Base Excision DNA Repair in Disease Susceptibility and Treatment

疾病易感性和治疗中的碱基切除 DNA 修复

• 批准号: 8552461
• 负责人: David Wilson
• 金额: $ 42.27万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8552461
• 关键词: Affect; Affinity; Amino Acid Sequence; Amino Acids; Animal Model; Antimetabolites; Antineoplastic Agents; Area; Base Excision Repairs; Biochemical; Biological Assay; Cancerous; Cell Death; Cells; Clinical; DNA; DNA Damage; DNA Ligases; DNA Repair; DNA Repair Gene; DNA Repair Pathway; DNA glycosylase; DNA lesion; DNA-(apurinic or apyrimidinic site) lyase; DNA-Directed DNA Polymerase; Data; Defect; Development; Disease; Disease Progression; Disease susceptibility; Excision; Exhibits; Fluorouracil; Gender; Genes; Genetic Polymorphism; Goals; Human; Immunologic Deficiency Syndromes; Individual; Investigation; Laboratories; Lesion; Ligation; Link; Lyase; Malignant Neoplasms; Methods; Modification; Neurodegenerative Disorders; Normal Cell; Nucleotides; Pathologic Mutagenesis; Pathway interactions; Pharmaceutical Preparations; Population; Predisposition; Procedures; Proteins; Research; Research Personnel; Role; Sampling; Site; Surgical incisions; Therapeutic; Treatment Efficacy; Variant; Vertebral column; Work; age related; base; cancer cell; cancer therapy; cohort; coping; cytotoxic; design; disorder risk; endonuclease; high throughput screening; human APEX1 protein; improved; inhibitor/antagonist; interest; miniaturize; neoplastic; novel; phosphodiester; prevent; protein function; repaired; seal; small molecule; stem; temozolomide; tumor

Studies indicate that significant inter-individual variation exists at the amino acid sequence level of BER proteins, and that variability exists in the BER capacity within the population. We have initiated efforts to delineate the impact of amino acid variants found in BER proteins and to develop assays to determine the extent of inter-individual variation in specific steps of BER. For example, we are currently investigating whether variants found in the major human abasic endonuclease (APE1), either within the normal population or that are tumor-associated, affect protein function and may therefore contribute to disease formation and/or progression. In addition, using established biochemical assays, we have begun to evaluate whether inter-individual variation in BER associates with disease susceptibility and/or clinical agent responsiveness; we are also assessing for age-dependent or gender-specific variation. Current biochemical methods have allowed us to interrogate the central steps of BER. Our results indicate that for AP site incision, the twenty-three individuals examined thus far exhibit an 1.9-fold inter-individual variation in repair capacity. For gap-filling and nick ligation, we observed an 1.3-fold and 3.4-fold inter-individual variation, respectively. The greater disparity in nick ligation stems at least in part from the low overall activity and the corresponding limited sensitivity of the assay. Like the AP endonuclease profile, the average repair capacity of each individual for gap-filling and nick sealing generally fell within the experimental variability of the population, suggesting a comparatively similar BER efficiency among this small group of samples. We are presently designing a high throughput BER pathway assay to more robustly evaluate the relationship of repair capacity with disease susceptibility. Current strategies to eradicate cancer cells typically employ agents that generate DNA lesions that induce cell death by blocking replication of rapidly dividing cells. Thus, a goal has been to regulate strategically the repair capacity of cancer and/or normal cells to improve the efficacy of specific therapeutic paradigms. In particular, inhibiting the DNA repair capacity of cancerous cells has been an area of promising interest. Our recent results indicate that APE1, and BER more generally, is a reasonable target for inactivation in anti-cancer treatment paradigms involving select alkylating drugs (e.g., temozolomide) and antimetabolites (e.g., 5-fluorouracil). We have recently described the development of a panel of complementary and improved miniaturized high-throughput screening and profiling assays, which will have a broad appeal to other research investigators, particularly those in the field of DNA repair. These assays have permitted the identification of novel, small molecule APE1-targeted bioactive inhibitors, which we are working to optimize with the long term goal of creating high affinity, selective inhibitors with therapeutic value. The establishment of small molecule probes will provide a platform for more extensive investigations on the therapeutic benefits of regulating cellular DNA repair capacity.

研究表明，在BER蛋白的氨基酸序列水平上存在显着的个体间变异，并且人群内的BER能力存在变异性。 我们开始努力描述BER蛋白中发现的氨基酸变体的影响，并开发测定，以确定BER特定步骤中个体间变异的程度。 例如，我们目前正在研究在正常种群内或与肿瘤相关的主要人体无碱性核酸内切酶（APE1）中发现的变体，会影响蛋白质功能，因此可能有助于疾病形成和/或进展。 此外，使用既定的生化测定法，我们已经开始评估疾病易感性和/或临床剂反应性的BER伴侣之间的个体差异；我们还评估了依赖年龄或性别特定的变异。 当前的生化方法使我们能够询问BER的中心步骤。 我们的结果表明，对于AP部位切口，迄今为止检查的23个个人表现出1.9倍的维修能力变化。 对于填充缝隙和划痕结扎，我们分别观察到一个个体间变化的1.3倍和3.4倍。 划痕连接的较大差异至少部分是由于总体活性低和相应的测定敏感性有限。 像AP核酸内核酸内切酶的轮廓一样，每个人的平均维修能力和划痕密封通常属于人群的实验变异性，这表明这一小部分样本中的BER效率相对相似。 我们目前正在设计高吞吐量的BER途径测定法，以更加牢固地评估修复能力与疾病易感性的关系。 消除癌细胞的当前策略通常采用通过阻断快速分裂细胞的复制来诱导细胞死亡的DNA病变的剂。 因此，一个目标是从战略上调节癌症和/或正常细胞的修复能力，以提高特定治疗范式的功效。 特别是，抑制癌细胞的DNA修复能力已成为有希望的领域。 我们最近的结果表明，APE1和BER更普遍地是涉及精选烷基化药物（例如，替莫唑胺）和抗替代代谢物（例如5-氟尿嘧啶）的抗癌治疗范式失活的合理靶标。 我们最近描述了一组互补和改进的小型高通量筛查和分析测定法的开发，这将对其他研究研究人员，尤其是DNA修复领域的研究人员具有广泛的吸引力。 这些测定方法允许鉴定出新型的小分子APE1靶向生物活性抑制剂，我们正在努力以创建具有治疗价值的高亲和力，选择性抑制剂的长期目标来进行优化。 小分子探针的建立将为调节细胞DNA修复能力的治疗益处提供更广泛的研究平台。