCRIPTION (provided by applicant): Nucleotides are natural products that are essential for all processes involving gene replication, maintenance, expression and protein synthesis (in transcription factor cycling), not to mention their importance in energy storage, group transfer reactions, chemical coupling, and signal transduction (e.g., G-proteins). The main goals of our research on modified nucleotides are: 1) the design, synthesis, and kinetic analysis of combined substrate, also called template-competitive, inhibitors of bacterial and viral DNA polymerases and viral reverse transcriptases. We propose in this application to expand our studies to inhibitors of cloned viral RNA-Dependent RNA polymerases (RDRP). The goal of these studies is to maximize inhibition by modifications in nucleotide, sugar (new to this application), side chain moieties, and phosphates (new to this application); 2) to determine the activity and selectivity of these inhibitors over a range of cloned bacterial and viral targets; 3) to expand the nucleotide analog libraries to study the binding and inhibition of modified nucleotides as combined substrate inhibitors of inosine monophosphate dehydrogenase (IMPDH), a target applicable to tissue rejection, cancer, bacterial, and parasite treatments (new to this application); and 4) to develop cloning methods for rapid expression and purification of the targets needed for these studies (new to this application). These studies offer numerous opportunities for undergraduate training, including modified nucleoside, NMP and NTP synthesis and purification using standard and combinatorial methods; development of continuous kinetic methods for analysis of polymerase and IMPDH activity with analysis of inhibition patterns; PCR for cDNA production, cloning, transformation and selection, bacterial cell growth, harvesting, and breakage, and enzyme purification, including affinity and ion-exchange chromatographies. These methods cover a wide variety of techniques in experimental design, execution, data collection, and analysis that can benefit undergraduates in chemistry and biology seeking post-baccalaureate education for careers in the biomedical sciences. Combined substrate inhibitors concept is a type of drug design that is being applied to find new entities that might be used to treat cancer and bacterial, parasite, and viral infections. The compounds specifically targets proteins that are key in the replication of these cell or particle types. These targets include DNA polymerase, reverse transcriptases, viral RNA dependent DNA polymerases, and inosine monophosphate dehydrogenase, a control enzyme in the cell synthesis of GMP.