Screening for Chemicals that Potentiate TRAIL-Induced Apoptosis of Cancer Cells

筛选增强 TRAIL 诱导癌细胞凋亡的化学物质

• 批准号: 7304465
• 负责人: DMITRI ROZANOV
• 金额: $ 2.5万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7304465
• 关键词: Address; Adopted; Adverse effects; Affect; Antineoplastic Agents; Apoptosis; Apoptotic; Automation; Biological Assay; Cancer Patient; Cell Count; Cell Death; Cell Survival; Cells; Cessation of life; Chemicals; Class; Clinical; Conditioned Culture Media; Cytokine Receptors; Cytotoxic Chemotherapy; Cytotoxic agent; Defect; Development; Dimethyl Sulfoxide; Doctor of Philosophy; Drug Design; Drug resistance; Drug usage; Excretory function; Failure; Family; Family member; Future; Generations; Goals; Hepatocyte; Hepatotoxicity; Human; Immune; Immune system; Immunotherapy; In Vitro; Incubated; Individual; Learning; Ligands; Malignant Neoplasms; Mediating; Metabolism; Mitochondria; Molecular Weight; Neoplasm Metastasis; Normal Cell; Numbers; Outcome; Pathway interactions; Patients; Personal Satisfaction; Pharmaceutical Preparations; Phenotype; Physiological; Pichia; Play; Principal Investigator; Proliferating; Promega; Property; Protocols documentation; Purpose; Reagent; Regulation; Relapse; Reproducibility; Research; Resistance; Risk; Role; Safety; Screening procedure; Sequential Treatment; Structure; Techniques; Temperature; Testing; Therapeutic; Therapeutic Agents; Therapeutic Effect; Toxic effect; Tumor Cell Line; Tumor Necrosis Factor-alpha; Tumor Necrosis Factors; Variant; Xenograft Model; Yeasts; absorption; base; cancer cell; cancer therapy; cell suicide; cell type; chemotherapeutic agent; chemotherapy; concept; cost; cytokine; cytotoxic; cytotoxicity; day; design; drug development; fibrosarcoma; human TNF protein; improved; in vivo; irradiation; killings; member; neoplastic cell; novel; programs; research study; small molecule libraries; tool; tumor; tumor xenograft

DESCRIPTION (provided by applicant): The purpose of this R03 application is to describe our proposed drug design effort designed to potentiate TRAIL-induced apoptosis in cancer cells. Apoptosis plays an essential role in the regulation of cell number in many physiological and pathological settings. Apoptosis mediates therapy-induced cytotoxicity in chemotherapy, ?-irradiation and immunotherapy. Many tumors, however, have proved to be drug- and treatment-resistant. TRAIL is a highly promising cancer therapeutic because it can induce apoptosis in a broad spectrum of cancer cell types but not in normal cells. Unfortunately, certain tumor cell lines have already acquired resistance to TRAIL. There are chemotherapeutic agents that augment TRAIL-mediated apoptosis when co-administered with TRAIL. These existing combined therapies, however, have demonstrated unacceptable side-effects when therapy affects both tumor and normal cells. We believe that the most realistic and practical means to employ TRAIL-induced apoptosis in cancer treatment is to identify chemical leads that specifically potentiate TRAIL-mediated anti-tumor cytotoxicity. We fully expect to identify these leads by using HTS techniques to screen comprehensive libraries of chemicals. Hypothesis/Aims. Because it is known that TRAIL induces apoptosis in a broad spectrum of cancer cells but not in normal cells and that available drugs used in combined therapy targeting the apoptotic pathways have serious deleterious side-effects, we hypothesize: (1) that it is of the utmost importance to discover and identify chemicals that can be developed into drugs that specifically augment TRAIL-mediated anti-tumor toxicity, and (2) that the use of HTS techniques within the MLSCN is the most efficient manner to accomplish this task. Our approach is based on the sequential treatment of human fibrosarcoma HT1080 cells with chemical library compounds followed by treatment with the trimeric, yeast-derived, TRAIL-LZ construct with a known low hepatotoxicity. The initial hits that augment TRAIL-induced apoptosis will be validated in an additional assay to select chemicals that are non-toxic to normal human hepatocytes. Our Specific Aims are: (1) To identify low molecular weight chemicals that can efficiently enhance TRAIL-mediated apoptosis in human fibrosarcoma HT1080 cells. (2) To identify, among selected hits, compounds that are non-toxic to normal cells in an additional cell-based assay. (3) To determine which of the hits specifically sensitize tumor cells to TRAIL (and other apoptosis inducing TNF family cytokines) without impacting the sensitivity of cells to other types of apoptosis pathways. The resulting compounds will be specific sensitizers of the "extrinsic" pathway and consequently will serve as valuable research tools for improving our understanding of the mechanisms of TRAIL resistance. In addition, these compounds can provide a starting point for the development of a novel class of less toxic and more powerful anticancer drugs. One liter of Pichia conditioned medium provides us with 5 mg of purified TRAIL-LZ. This amount is sufficient for the analysis of 500,000 individual chemicals. Future Plans include: (1)To identify the putative targets and to determine the efficiency and selectivity of the apoptosis activation mechanism exerted by initial hits in cell-based assays; (2) Optimize the structure of the selected hits to improve their properties such as potency and ADMET (absorption, distribution, metabolism, excretion, and toxicity) attributes; (3) Confirm the selectivity and efficiency of the derivatized compounds to potentiate TRAIL-mediated apoptosis employing multiple cancer cell types; (4) Confirm the efficiency and safety of the derivatized hits in vivo in tumor xenograft models. Assays. We propose to use the cell-based assays employing HT1080 cells (primary screen) and human hepatocytes (secondary screen). The cells will be incubated with chemicals followed by TRAIL-LZ treatment. This assay with a Z'-factor equal to 0.6-0.9 is readily adaptable to automation to fit 384-well or 1536-well plates. We are confident that reproducibility between plates and day-to-day experiments will be greater than 95% and the coefficient of variation (CV) will not exceed 5%. To select compounds non-toxic to normal cells, we will evaluate the toxicity of the identified hits in human primary hepatocytes. The normal human primary hepatocytes, TRAIL-LZ, and the detailed experimental protocol will be provided to the screening center. The main technical parameters of the primary assay are as follows: assay, CellTiter-Glo Luminescent Cell Viability Assay (Promega); assay volume, 0.1 ml; number of cells/well, 50,000; plates, 96-well transparent, flat bottom; temperature, ambient; cost, unless a bulk purchase is arranged, the $2,159.0 costs of the commercially available CellTiter-Glo Cell Viability Assay will be sufficient for the analysis of 10,000 compounds in a 96-well plate format. The cost of all other assay reagents is minimal. Stability of the cells and the substrate: DMSO up to a 1% concentration does not affect the viability of HT1080 target cells. The HT1080 cells, human primary hepatocytes, TRAIL-LZ, and the detailed experimental protocol will be provided to the screening center.

