DNA Repair Inhibition and Cancer Therapy

描述(由申请人提供)：修复DNA损伤的能力对细胞的生存至关重要，无论是正常细胞还是癌症细胞。DNA损伤后，DNA修复基因和其他因子被激活，以消除损伤，或者，如果DNA损伤太大，就会启动细胞程序性死亡。目前的大多数抗癌治疗依赖于它们造成不可修复的DNA损伤，导致细胞凋亡/细胞死亡的能力。虽然化疗往往对快速分裂的癌细胞毒性更大，但它往往缺乏特异性，因此也会影响正常生长的细胞。因此，由于临床医生必须避免超过最大耐受量(MTD)，患者经常会出现使人虚弱的副作用，从而限制治疗的有效性。一个额外的并发症是患者对DNA损伤剂产生抗药性，耐药癌细胞能够修复对其DNA造成的损伤，从而降低治疗效果。癌症治疗中最广泛使用的药物是以铂类化合物为基础的药物，其中最常用的是顺铂。顺铂的细胞毒性机制是DNA加合物的形成，其中相邻两个鸟嘌呤之间的共价1，2链内交联键最丰富。有大量证据表明，核苷酸切除修复(NER)是铂类化合物耐药的重要来源。顺铂-DNA加合物被NER去除，这是已知的从DNA中去除铂-DNA链内加合物的唯一机制。尽管NER机制很复杂(目前已在该途径中发现了20种蛋白质)，但NER机制可以概括为五个基本步骤：识别DNA损伤；切割受损的链和损伤；切除受损的链，产生缺口；合成新的DNA来填补缺口；以及连接最终的缺口。研究表明，抑制NER将为增强顺铂的细胞毒性和克服顺铂耐药性提供一种合理的途径。当这种抑制剂与顺铂联合使用时，应该会产生协同的细胞毒性。我们有兴趣开发这一领域，以提供一种有效、灵敏、可靠但成本效益高的方法，用于筛选大量化合物以确定NER的抑制剂。有几种方法可以测量DNA修复，尽管所有方法都被广泛用于研究DNA修复途径的细节，但精确和特异的分析既繁琐又昂贵，而且很难开发出可靠的药物发现读数。我们有兴趣开发这一领域的高通量筛选，在那里测试大量的化合物。第一阶段将是设计、开发和验证适合于在中高通量(96孔微滴定板)中筛选化合物文库的分析方法(坚固、灵敏、成本效益和安全)。当使用NER抑制、XPG结合和XPG核酸酶活性的检测来筛选化合物集合并鉴定和确认HITS时，将达到第一阶段的特定目标。

DESCRIPTION (provided by applicant): The ability to repair DNA damage is critical to the survival of a cell, whether normal or cancerous. Upon damage, DNA repair genes and other factors are activated either to remove the damage, or, if the DNA damage is too extensive, to initiate programmed cell death. The majority of current anticancer therapies rely on their ability to create unrepairable DNA lesions, leading to apoptosis/cell death. While chemotherapy tends to be more toxic to rapidly dividing cancer cells, it is often insufficiently specific and thus also affects normally growing cells. As a result, patients frequently experience debilitating side effects that can limit the effectiveness of the therapy because the clinician must avoid exceeding the maximum tolerated dose (MTD). An added complication is that patients acquire resistance to the DNA-damaging agent, resistant cancer cells being able to repair the damage inflicted to their DNA and thus reducing the effect of treatment. The most widely used drugs in cancer therapy are those based on platinum compounds of which the most commonly used is Cisplatin. Cisplatin's mechanism of cytotoxicity is the formation of DNA adducts, the covalent 1,2 intrastrand cross-link between two adjacent guanines being the most abundant. There is a large body of evidence that Nucleotide Excision Repair (NER) is an important source of resistance to platinum compounds. Cisplatin-DNA adducts are removed by NER, the only mechanism known by which platinum-DNA intrastrand adducts are removed from DNA. Although complex (>20 proteins have so far been identified in the pathway), NER mechanisms can be summarized by five basic steps: recognition of the DNA lesion; cleavage of the damaged strand and of the lesion; excision of the damaged strand, creating a gap; synthesis of new DNA to fill the gap; and ligation of the final nick. Studies indicate that inhibition of NER would provide a rational approach to enhance cisplatin's cytotoxicity and to overcome cisplatin resistance. Such an inhibitor should produce synergistic cytotoxicities when administered in combination with cisplatin. We are interested in developing this area to provide an effective, sensitive, reliable yet cost-effective assay to use to screen large numbers of compounds to identify an inhibitor of NER. There are several ways to measure DNA repair, and although all have been used extensively to investigate the details of DNA repair pathways, precise and specific assays are cumbersome and expensive, and reliable readouts for Drug Discovery have been difficult to develop. We are interested in developing this area for High Throughput Screening where large numbers of compounds are tested. Phase I will be the design, development and validation of assays suitable (robust, sensitive, cost-effective and safe) for screening a compound library in medium or high throughput (96-well microtitre plates). The specific aims of Phase I will be met when the assays for NER inhibition, XPG binding and XPG nuclease activity are used to screen a compound collection and hits are identified and confirmed.