Recognition and Repair of Cisplatin-DNA Damage

描述(由申请人提供)：铂(铂)为基础的疗法已被证明是治疗癌症的子集，包括大多数睾丸癌。许多其他类型的癌症，包括卵巢癌和肺癌，对铂类药物有反应，这些药物通常使用顺铂或卡铂。尽管这些癌症的初期反应良好，但肿瘤复发和耐药仍是一个重大且持续的临床问题。这些基于铂的疗法的疗效是形成铂-DNA加合物的功能，而不是通过DNA修复途径移除这些加合物。从基因组中移除顺铂-DNA损伤是由核苷酸切除修复(NER)途径催化的，不利于治疗效果。此外，虽然对铂类药物的耐药性通常是多因素的，但临床耐药性往往包含DNA修复成分。本工作的目的是阐明NER途径识别和修复顺铂-DNA损伤的分子机制，并确定干扰该途径如何影响顺铂的疗效。为实现所述目标，提出了三个具体目标。在目标1中，我们将继续研究NER蛋白对DNA损伤的识别过程。我们将扩大我们的重点，包括损伤DNA结合蛋白(DDB)和TFIIH复合体。在以前赠款期间完成的复制蛋白A(RPA)、XPA和最近的XPC/hHR23B工作的基础上，我们将使用体外方法的新组合来构建NER蛋白识别顺铂-DNA过程的综合结构、动力学和生化模型。在目标2中，我们将使用化学遗传学方法，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，我们将确定干扰DNA损伤识别蛋白如何影响体外DNA复制、修复和重组途径。在第三个也是最后一个目标中，我们将评估这些参与损伤识别过程的蛋白质的抑制剂和扰动如何影响细胞增殖、细胞周期进展，并最终影响顺铂的活性。这种新颖、创新的方法所产生的知识和分子工具可能会影响针对这些途径的治疗的发展，以克服临床对顺铂的耐药性。这项研究的最终目标是将某些癌症中明显基于铂的根治性疗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.