描述（由申请人提供）：本R 03申请的目的是描述我们提出的旨在增强癌细胞中TRAIL诱导的细胞凋亡的药物设计工作。细胞凋亡在许多生理和病理环境中的细胞数量调节中起着重要作用。细胞凋亡介导化疗诱导的细胞毒性，放疗和免疫治疗。然而，许多肿瘤已被证明是耐药和耐治疗的。TRAIL是一种非常有前途的癌症治疗剂，因为它可以在广泛的癌细胞类型中诱导凋亡，而不是在正常细胞中。不幸的是，某些肿瘤细胞系已经获得了对TRAIL的抗性。当与TRAIL共同施用时，存在增强TRAIL介导的细胞凋亡的化疗剂。然而，当治疗影响肿瘤和正常细胞时，这些现有的组合疗法已经证明了不可接受的副作用。我们认为，在癌症治疗中采用TRAIL诱导的细胞凋亡的最现实和实用的方法是鉴定特异性增强TRAIL介导的抗肿瘤细胞毒性的化学先导物。我们完全期望通过使用HTS技术筛选化学品的综合库来识别这些线索。假设/目标。因为已知TRAIL在广谱癌细胞中诱导凋亡，但在正常细胞中不诱导凋亡，并且靶向凋亡途径的联合治疗中使用的可用药物具有严重的有害副作用，所以我们假设：（1）最重要的是发现和鉴定可以开发成特异性增强TRAIL介导的抗肿瘤毒性的药物的化学物质，以及（2）在MLSCN内使用HTS技术是完成该任务的最有效方式。我们的方法是基于顺序处理的人纤维肉瘤HT 1080细胞与化学库化合物，然后用三聚体，酵母衍生的，TRAIL-LZ构建与已知的低肝毒性的治疗。增加TRAIL诱导的细胞凋亡的初始命中将在额外的测定中进行验证，以选择对正常人肝细胞无毒的化学物质。我们的具体目标是：（1）鉴定能够有效增强TRAIL介导的人纤维肉瘤HT 1080细胞凋亡的低分子量化学物质。(2)在选定的命中中，在额外的基于细胞的测定中鉴定对正常细胞无毒的化合物。(3)确定哪些命中物特异性地使肿瘤细胞对TRAIL（和其他凋亡诱导TNF家族细胞因子）敏感，而不影响细胞对其他类型的凋亡途径的敏感性。产生的化合物将成为“外源性”途径的特异性致敏剂，因此将作为有价值的研究工具，用于提高我们对肿瘤坏死因子相关凋亡诱导配体（TRAIL）耐药机制的理解。此外，这些化合物可以为开发一类毒性较小、作用更强的新型抗癌药物提供起点。1升毕赤酵母条件培养基为我们提供了5 mg纯化的TRAIL-LZ。这一数量足以分析50万种化学品。未来计划包括：（1）鉴定推定的靶点并确定在基于细胞的测定中由初始命中所施加的凋亡激活机制的效率和选择性;（2）优化所选命中的结构以改善其性质，例如效力和ADMET。（吸收、分布、代谢、排泄和毒性）属性;（3）确认衍生化合物使用多种癌细胞类型增强TRAIL介导的细胞凋亡的选择性和效率;（4）确认衍生命中物在肿瘤异种移植模型中的体内效率和安全性。试验。我们建议使用基于细胞的试验，采用HT 1080细胞（初筛）和人肝细胞（二次筛选）。将细胞与化学品一起孵育，然后进行TRAIL-LZ处理。Z '因子等于0.6-0.9的该测定法易于适应自动化以适合384孔或1536孔板。我们确信平板和日常实验之间的重现性将大于95%，变异系数（CV）将不超过5%。为了选择对正常细胞无毒的化合物，我们将在人原代肝细胞中评价鉴定的命中物的毒性。将向筛选中心提供正常人原代肝细胞、TRAIL-LZ和详细的实验方案。初步测定的主要技术参数如下：测定，CellTiter-Glo Luminescent Cell Viability Assay（Promega）;测定体积，0.1ml;细胞数/孔，50，000;板，96孔透明，平底;温度，环境温度;成本，除非安排批量购买，否则市售CellTiter-Glo细胞活力测定的2，159.0美元成本将足以分析10，000种化合物在96孔板格式中。所有其他检测试剂的成本都很低。细胞和底物的稳定性：浓度高达1%的DMSO不会影响HT 1080靶细胞的活力。将向筛选中心提供HT 1080细胞、人原代肝细胞、TRAIL-LZ和详细的实验方案。