DRUG-INDUCED ATM READTHROUGH OF PTC MUTATIONS

描述(申请人提供)：这个项目源于我们治疗ATM缺乏症的长期目标，无论是癌症患者还是患有共济失调-毛细血管扩张(A-T)的患者，A-T是一种罕见的儿童神经退行性疾病，目前还没有治疗方法。最近，我们获得了令人兴奋的证据，某些抗生素氨基糖苷类，如庆大霉素和遗传素，可以诱导ATM基因的提前终止密码子(PTC)突变。合成的ATM蛋白具有功能性，它纠正了A-T细胞的辐射敏感性，使ATM靶点磷酸化，并部分恢复了S时相检查点，如辐射抗性DNA合成的测量所证明的那样。有理由相信，在抗生素氨基糖苷类化合物的发展过程中，在过去的40年里，许多化合物被开发并进行了抗菌活性的筛选，但没有进行PTC阅读能力的筛选。将开发高通量筛选，使用基于蛋白质截断测试的分析来筛选新的直读药物。然后，将使用突变和与患者突变相对应的细胞系，在基于二次细胞的ATM功能测试中进一步评估有希望的化合物。第三次测试将调查各种终止密码子的通读效率以及每个终止密码子的+4核苷酸的影响。将与ATM启动子诱导剂一起测试氨基糖苷诱导的通读，以进一步提高细胞内ATM水平。将产生一个携带ATM基因PTC突变的小鼠模型，用于选定药物的动物测试。生物标志物将被开发用于跟踪氨基糖苷类药物的体内反应。ATM杂合子基本上过着正常的生活，ATM蛋白水平低于正常水平的50%，这一理论鼓励了这些努力。此外，具有15%正常ATM蛋白的A-T患者的子集往往表现出不那么严重的疾病。这些研究可能也会对其他遗传疾病以及ATM蛋白水平低的癌症患者产生影响。

DESCRIPTION (provided by applicant): This project stems from our long-standing goal to treat ATM deficiency, whether this be in cancer patients or in those suffering from ataxia-telangiectasia (A-T), a rare neurodegenerative disorder of children for which no treatment exists. Recently, we have obtained exciting evidence that certain antibiotic aminoglycosides, such as gentamicin and geneticin, induce the readthrough of premature termination codon (PTC) mutations in the ATM gene. The resulting ATM protein is functional, in that it corrects the radiosensitivity of A-T cells, phosphorylates ATM targets, and partially restores the S phase checkpoint, as demonstrated by measurement of radioresistant DNA synthesis. There is reason to believe that during the development of antibiotic aminoglycosides, over the past 40 years, many compounds were developed and screened for antimicrobial activity but not for PTC readthrough ability. High throughput screening will be developed, using assays based on protein truncation testing, to screen for new readthrough drugs. Promising compounds will then be further evaluated in secondary cell-based assays for ATM function, using mutations and cell lines that correspond to patient mutations. Tertiary testing will investigate the readthrough efficiency of various stop codons and the effect of the +4 nucleotide of each termination codon. Aminoglycoside-induced readthrough will be tested in combination with ATM promoter inducers in an effort to further boost intracellular ATM levels. A mouse model carrying a PTC mutation in the ATM gene will be generated for animal testing of selected drugs. Biomarkers will be developed for following in vivo responses to aminoglycoside treatment. These efforts are encouraged by the rationale that ATM heterozygotes live essentially normal lives with less than 50 percent of normal ATM protein levels. Furthermore, a subset of A-T patients with <15 percent of normal ATM protein tends to manifest less severe disease. These studies may impact upon other genetic disorders as well, and upon cancer patients with low levels of ATM protein.