Salvage Transporter as a Target for Drug Discovery

说明（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。抗代谢药物临床有效性的一个主要限制因素是由于固有和获得性耐药导致的肿瘤无反应性。因为癌细胞可以通过补救细胞外预先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，所以介导嘌呤的细胞摄取的核碱基转运蛋白代表了克服肿瘤抗性和提高抗嘌呤抗代谢物的临床功效的有希望的靶标。我们假设，无毒和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和补救途径，从而提高治疗效果和降低耐药性。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，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，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至关重要的。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核碱基转运蛋白基因。在特定目标1下提出的研究中，我们将使用这种方法从两种人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性，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表达和测定系统，通过对可疑化合物的小规模分析和对几种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。这些研究将提供癌细胞中核碱基转运的机制理解。此外，它还将提供分子和化学工具来进一步开发这些转运蛋白，作为抗癌药物发现的目标。

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.