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治疗ATM缺陷的目标，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，我们已经获得了令人兴奋的证据，某些抗生素氨基糖苷类，如庆大霉素和遗传霉素，诱导通读提前终止密码子（PTC）突变的ATM基因。所得到的ATM蛋白是功能性的，因为它校正A-T细胞的辐射敏感性，磷酸化ATM靶，并部分恢复S期检查点，如通过测量抗辐射DNA合成所证明的。有理由相信，在抗生素氨基糖苷类的开发过程中，在过去的40年里，开发和筛选了许多化合物的抗微生物活性，但没有PTC通读能力。将开发高通量筛选，使用基于蛋白质截短测试的测定，以筛选新的通读药物。然后，将使用与患者突变相对应的突变和细胞系，在基于二级细胞的ATM功能测定中进一步评估有前景的化合物。三级测试将研究各种终止密码子的通读效率和每个终止密码子的+4核苷酸的影响。氨基糖苷类诱导的通读将与ATM启动子诱导剂联合检测，以进一步提高细胞内ATM水平。将生成在ATM基因中携带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，用于跟踪对氨基糖苷类药物治疗的体内反应。ATM杂合子基本上过着正常的生活，其ATM蛋白水平低于正常的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患者的子集倾向于表现出不太严重的疾病。这些研究也可能影响其他遗传疾病，以及ATM蛋白水平低的癌症患者。

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.