Selective Inhibitors of Human HSF2

描述(申请人提供)：最近的科学发展表明，分子伴侣系统积极参与癌症发生、神经变性和心血管疾病。热休克因子(HSF)是转录水平上分子伴侣蛋白表达的主要调节因子。这项研究的具体目的是建立和实施人类HSF2感染者的筛查系统。HSF的抑制剂(在癌症的情况下)或激活剂(在神经退行性疾病和心血管疾病的情况下)可以被用作未来药理学中重要的药物先导。筛选系统的设计是基于利用我们实验室创造的一种新的酵母菌株，在该菌株中，人HSF2在Gal1启动子下的条件过表达产生了缓慢生长的表型。提高测试菌株生长速度的潜在化合物将得到验证，并用作新药开发的先导。通过在含有半乳糖作为主要碳源营养的生长介质中加入葡萄糖，可以很容易地调整测试菌株的初始生长速度，以适应不同严格程度的筛选。细胞生长恢复的检测将通过记录细胞培养的光吸收的变化来完成，此外，使用商业上可买到的BacTiter-Glo微生物细胞活力分析(Promega)来记录发光。将在初始筛选周围的一系列浓度范围内对复合HITS进行评估，并确定IC50。HITS还将通过在非热休克和热休克条件下使用实时定量RT-PCR测量一组报告热休克基因的信使核糖核酸水平的变化来进行验证，并将其与来自未处理细胞的类似样本进行比较。这种方法的优点是可以识别和确认hHSF2在体内的真实影响因素。

DESCRIPTION (provided by applicant): Recent scientific developments indicate that the molecular chaperone system is actively involved in cancerogenesis, neurodegeneration and cardiovascular disorders. The master regulator of molecular chaperone expression at the level of transcription is the heat shock factor (HSF). The specific aim of the proposed research is to setup and implement the screening system for affecters of human HSF2. Inhibitors (in case of cancer) or activators (in case of neurodegenerative and cardiovascular disorders) of HSF can be used as important future drug leads in pharmacology. The screening system design is based on the utilization of a new yeast strain created by our laboratory, in which the conditional overexpression of human HSF2 under Gal1 promoter creates a slow growth phenotype. Potential compounds increasing the growth rate of the tester strain will be verified and used as leads for new drug development. The initial growth rate of the tester strain will be easily adjusted for screens of different stringencies by the incorporation of glucose in the growth media containing galactose as a major carbon source nutrient. The detection of cell growth restoration will be done by registering a change in the light absorption of the cell culture and, in addition, y registering luminescence using the commercially available BacTiter-Glo" Microbial Cell Viability Assay (Promega). Compound hits will be evaluated over a range of concentrations around the initial screening and the IC50 will be determined. Hits will be verified additionally by measuring change in the mRNA level of a set of reporter heat shock genes using real-time quantitative RT-PCR for non-heat shock and heat shock conditions in the presence of the identified compound and compared to similar samples from untreated cells. The advantage of this approach is that the true in vivo affecters of hHSF2 will be identified and confirmed.