Salvage Transporter as a Target for Drug Discovery

补救转运蛋白作为药物发现的目标

• 批准号: 6575007
• 负责人: Joanne Wang
• 金额: $ 13.53万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-01-17 至 2004-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6575007
• 关键词: Saccharomyces cerevisiae; antimetabolites; cell line; cell transformation; drug discovery /isolation; gene deletion mutation; gene expression; genetic library; genetic screening; hypoxanthines; membrane transport proteins; molecular cloning; neoplastic cell; northern blottings; nucleic acid sequence; nucleobase; phenotype; plasmids; polymerase chain reaction; protein localization; protein protein interaction; protein structure function; transfection /expression vector; transport inhibitor; yeast two hybrid system

DESCRIPTION (provided by applicant) Antimetabolites targeting the key enzymes of purine de novo synthesis are important drugs used in cancer chemotherapy. A major limiting factor in the clinical effectiveness of antimetabolites is tumor nonresponsiveness due to inherent and acquired resistance. Because cancer cells can circumvent the growth-inhibitory effect of antipurine antimetabolites via the salvage of extracellular preformed purines such as hypoxanthine, nucleobase transporter's that mediate cellular uptake of purines represent a promising target for overcoming tumor resistance and improving the clinical efficacy of antipurine antimetabolites. We hypothesized that a combination of nontoxic and high potency inhibitors of nucleobase transporters with antipurine antimetabolites can block both purine de novo and salvage pathways, leading to enhanced therapeutic afficacy and decreased drug resistance. To date, mammalian nucleobase transporters have not been identified at the molecular level and there are no specific inhibitors available for these transporters. To pave the way for utilizing nucleobase transporters for anticancer therapy, it is essential to isolate genes encoding these transporters from cancer cells and identify specific inhibitors for these transporters. Recently, we developed a complementation cloning approach in yeast that allows us to isolate nucleobase transporter genes from other organisms. In studies proposed under Specific Aim 1, we will use this approach to isolate nucleobase transporter genes from two human cancer cell lines where the presence of distinct nucleobase transporters has been demonstrated. Once clone, we will elucidate the functional characteristics of these transporters and determine their expression in normal tissues and neoplastic cells. In studies proposed under Specific Aim 2, we will use yeast as an expression and assay system to identify small molecule inhibitors for these transporters through both small-scale analysis of suspected compounds and large-scale screening of several compound libraries. These studies will provide a mechanistic understanding of nucleobase transport in cancer cells. Furthermore, it will make molecular and chemical tools available for further developing these transporters as a target for anticancer drug discovery.

描述（由申请人提供）针对新生合成的关键酶的反代谢物是癌症化学疗法中使用的重要药物。抗代谢产物临床有效性的主要限制因素是由于固有和获得的耐药性引起的肿瘤无反应性。因为癌细胞可以通过挽救细胞外预形的嘌呤（例如低黄嘌呤）（核碱性转运蛋白）的细胞外预制嘌呤来避免抗磷灰碱抗代谢物的生长抑制作用，从而介导细胞的嘌呤摄取代表了克服肿瘤耐药性的有希望的靶标，以克服肿瘤抗性，并提高抗二耐酸酯反尿素反尿素的临床效率。我们假设，核碱基转运蛋白与抗磷脂抗代谢物的无毒和高效力抑制剂的结合可以阻止从头和挽救途径，从而增强治疗性宣传和降低药物耐药性。迄今为止，尚未在分子水平上鉴定哺乳动物核碱酶转运蛋白，并且这些转运蛋白尚无特定抑制剂。为了为利用核碱酶转运蛋白进行抗癌治疗铺平道路，必须分离从癌细胞中编码这些转运蛋白的基因并确定这些转运蛋白的特定抑制剂。最近，我们在酵母中开发了一种互补克隆方法，使我们能够将核碱基转运蛋白基因与其他生物分离。在特定目标1下提出的研究中，我们将使用这种方法将核碱基转运蛋白基因与两个人类癌细胞系分离，在这些人类癌细胞系中已经证明了不同的核碱酶转运蛋白。克隆后，我们将阐明这些转运蛋白的功能特征，并确定它们在正常组织和肿瘤细胞中的表达。在特定目标2下提出的研究中，我们将使用酵母作为一种表达和测定系统，通过对可疑化合物的小规模分析和对几个化合物文库的大规模筛选进行小规模分析来鉴定这些转运蛋白的小分子抑制剂。这些研究将提供对癌细胞中核酶转运的机械理解。此外，它将使分子和化学工具可用于进一步开发这些转运蛋白作为抗癌药物发现的目标。