Targeting the TLS DNA Damage Tolerance Pathway for Cancer Therapy

描述(由申请人提供)：顺铂(顺铂，CDDP)等铂类药物是用于治疗许多癌症的重要遗传毒性(DNA损伤)药物。不幸的是，用铂类药物成功治疗癌症有很大的局限性，包括出现耐药肿瘤细胞和毒副作用。为了充分发挥铂类药物的治疗潜力，设计更好的靶向顺铂耐药癌细胞和减少顺铂毒物的方法至关重要。跨损伤合成(TLS)是癌细胞获得对铂类药物DNA损伤耐受性的主要机制。因此，该项目的长期目标是抑制癌症患者的TLS，从而 改进我们对抵抗传统化疗的肿瘤的治疗。这项应用的目的是开发小分子，以抑制TLS蛋白DNA聚合酶ETA(POLη)和增殖细胞核抗原之间的相互作用，这对DNA损伤耐受至关重要。中心假设是，治疗性抑制POLη-增殖细胞核抗原的相互作用将使癌细胞对铂类药物的杀伤敏感，并降低顺铂的治疗剂量。其基本原理是，由TLS抑制剂和铂类药物组成的联合疗法将为改善大剂量顺铂治疗的化疗耐药性和毒副作用提供一种强有力的策略。基于强有力的初步研究，我们的假设将使用三个特定目标(SA)进行验证：(1)开发并验证POLη-增殖细胞核抗原相互作用抑制剂的高通量筛选(HTS)。(2)建立和验证特异性抑制Pol-η-增殖细胞核抗原相互作用的二级检测方法。(3)研究活性化合物对癌细胞顺铂敏感性的影响。我们已经成功地在体外重建了POL、η和增殖细胞核抗原的相互作用，并将在SA1中优化和适应高温超导。我们将使用商业上可用的小(1280)药理活性化合物库和从北卡罗来纳大学多样性库中随机选择的5000种化合物进行中试筛选。在SA2中，通过试点筛选确定的候选抑制剂将在效力曲线中得到确认和重新测试，然后接受一组独立的二级测试，以消除假阳性。在SA3中，我们将确定我们确定的抑制剂对一组人类癌细胞株和原代人类细胞的活性和顺铂耐受性的影响。在完成拟议的研究并验证我们的分析后，我们将准备使用大型(&gt；200,000)多样性文库进行HTS。这项拟议的工作将针对POLη和单一泛素化形式的增殖细胞核抗原之间的受调控的相互作用。因此，识别抑制单一泛素化蛋白相互作用的化合物将为涉及泛素信号转导的治疗策略提供新的范例，并无疑将促进未来的药物开发工作。因此，提出的战略具有很强的创新性。这项拟议的研究意义重大，因为它旨在减少美国因癌症而导致的死亡率问题。

DESCRIPTION (provided by applicant): Platinating agents such as cis-platinum (cisplatin, CDDP) are important genotoxic (DNA-damaging) drugs used for the treatment of many cancers. Unfortunately, there are major limitations to the successful treatment of cancer with platinating agents including the emergence of drug-resistant tumor cells and toxic side effects. It is crucial to devise better means of targeting cisplatin-resistant cancer cells and reducing cisplatin toxicit in order to attain the full therapeutic potential of platinum drugs. Trans-Lesion Synthesis (TLS) i a major mechanism by which cancer cells acquire tolerance to DNA damage from platinating agents. Therefore, the long-term goal of this project is to inhibit TLS in cancer patients, thereby improving our treatment of tumors that resist conventional chemotherapies. The objective in this application is to develop small molecules that inhibit an interaction between the TLS proteins DNA Polymerase eta (Polη) and PCNA that is crucial for DNA damage tolerance. The central hypothesis is that therapeutic inhibition of Polη-PCNA interactions will sensitize cancer cells to killing by platinating agents and lower the therapeutic doses of cisplatin. The rationale is that combination therapies comprising TLS inhibitors and platinating agents will provide a powerful strategy for ameliorating chemoresistance and toxic side effects of high-dose cisplatin therapy. Based on strong preliminary studies, our hypothesis will be tested using three Specific Aims (SAs): (1) Develop and Validate a High-Throughput Screen (HTS) for inhibitors of the Polη-PCNA interaction. (2) Develop and Validate Secondary Assays for specific inhibition of the Polη-PCNA interaction. (3) Characterize the effects of active compounds on cisplatin-sensitivity of cancer cell lines. We have successfully reconstituted the Polη-PCNA interaction in vitro and in SA1 this biochemical assay will be optimized and adapted for HTS. We will perform a pilot screen using a commercially-available small (1280) Library of Pharmacologically Active Compounds and 5000 randomly-selected compounds from the UNC diversity library. In SA2 candidate inhibitors identified by the pilot screen will be confirmed and retested in potency curves, then subject to a panel of independent secondary assays to eliminate false positives. In SA3, we will determine the effects of inhibitors we identify on viability and CDDP-tolerance of a panel of human cancer cell lines and primary human cells. Following completion of the proposed studies and validation of our assays we will be poised to conduct a HTS using large (>200,000) diversity libraries. The proposed work will target a regulated interaction between Polη and the mono-ubiquitinated form of PCNA. Thus, identification of compounds that inhibit interactions involving mono- ubiquitinated proteins would provide a new paradigm for therapeutic strategies involving ubiquitin signaling and would undoubtedly facilitate future drug discovery efforts. Therefore the proposed strategy is highly innovative. The proposed research is significant because it aims to reduce the problem of mortality due to cancer in the United States.