Selective Inhibitors of Human HSF2

描述（由申请人提供）：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。转录水平上分子伴侣表达的主要调节因子是热休克因子（HSF）。本研究的具体目标是建立和实施人类HSF 2影响者的筛选系统。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）可以用作药理学中重要的未来药物先导。筛选系统的设计是基于利用一个新的酵母菌株创建我们的实验室，其中的条件下过表达的人HSF 2的Gal 1启动子创建一个缓慢的生长表型。将验证增加试验菌株生长速率的潜在化合物，并将其用作新药开发的先导化合物。通过在含有半乳糖作为主要碳源营养物的生长培养基中掺入葡萄糖，可以很容易地调整试验菌株的初始生长速率以筛选不同的严格性。通过记录细胞培养物光吸收的变化，以及使用市售BacTiter-Glo微生物细胞活力测定（Promega）记录发光，检测细胞生长恢复。将在初始筛选前后的浓度范围内评价化合物命中，并测定IC 50。在存在已鉴别化合物的情况下，使用实时定量RT-PCR测量非热休克和热休克条件下一组报告热休克基因的mRNA水平变化，并与来自未处理细胞的类似样品进行比较，从而额外验证命中。这种方法的优点是，真正的hHSF 2的体内影响者将被鉴定和确认。

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.