Recognition and Repair of Cisplatin-DNA Damage

描述（由申请人提供）：铂（Pt）为基础的治疗已被证明是一个癌症子集，包括大多数睾丸癌的治愈性治疗。包括卵巢癌和肺癌在内的大量其他癌症类型对基于铂的疗法有反应，这些疗法通常采用顺铂或卡铂。尽管在这些癌症中有良好的初始反应，但肿瘤复发和抗性代表了显著且持续的临床问题。这些基于铂的疗法的功效是Pt-DNA加合物的形成与通过DNA修复途径去除这些加合物的函数。从基因组中去除顺铂-DNA损伤是由核苷酸切除修复（NER）途径催化的，并且对治疗效果有害。此外，虽然对铂类疗法的耐药性通常是多因素的，但临床耐药性通常包含DNA修复成分。这项工作的目标是阐明顺铂-DNA损伤的识别和修复的NER途径的分子机制，并确定如何干扰的途径影响顺铂的疗效。为实现上述目标，提出了三个具体目标。在目标1中，我们将继续研究NER蛋白的DNA损伤识别过程。我们将扩大我们的重点，包括损伤DNA结合蛋白（DDB）和TFIIH复合物。基于在以前的授权期间完成的工作与复制蛋白A（RPA），XPA和最近的XPC/hHR 23 B，我们将使用一种新的体外方法的组合，构建一个全面的结构，动力学和生化模型的顺铂-DNA识别过程的NER蛋白。在目标2中，我们将采用化学遗传学方法并开发NER DNA损伤识别蛋白的小分子抑制剂。使用这些分子工具，我们将确定如何干扰DNA损伤识别蛋白影响体外DNA复制，修复和重组途径。在第三个也是最后一个目标中，我们将评估这些抑制剂和参与损伤识别过程的蛋白质的扰动如何影响细胞增殖，细胞周期进展，并最终影响顺铂活性。这种新颖的创新方法产生的知识和分子工具可能会影响针对这些途径的治疗方法的发展，以克服顺铂的临床耐药性。这项研究的最终目标是将某些癌症中明显的基于铂的治疗方法转化为更广泛的癌症，包括卵巢癌和肺癌。

DESCRIPTION (provided by applicant): Platinum (Pt) based therapies have proven to be curative treatments for a subset of cancers including the majority of testicular cancers. A large number of other cancer types including ovarian and lung, respond to Pt-based therapies which typically employ either cisplatin or carboplatin. Despite good initial responses in these cancers, tumor recurrence and resistance represent a significant and continuing clinical problem. The efficacy of these Pt-based therapies is a function of the formation of Pt-DNA adducts versus the removal of these adducts via DNA repair pathways. Removal of cisplatin-DNA lesions from the genome is catalyzed by the nucleotide excision repair (NER) pathway and is detrimental to treatment efficacy. In addition, while resistance to Pt-based therapies is typically multifactorial, clinical resistance often contains a DNA repair component. The goals of this work are to elucidate the molecular mechanism by which cisplatin-DNA damage is recognized and repaired by the NER pathway and to determine how perturbing the pathway influences cisplatin efficacy. Three Specific Aims are proposed to achieve the stated goals. In Aim 1 we will continue our study of the DNA damage recognition process by NER proteins. We will expand our focus to include the damage DNA binding protein (DDB) and the TFIIH complex. Building on the work accomplished in the previous grant periods with replication protein A (RPA), XPA and more recently XPC/hHR23B, we will use a novel combination of in vitro methodologies to construct a comprehensive structural, kinetic and biochemical model of the cisplatin-DNA recognition process by NER proteins. In Aim 2 we will employ a chemical genetics approach and develop small molecule inhibitors of NER DNA damage recognition proteins. Using these molecular tools we will determine how perturbing DNA damage recognition proteins influence in vitro DNA replication, repair and recombination pathways. In the third and final Aim we will assess how these inhibitors and perturbations of proteins involved in the damage recognition process influence cell proliferation, cell cycle progression, and ultimately cisplatin activity. The knowledge and molecular tools generated by this novel, innovative approach will likely impact the development of therapies targeting these pathways to overcome clinical resistance to cisplatin. The ultimate goal of this research is to translate the curative Pt-based therapies evident in certain cancers, to a wider array of cancers, including ovarian and lung.