Identification of novel system xc- inhibitors

描述（由申请人提供）：本提案的总体目标是开发一种96孔板筛选试验，该试验将允许筛选胱氨酸-谷氨酸反向转运蛋白（也称为系统XC-）的小分子抑制剂。XC-系统是一个Na+非依赖性氨基酸转运蛋白，它将细胞内L-谷氨酸的输出与细胞外L-谷氨酸的输入耦合起来， 胱氨酸。胱氨酸转运对谷胱甘肽合成至关重要，谷胱甘肽是一种细胞内抗氧化剂，可保护细胞免受活性氧的侵害。谷胱甘肽水平降低导致细胞生长停滞，这使得系统XC成为癌症治疗的目标，包括脑癌。在脑癌中，通过反向转运蛋白释放谷氨酸引起了额外的担忧，因为高水平的细胞外谷氨酸导致肿瘤周围神经元细胞死亡，通过清除周围组织空间帮助肿瘤扩张。除了脑癌之外，通过系统XC-的过量谷氨酸释放也已显示在几种神经变性疾病中起重要作用，其中过量谷氨酸被认为是致病性的，包括帕金森病、阿尔茨海默病、多发性硬化症和癫痫，这使得系统XC-成为神经变性疾病的广泛治疗靶标。目前还没有公众可获得的选择性和有效的小分子系统Xc-抑制剂，并且据我们所知，还没有对该靶标进行高通量筛选。因此，我们建议开发一种HTS就绪的96孔测定法来筛选抑制系统XC活性的新型化学实体。我们将通过筛选由临床批准的药物以及非临床化合物组成的四个化学库来验证该测定。命中将在二次筛选测定中确认，并且活性新化合物将被转移到由Xc-效力和代谢、毒性和靶标选择性的体外药物能力测定指导的结构活性关系（SAR）研究中。作为未来的目标，将在神经胶质瘤和多发性硬化症的动物模型中测试这些研究中产生的先导化合物，并且将验证的96孔测定进一步小型化为384孔格式，用于NIH>300 K化合物库的高通量筛选。 公共卫生相关性：系统XC-在多种癌症和神经退行性疾病中上调，并且其通过原型小分子或遗传缺失的抑制抑制体内肿瘤生长并在神经退行性疾病的动物模型中提供保护。没有公众可获得的选择性和有效的小分子系统XC-抑制剂调节剂，并且据我们所知，还没有对该靶标进行高通量筛选（HTS）。因此，我们建议开发一种HTS就绪的96孔测定法，筛选多个化学文库，并将命中物推进到由Xc效力和代谢、毒性和靶标选择性的体外药物能力测定指导的结构活性关系（SAR）研究中。

DESCRIPTION (provided by applicant): The overall aim of this proposal is to develop a 96-well plate screening assay that will allow screening for small molecule inhibitors of the cystine-glutamate antiporter, also known as system Xc-. System Xc- is a Na+-independent amino acid transporter that couples the export of intracellular L-glutamate with the import of extracellular L cystine. Cystine transport is critical for glutathione synthesis, an intracellular antioxidant whic protects cells from reactive oxygen species. Decreased glutathione levels lead to cellular growth arrest which has made system Xc- a target in cancer therapy, including brain cancer. In brain cancers, the release of glutamate via the antiporter raises additional concerns, as high levels of extracellular glutamate leads to peritumoral neuronal cell death aiding in tumor expansion by clearing surrounding tissue space. In addition to brain cancer, excessive glutamate release via system Xc- has also been shown to play a significant role in several neurodegenerative diseases wherein excess glutamate is presumed pathogenic, including Parkinson's disease, Alzheimer's disease, Multiple Sclerosis and epilepsy, which makes system Xc- a broad therapeutic target for neurodegenerative disorders. There are no selective and potent small molecule system Xc- inhibitors available to the public, and to our knowledge no high throughput screening for this target has been performed. As such, we propose to develop an HTS-ready 96-well assay to screen for novel chemical entities that inhibit system Xc- activity. We will validate this assay by screening four chemical libraries composed of clinically approved drugs as well as non-clinical compounds. The hits will be confirmed in a secondary screening assay and active novel compounds will be moved into structure activity relationship (SAR) studies guided by Xc- potency and in vitro drug- ability assays of metabolism, toxicity and target selectivity. As a future goal, lead compounds arising from these studies will be tested in animal models of glioma and multiple sclerosis and the validated 96-well assay will be further miniaturized to a 384-well format for high throughput screening of the NIH's >300 K compound library. PUBLIC HEALTH RELEVANCE: System Xc- is up-regulated in a variety of cancers and neurodegenerative diseases, and its inhibition with prototype small molecules or genetic deletion inhibits tumor growth in vivo and provides protection in animal models of neurodegeneration. There are no selective and potent small molecule system Xc- inhibitors modulators available to the public, and to our knowledge no high throughput screening (HTS) for this target has been performed. As such, we propose to develop an HTS-ready 96-well assay, screen multiple chemical libraries, and advance hits into a structure activity relationship (SAR) studies guided by Xc- potency and in vitro drug-ability assays of metabolism, toxicity and target selectivity.