Salvage Transporter as a Target for Drug Discovery

描述（由申请人提供）靶向嘌呤从头合成关键酶的抗代谢药物是用于癌症化疗的重要药物。抗代谢物临床有效性的一个主要限制因素是肿瘤由于固有和获得性耐药而无反应性。由于癌细胞可以通过挽救细胞外预形成的嘌呤（如次黄嘌呤）来规避抗嘌呤抗代谢物的生长抑制作用，因此介导嘌呤细胞摄取的核碱基转运体是克服肿瘤耐药性和提高抗嘌呤抗代谢物临床疗效的一个有希望的靶点。我们假设核碱基转运蛋白的无毒高效抑制剂与抗嘌呤抗代谢物的组合可以阻断嘌呤新生和挽救途径，从而提高治疗效果并降低耐药性。迄今为止，哺乳动物核碱基转运蛋白尚未在分子水平上被鉴定出来，也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，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是必不可少的。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出核碱基转运体基因。在Specific Aim 1下提出的研究中，我们将使用这种方法从两种人类癌细胞系中分离出核碱基转运蛋白基因，其中已经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Specific Aim 2下提出的研究中，我们将使用酵母作为表达和检测系统，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，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核碱基转运机制的理解。此外，它将为进一步开发这些转运蛋白作为抗癌药物发现的靶点提供分子和化学工具。

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.