Identification of chemotherapeutic sensitizers

化疗增敏剂的鉴定

• 批准号: 8750708
• 负责人: Kyungjae Myung
• 金额: $ 50.17万
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8750708
• 关键词: Adverse effects; Antineoplastic Agents; Antioxidants; Biochemical; Bioinformatics; Biological Assay; Biological Markers; Categories; Cell Death; Cell Line; Cells; Chemicals; Chimeric Proteins; Collaborations; Collection; DNA Damage; DNA Repair Pathway; DNA repair protein; Dose; Exhibits; Future; Gene Targeting; Genes; Genistein; Genomics; Human Cell Line; Inhibitory Concentration 50; Laboratories; Lead; Libraries; Luciferases; Malignant Neoplasms; Mismatch Repair; Molecular; Molecular Bank; Mus; Mutagenesis; National Institute of Environmental Health Sciences; National Toxicology Program; Pathway interactions; Pharmaceutical Preparations; Phosphotransferases; Production; Proliferating; Proteins; Proteolysis; Radiation; Radiation therapy; Reporting; Resveratrol; Signal Transduction; Small Interfering RNA; Specificity; Testing; Translational Research; Tumor Burden; United States National Institutes of Health; Xenograft procedure; baicalein; base; cancer cell; cell killing; chemotherapeutic agent; chemotherapy; compound 30; fighting; genome-wide; high throughput screening; improved; in vivo; killings; knockout gene; mouse model; novel; response; screening; small molecule; small molecule libraries; tool; tumor

Chemotherapeutic and radiation treatments cause a variety of genotoxic insults that lead to cell death in rapidly proliferating cancer cells. To survive genotoxic insults, cancer cells depend on multiple DNA repair pathways. Depending on the types of genotoxic insult, cells use a specific DNA repair pathway. When a DNA repair pathway is compromised, cancer cells become more sensitive to certain genotoxic insults. The identification of chemotherapeutic agents acting on compromised DNA repair pathways in cancer cells would result in more efficient treatment of cancer cells. Such agents are potential sensitizers for radiation therapy. We found that ATAD5 protein is stabilized in response to almost all genotoxic insults. Thus, we hypothesized that ATAD5 would be a good biomarker to detect genotoxic insults. We generated a cell line expressing the ATAD5-luciferase fusion protein and showed that the fusion protein is also stabilized in response to genotoxic insults. Previously, we used this novel cell-based quantitative high-throughput ATAD5-luciferase assay and successfully screened over 4,000 compounds from commercially available compound library as well as National Toxicology Program (NTP) library in collaboration with the NIH Chemical Genomics Center (NCGC) that is now part of National Center for Advancing Translational Science (NCATS) and National Institute of Environmental Health Sciences (NIEHS). In this pilot screen, we reported 22 antioxidants, including resveratrol, genistein, and baicalein induced DNA damage and resulted in cell death. Despite their genotoxic effects, resveratrol, genistein, and baicalein did not cause mutagenesis, which is a major side effect of conventional anti-cancer drugs. We therefore propose that resveratrol, genistein, and baicalein are attractive candidates for improved chemotherapeutic agents. Furthermore, we identified 200 compounds that stabilized ATAD5-luciferase in a dose dependent-manner from 300,000 compounds in the NIH chemical library in collaboration with the NCGC. To identify DNA repair pathways targeted by the genotoxic compounds, we used 8 isogenic human cell lines with targeted gene knockouts in specific DNA repair pathways. Approximately 200 compounds were tested in survival assays on these cells and group into sub-categories based on their IC50 to kill these cells. We found a small molecule that killed a mismatch repair deficient cancer cells and two small molecules that killed parp1 deficient cancer cells more efficiently. We are investigating whether these compounds can reduce tumor burden in vivo using xenograft mice as well as gene targeted mice models. Each compound will become a good tool to dissect molecular functions of different DNA repair pathways. In collaboration with NCATS, we also used the same ATAD5-luciferase cell line to identify compounds and siRNAs that inhibit the ATAD5 stabilization in response to genotoxic insults and have identified >80 compounds and >30 siRNAs. Genes identified from these siRNA screens will unveil the unknown mechanisms that inhibit proteolysis of DNA repair proteins in response to genotoxic insults. Two compounds from initial hits could be potential radiation and chemotherapeutic sensitizers in tumors. We found one compound inhibits general DNA damage response by destabilizing DNA damage response kinase(s). We are currently studying to identify targets of these compounds among genes identified from siRNA screening using bioinformatic analysis, epistatic analysis, as well as biochemical interactions.

