Targeting the TLS DNA Damage Tolerance Pathway for Cancer Therapy

针对癌症治疗的 TLS DNA 损伤耐受途径

• 批准号: 8786895
• 负责人: Cyrus Vaziri
• 金额: $ 28.64万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-01-01 至 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8786895
• 关键词: Adverse effects; Binding; Biochemical; Biological Assay; Bypass; Cancer Patient; Cancer cell line; Carboplatin; Cell Survival; Cell physiology; Cells; Chemicals; Cherry - dietary; Cisplatin; Combination Drug Therapy; Combined Modality Therapy; DNA Damage; DNA Replication Damage; DNA-Directed DNA Polymerase; Diversity Library; Dose; Drug resistance; Drug usage; Future; Genome; Genomic Instability; Goals; Health; Human; In Vitro; Industry; Knowledge; Lead; Lesion; Libraries; Life; Maintenance; Malignant Neoplasms; Mission; Mono-S; Neoplasms; Pathologic Mutagenesis; Pathway interactions; Pharmaceutical Preparations; Platinum; Polymerase; Population; Protein Biosynthesis; Proteins; Public Health; Rad30 protein; Research; Resistance; Role; S Phase; Signal Transduction; Site; Structure; Testing; Therapeutic; Toxic effect; Ubiquitin; United States; Work; analog; base; cancer cell; cancer therapy; chemotherapy; combat; conventional therapy; drug discovery; high throughput screening; human disease; improved; in vitro Assay; inhibitor/antagonist; innovation; killings; liquid chromatography mass spectrometry; mortality; neoplastic cell; novel; reconstitution; small molecule; tumor; validation studies

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.

描述（由申请人提供）：铂类药物如顺铂（顺铂，CDDP）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，用铂剂成功治疗癌症存在主要限制，包括耐药肿瘤细胞的出现和毒副作用。关键是设计出更好的靶向顺铂耐药癌细胞和降低顺铂毒性的方法，以实现铂类药物的全部治疗潜力。跨损伤合成（TLS）是癌细胞获得对铂化剂的DNA损伤的耐受性的主要机制。因此，该项目的长期目标是抑制癌症患者的TLS，从而 改善我们对抵抗常规化疗的肿瘤的治疗。本申请的目的是开发抑制TLS蛋白DNA聚合酶eta（Polη）和PCNA之间相互作用的小分子，这对DNA损伤耐受性至关重要。中心假设是，治疗性抑制Polη-PCNA相互作用将使癌细胞对铂剂的杀伤敏感，并降低顺铂的治疗剂量。基本原理是，包含TLS抑制剂和铂化剂的组合疗法将提供改善高剂量顺铂疗法的化学抗性和毒副作用的有力策略。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）进行测试：（1）开发并建立高通量筛选（HTS），以寻找Polη-PCNA相互作用的抑制剂。(2)开发和验证特异性抑制Polη-PCNA相互作用的二级试验。(3)表征活性化合物对癌细胞系顺铂敏感性的影响。我们已经成功地在体外重建了Polη-PCNA相互作用，并且在SA 1中，该生化测定将被优化并适用于HTS。我们将使用市售的小型（1280）药理学活性化合物库和从生物多样性库中随机选择的5000种化合物进行中试筛选。在SA 2中，将在效价曲线中确认并重新检测通过中试筛选鉴定的候选抑制剂，然后进行一组独立的二次试验以消除假阳性。在SA 3中，我们将确定我们鉴定的抑制剂对一组人类癌细胞系和原代人类细胞的活力和CDDP耐受性的影响。在完成拟议的研究和验证我们的测定后，我们将准备使用大型（> 200，000）多样性文库进行HTS。拟议的工作将针对Polη和PCNA的单泛素化形式之间的调节相互作用。因此，鉴定抑制涉及单泛素化蛋白的相互作用的化合物将为涉及泛素信号传导的治疗策略提供新的范例，并且无疑将促进未来的药物发现努力。因此，提出的战略具有很强的创新性。这项拟议中的研究意义重大，因为它旨在减少美国癌症死亡率的问题。