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） 针对嘌呤从头合成的关键酶的抗代谢物是癌症化疗中使用的重要药物。抗代谢药物临床有效性的一个主要限制因素是由于固有的和获得性的耐药性导致的肿瘤无反应。由于癌细胞可以通过挽救细胞外预先形成的嘌呤（例如次黄嘌呤）来规避抗嘌呤抗代谢药的生长抑制作用，因此介导细胞摄取嘌呤的核碱基转运蛋白代表了克服肿瘤耐药性和提高抗嘌呤抗代谢药临床疗效的有希望的靶标。我们假设无毒且高效的核碱基转运蛋白抑制剂与抗嘌呤抗代谢物的组合可以阻断嘌呤从头途径和补救途径，从而增强治疗功效并降低耐药性。迄今为止，哺乳动物核碱基转运蛋白尚未在分子水平上得到鉴定，并且没有针对这些转运蛋白的特异性抑制剂。为了为利用核碱基转运蛋白进行抗癌治疗铺平道路，有必要从癌细胞中分离编码这些转运蛋白的基因并鉴定这些转运蛋白的特异性抑制剂。最近，我们在酵母中开发了一种互补克隆方法，使我们能够从其他生物体中分离核碱基转运蛋白基因。在具体目标 1 下提出的研究中，我们将使用这种方法从两个人类癌细胞系中分离核碱基转运蛋白基因，其中已证明存在不同的核碱基转运蛋白。一旦克隆，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。在具体目标 2 下提出的研究中，我们将使用酵母作为表达和测定系统，通过对可疑化合物的小规模分析和对多个化合物库的大规模筛选来鉴定这些转运蛋白的小分子抑制剂。这些研究将为癌细胞中核碱基运输提供机制上的理解。此外，它将提供分子和化学工具，用于进一步开发这些转运蛋白作为抗癌药物发现的目标。