D-ARABINOSE SYNTHESIS AS A TARGET SITE FOR CHEMOTHERAPY

D-阿拉伯糖合成作为化疗的靶点

• 批准号: 2661146
• 负责人: Alan D Elbein
• 金额: $ 7.3万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2661146
• 关键词: Mycobacterium smegmatis; Mycobacterium tuberculosis; antineoplastics; arabinose; carbohydrate biosynthesis; carbohydrate metabolism; carbohydrate structure; cell free system; enzyme activity; high performance liquid chromatography; inhibitor /antagonist; microorganism culture; monosaccharides; nucleotides; phosphomutase; protein purification

DESCRIPTION (adapted from application abstract): D-Arabinose is a major sugar of the mycobacterial cell wall and occurs in two important cell wall polymers, an arabinogalactan and a lipoarabinomannan. In spite of the widespread occurrence of this sugar in this group of organisms, very little is known about its pathway of biosynthesis. Previous studies from our laboratory and that of McNeil et al, in which mycobacterial cells were incubated with [1-(14)C]- or - [6-(14)C] glucose indicated that both labels were equally good precursors of radioactive arabinose, suggesting that this sugar arises via the degradation and reassembly of a hexose precursor, rather than directly by the hexose-monophosphate shunt. However, the specific steps leading to an arabinose-phosphate or an activated form of D-arabinose are unknown, or not well documented. Thus, while the Brennan group have definitely identified an undecaprenyl-P-arabinose as the immediate precuror of arabinose polymers, steps prior to formation of the lipid are unclear. Published reports on the nature of the nucleoside diphosphate arabinose are conflicting. Since D-arabinose does not occur in any animal cells, any of the steps in its synthesis or activation represent excellent target sites for chemotherapy. Thus, in this pilot project, we will explore the reactions involved in synthesis and activation of D-arabinose. First, we will isolate and characterize the nucleoside diphosphate D-arabinose from whole cells of mycobacteria, in order to determine the nature of the nucleotide base (one report in the literature indicates it is guanosine, another that it is uridine). In these cell extracts, we will also look for the presence of arabinose-5-P and arabinose-1-P to verify the pathway of synthesis. Then, we will incubate cell free extracts of mycobacteria with radioactive arabinose-5-P and arabinose-1-P to assay for the presence of a phosphoarabinose mutase and an XDP-arabinose pyrophosphorylase. These enzymes will be purified and characterized and tested with various potential inhibitors that may be useful chemotherapeutic agents.

描述（改编自申请摘要）：D-阿拉伯糖是一种主要的 糖的分枝杆菌细胞壁和发生在两个重要的细胞壁 聚合物、阿拉伯半乳聚糖和脂阿拉伯甘露聚糖。 尽管有 这种糖在这组生物中广泛存在， 已知其生物合成途径。 我们以前的研究 实验室和McNeil等人的实验，其中分枝杆菌细胞被 与[1-（14）C]-或- [6-（14）C]葡萄糖孵育表明， 同样是放射性阿拉伯糖的前体，这表明， 糖通过己糖前体的降解和重组而产生， 而不是直接通过单磷酸己糖分流。 但 产生阿拉伯糖-磷酸或其活化形式的特定步骤 D-阿拉伯糖是未知的，或没有很好的记录。 因此，虽然布伦南 研究小组已经明确确定十一戊烯基-β-阿拉伯糖为 阿拉伯糖聚合物的直接前体，在形成阿拉伯糖聚合物之前的步骤， 血脂不清。 已发表的关于核苷性质的报告 二磷酸阿拉伯糖是矛盾的。 由于D-阿拉伯糖不存在于 任何动物细胞，其合成或活化的任何步骤都代表 化疗的理想靶点。 在这个试点项目中，我们 将探讨参与合成和激活的反应， 阿拉伯糖 首先，我们将分离和表征核苷 二磷酸D-阿拉伯糖，来自分枝杆菌的全细胞，以 确定核苷酸碱基的性质（文献中的一个报告 表示它是鸟苷，另一个表示它是尿苷）。 在这些细胞中 提取物，我们还将寻找阿拉伯糖-5-P和 阿拉伯糖-1-P，以验证合成途径。 然后，我们将孵化 含有放射性阿拉伯糖-5-P的分枝杆菌无细胞提取物， 阿拉伯糖-1-P，以测定磷酸阿拉伯糖链和磷酸阿拉伯糖链的存在。 XDP-阿拉伯糖焦磷酸化酶。 这些酶将被纯化， 用各种可能的抑制剂进行表征和测试， 有用的化疗剂。