Identification of small molecule activators of procaspase-7 to caspase-7

描述（由申请人提供）：癌症的一个标志是逃避程序性细胞死亡或凋亡的能力;这允许不受控制的增殖和肿瘤生长。我们的目标是使用一种小分子通过靶向细胞凋亡途径中的关键蛋白质来激活并触发癌细胞的细胞凋亡：刽子手前半胱氨酸蛋白酶。我们希望开发一种个性化癌症治疗策略，其中具有升高水平的前半胱氨酸蛋白酶的癌症将用专门的小分子治疗。已知在某些癌症如前列腺癌中存在升高水平的半胱天冬酶原-7。我们假设，细胞凋亡可以选择性地诱导在癌细胞中含有高水平的procaspase-7通过一个直接的小分子活化剂的procaspase- 7到caspase-7。这一假设得到了我们小组最近研究的支持，在我们的研究中，我们能够通过小分子活化半胱天冬酶-3至caspase-3来诱导癌细胞凋亡。 具体目标1。活化半胱天冬酶-7至半胱天冬酶-7的小分子的鉴定。在该具体目标中，我们打算A）进行时程分析以阐明半胱氨酸天冬氨酸蛋白酶原-7如何随时间活化，B）从文库筛选中鉴定半胱氨酸天冬氨酸蛋白酶原-7的小分子活化剂，和C）验证在文库筛选中发现的半胱氨酸天冬氨酸蛋白酶原-7活化化合物。 具体目标2。使用经鉴定的半胱天冬酶原-7的小分子激活剂诱导凋亡性死亡。在细胞凋亡过程中，细胞显示出多种特征性性状。我们将使用各种技术来监测由特异性目的1中鉴定的小分子诱导的HL 60和MCF-7细胞中的凋亡标志。技术包括流式细胞术以检测凋亡细胞膜上的磷脂酰丝氨酸暴露，Hoescht染色以检测凋亡细胞中的染色质凝聚，用泛半胱天冬酶抑制剂拯救凋亡性死亡， 具体目标3。鉴定其中Executioner procaspase水平升高的特定癌症。Hergenrother实验室可获得各种石蜡包埋的原发癌组织。使用蛋白质印迹和免疫组织化学，我们可以确定在前列腺癌和乳腺癌组织中的刽子手procaspase-3，-6和-7水平。

DESCRIPTION (provided by applicant): One hallmark of cancer is the ability to evade programmed cell death, or apoptosis; this allows uncontrolled proliferation and tumor growth. Our goal is to use a small molecule to activate to trigger apoptosis in cancer cells by targeting key proteins in the apoptotic pathway: executioner procaspases. We hope to develop a strategy for personalized cancer treatment in which cancers with elevated levels of procaspases will be treated with specialized small molecules. It is known that there are elevated levels of procaspase-7 in certain cancers, such as prostate cancer. We hypothesize that apoptosis can be selectively induced in cancer cells containing elevated levels of procaspase-7 via a direct small molecule activator of procaspase- 7 to caspase-7. This hypothesis is supported by recent research in our group in which we were able to induce apoptosis in cancer cells by small molecule activation of procaspase-3 to caspase-3. Specific Aim 1. Identification of small molecules which activate procaspase-7 to caspase-7. In this specific aim we intend to A) perform timecourse analyses to elucidate how procaspase-7 activates over time, B)identify a small molecule activator of procaspase-7 from a library screen, and C) validate procaspase-7-activating compounds found in the library screen. Specific Aim 2. Induction of apoptotic death using the identified small molecule activator of procaspase-7. During apoptosis, cells display a variety of characteristic traits. We will use various techniques to monitor the apoptotic hallmarks in HL60 and MCF-7 cells induced by the identified small molecule in Specific Aim 1. Techniques include Flow cytometry to detect phosphatidyl serine exposure on apoptotic cell membranes, Hoescht staining to detect chromatin condensation in apoptotic cells, Rescue from apoptotic death with a pan-caspase inhibitor Specific Aim 3. Identification of specific cancers in which executioner procaspase levels are elevated. A variety of paraffin-embedded primary cancer tissues are available to the Hergenrother laboratory. Using Western blotting and immunohistochemistry, we can determine executioner procaspase-3, -6, and -7 levels in prostate and breast cancer tissues.