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.
By screening large sets of compounds called chemical libraries, we will identify new molecules, which will be used as leads for new drug developments. These drugs will potentially work by crippling the cellular system of molecular chaperones, thus promoting cellular apoptosis and/or exposing cancer cells expressing mutated, misfolded and chimeric proteins to the immune system.
|Erkina, Tamara Y; Erkine, Alexandre M (2016) Nucleosome distortion as a possible mechanism of transcription activation domain function. Epigenetics Chromatin 9:40|
|Smith, Levi M; Bhattacharya, Dwipayan; Williams, Daniel J et al. (2015) High-throughput screening system for inhibitors of human Heat Shock Factor 2. Cell Stress Chaperones 20:833-41|
|Erkina, Tamara Y; Erkine, Alexandre (2015) ASF1 and the SWI/SNF complex interact functionally during nucleosome displacement, while FACT is required for nucleosome reassembly at yeast heat shock gene promoters during sustained stress. Cell Stress Chaperones 20:355-69|