DRUG-INDUCED ATM READTHROUGH OF PTC MUTATIONS

描述（由申请人提供）：该项目源于我们长期以来的目标，即治疗ATM缺乏症，无论是癌症患者还是患有共济失调-毛细血管扩张症（a-t）的患者，这是一种罕见的儿童神经退行性疾病，目前尚无治疗方法。最近，我们获得了令人兴奋的证据，某些抗生素氨基糖苷，如庆大霉素和遗传素，诱导ATM基因的过早终止密码子（PTC）突变的读取。由此产生的ATM蛋白是功能性的，因为它纠正了A-T细胞的放射敏感性，磷酸化了ATM靶点，并部分恢复了S期检查点，正如辐射抗性DNA合成的测量所证明的那样。有理由相信，在抗生素氨基糖苷的发展过程中，在过去的40年里，许多化合物被开发出来并进行了抗菌活性筛选，但没有进行PTC读通能力的筛选。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可读药物。有希望的化合物将在基于二级细胞的ATM功能分析中进一步评估，使用与患者突变相对应的突变和细胞系。三级测试将研究各种终止密码子的读通效率和每个终止密码子的+4核苷酸的影响。氨基糖苷诱导的读透将与ATM启动子诱导剂联合进行测试，以进一步提高细胞内ATM水平。将产生携带ATM基因PTC突变的小鼠模型，用于选定药物的动物试验。将开发生物标志物来跟踪对氨基糖苷治疗的体内反应。这些努力受到ATM杂合子基本正常生活的基本原理的鼓励，其蛋白质水平低于正常ATM的50%。此外，一组α - t患者的ATM蛋白水平低于正常水平的15%，往往表现出较轻的疾病。这些研究也可能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.