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

描述（由申请人提供）：本R03申请的目的是描述我们提议的药物设计工作，旨在增强trail诱导的癌细胞凋亡。细胞凋亡在许多生理和病理环境中对细胞数量的调节起着重要作用。细胞凋亡介导化疗中治疗诱导的细胞毒性-辐照和免疫治疗。然而，许多肿瘤已被证明对药物和治疗具有耐药性。TRAIL是一种非常有前途的癌症治疗药物，因为它可以在广泛的癌细胞类型中诱导细胞凋亡，而不是在正常细胞中。不幸的是，某些肿瘤细胞系已经对TRAIL产生了耐药性。当化疗药物与TRAIL联合使用时，会增加TRAIL介导的细胞凋亡。然而，当治疗同时影响肿瘤细胞和正常细胞时，这些现有的联合疗法已显示出不可接受的副作用。我们认为，在癌症治疗中使用trail诱导的细胞凋亡最现实和实用的方法是识别特异性增强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技术筛选化学物质的综合文库来确定这些线索。假设/目标。由于已知TRAIL在广泛的癌细胞中诱导细胞凋亡，而在正常细胞中不诱导细胞凋亡，并且针对凋亡通路的联合治疗中使用的现有药物具有严重的有害副作用，我们假设：(1)发现和鉴定可以开发成特异性增强trail介导的抗肿瘤毒性药物的化学物质至关重要，(2)在MLSCN中使用HTS技术是完成这一任务的最有效方式。我们的方法是基于化学文库化合物对人纤维肉瘤HT1080细胞的顺序治疗，然后用三聚体、酵母衍生的TRAIL-LZ结构物治疗，已知具有低肝毒性。增加trail诱导的细胞凋亡的初始靶点将在另一项试验中进行验证，以选择对正常人类肝细胞无毒的化学物质。我们的具体目标是：(1)鉴定能够有效促进trail介导的人纤维肉瘤HT1080细胞凋亡的低分子量化学物质。(2)在选定的靶点中，通过额外的基于细胞的检测，鉴定对正常细胞无毒的化合物。(3)确定哪些命中特异性地使肿瘤细胞对TRAIL（以及其他诱导凋亡的TNF家族细胞因子）敏感，而不影响细胞对其他类型凋亡途径的敏感性。由此产生的化合物将是“外在”途径的特异性增敏剂，因此将成为提高我们对TRAIL抗性机制理解的有价值的研究工具。此外，这些化合物可以为开发一类毒性更小、效力更强的新型抗癌药物提供一个起点。一升毕赤酵母条件培养基为我们提供5mg纯化TRAIL-LZ。这个数量足以分析50万种单独的化学物质。未来的计划包括：(1)在基于细胞的实验中确定可能的靶点，并确定初始命中所产生的细胞凋亡激活机制的效率和选择性；(2)优化所选hit的结构，提高其效价和ADMET（吸收、分布、代谢、排泄和毒性）属性；(3)证实衍生化合物对trail介导的多种肿瘤细胞凋亡的选择性和效率；(4)在异种肿瘤移植模型体内验证衍生hit的有效性和安全性。化验。我们建议使用HT1080细胞（初级筛选）和人肝细胞（次级筛选）的基于细胞的检测。细胞将与化学物质孵育，然后进行TRAIL-LZ处理。Z′因子等于0.6-0.9，易于自动化适应384孔或1536孔板。我们有信心，平板和日常实验之间的重复性将大于95%，变异系数（CV）不会超过5%。为了选择对正常细胞无毒的化合物，我们将评估已确定的撞击对人类原代肝细胞的毒性。正常人原代肝细胞、TRAIL-LZ和详细的实验方案将提供给筛选中心。本实验主要技术参数如下：CellTiter-Glo Luminescent Cell Viability assay (Promega)；检测体积：0.1 ml；细胞数/孔，50,000；平板，96孔透明，平底；温度、环境;除非安排批量购买，否则市售CellTiter-Glo细胞活力测定试剂盒的2,159.0美元的成本将足以分析96孔板格式的10,000种化合物。所有其他化验试剂的成本是最低的。细胞和底物的稳定性：高达1%浓度的DMSO不影响HT1080靶细胞的活力。HT1080细胞、人原代肝细胞、TRAIL-LZ及详细的实验方案将提供给筛选中心。

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.