化学治疗和辐射处理引起多种遗传毒性损伤，导致迅速增殖的癌细胞中细胞死亡。为了生存遗传毒性损伤，癌细胞依赖于多个DNA修复途径。根据遗传毒性损伤的类型，细胞使用特定的DNA修复途径。当DNA修复途径受到损害时，癌细胞对某些遗传毒性损伤变得更加敏感。鉴定作用于癌细胞中DNA修复途径的化学治疗剂将导致对癌细胞的更有效治疗。这样的药物是对放射治疗的潜在敏感性。我们发现ATAD5蛋白几乎响应所有遗传毒性损伤而稳定。因此，我们假设ATAD5将是检测遗传毒性侮辱的好生物标志物。我们产生了表达ATAD5-荧光素酶融合蛋白的细胞系，并表明融合蛋白还响应于遗传毒性损伤而稳定。 Previously, we used this novel cell-based quantitative high-throughput ATAD5-luciferase assay and successfully screened over 4,000 compounds from commercially available compound library as well as National Toxicology Program (NTP) library in collaboration with the NIH Chemical Genomics Center (NCGC) that is now part of National Center for Advancing Translational Science (NCATS) and National Institute of Environmental Health Sciences (NIEHS).在此试验筛选中，我们报道了222种抗氧化剂，包括白藜芦醇，染料木黄酮和黄胶诱导DNA损伤，并导致细胞死亡。 尽管它们具有遗传毒性作用，但白藜芦醇，染料木黄酮和黄氨酸并未引起诱变，这是常规抗癌药物的主要副作用。因此，我们建议白藜芦醇，染料木黄酮和黄铜素是改善化学治疗剂的有吸引力的候选者。 此外，我们确定了200种化合物，这些化合物稳定了与NCGC合作的NIH化学文库中300,000种化合物的剂量依赖性曼纳中的ATAD5-荧光素酶。为了鉴定遗传毒性化合物靶向的DNA修复途径，我们在特定的DNA修复途径中使用了8个具有靶向基因敲除的等源性人类细胞系。在这些细胞上的生存测定中测试了大约200种化合物，并根据其IC50杀死这些细胞的子类别。我们发现了一个小分子，该分子杀死了不匹配的维修缺乏癌细胞和两个小分子，可更有效地杀死PARP1缺乏癌细胞。我们正在研究这些化合物是否可以使用异种移植小鼠以及基因靶向小鼠模型减轻体内肿瘤负担。每种化合物将成为剖析不同DNA修复途径的分子功能的好工具。 通过与NCAT合作，我们还使用了相同的ATAD5-荧光素酶细胞系来识别抑制ATAD5稳定化的化合物和siRNA，以响应遗传毒性损伤，并鉴定出> 80种化合物和> 30 siRNA。从这些siRNA筛选中鉴定出的基因将揭示出对遗传毒性损伤抑制DNA修复蛋白蛋白质水解的未知机制。初始命中的两种化合物可能是肿瘤中潜在的辐射和化学治疗敏化剂。 我们发现一种化合物通过破坏DNA损伤反应激酶（S）的稳定而抑制了一般的DNA损伤反应。我们目前正在研究使用生物信息学分析，上皮分析以及生化相互作用从siRNA筛选中鉴定出的基因中这些化合物的靶标。