ATM KINASE AS A NOVEL TARGET FOR RADIOSENSITIZING AGENTS

由未复制或受损DNA引起的细胞周期检查点激活触发了转导级联，其协调了多种细胞反应，包括细胞周期停滞、DNA修复和凋亡性死亡。磷脂酰肌醇3-激酶相关激酶（PIKK）家族的几个成员，包括共济失调-毛细血管扩张（A-T）综合征，是由ATM基因的两个等位基因的遗传缺陷引起的。基于受A-T影响的个体的极端辐射超敏性，TM催化活性的小分子抑制剂可能用作新型辐射增敏剂。为了支持这一点，我们最近表明，真菌代谢产物渥曼青霉素，抑制ATM激酶活性的浓度，诱导显着的放射增敏。目前研究项目的长期目标是推进ATM激酶抑制剂作为增敏剂用于癌症治疗的临床前开发。在初步的实验中，我们发现，渥曼青霉素治疗前照射的S期同步化细胞导致G2延迟的显着延长。通过比较Wortmannin处理的细胞和A-T患者来源的细胞中G2检查点和相关信号转导通路的缺陷，将进一步了解Wortmannin介导的放射增敏作用的机制。为了证明在临床环境中使用ATM抑制剂的原理证明，将检查渥曼青霉素作为异种移植系统中的放射增敏剂的功效。为了加速鉴定新型ATM抑制剂，将评估一系列ATM截短和缺失突变体的催化活性，以鉴定催化活性蛋白片段。这样的片段然后将用于开发ATM激酶抑制剂的高通量筛选。有效的ATM特异性抑制剂的鉴定可能导致癌症治疗中诺埃尔治疗剂的开发。

The cell cycle checkpoint activation by un-replicated or damaged DNA triggers a transduction cascade that orchestrates a variety of cellular responses including cell-cycle arrest, DNA repair, and apoptotic death. Several members of the phosphatidylinositol 3-kinase related kinase (PIKK) family, including the Ataxia-Telangiectasia (A-T) syndrome is caused by an inherited defect in both alleles of the ATM gene. Based on the extreme radiation hypersensitivity of individuals affected by A-T small-molecule inhibitors of TM catalytic activity may be useful as a novel radiosensitizing agents. In support of this, we have recently shown that the fungal metabolite, wortmannin, inhibits ATM kinase activity at concentrations that induce significant radiosensitization. The long-term goal of the current research project is to advanced the pre-clinical development of ATM kinase inhibitors as sensitizing agents for use in cancer therapy. In preliminary experiments, we found that wortmannin treatment prior to irradiation of S-phase synchronized cells resulted in a significant prolongation of the G2 delay. By comparing the defects in the G2 checkpoint and associated signal transduction pathways in wortmannin treated cells and cells derived from A-T patients, further insight into the mechanism of wortmannin-mediated radiosensitization will be gained. To demonstrate proof-of-principle for the use of ATM inhibitors in the clinical setting, the efficacy of wortmannin as a radiosensitizer in xenograft system will be examined. To accelerate the identification of novel ATM inhibitors, the catalytic activity of a series of ATM truncation and deletion mutants will be assessed in an effort to identify a catalytically active protein fragment. Such a fragment will then be used in the development of high-throughput screen for ATM kinase inhibitors. The identification of potent, specific inhibitors of ATM may lead to the development of noel therapeutic agents in the treatment of cancer.