High-throughput screening system for inhibitors of human Heat Shock Factor 2
| Autor: | Daniel J. Williams, Levi M. Smith, Nicholas R. Powell, Ivan Dixon, Alexandre M. Erkine, Dwipayan Bhattacharya, Tamara Y. Erkina |
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| Rok vydání: | 2015 |
| Předmět: |
Genetics
Original Paper Organisms Genetically Modified Cell growth High-throughput screening Saccharomyces cerevisiae Cell Biology Biology Biochemistry Hsp90 Phenotype High-Throughput Screening Assays Cell biology Heat shock factor Heat shock protein biology.protein Humans HSF1 Transcription factor Heat-Shock Proteins Molecular Chaperones Transcription Factors |
| Zdroj: | Cell Stress and Chaperones. 20:833-841 |
| ISSN: | 1466-1268 1355-8145 |
| DOI: | 10.1007/s12192-015-0605-0 |
| Popis: | Development of novel anti-cancer drug leads that target regulators of protein homeostasis is a formidable task in modern pharmacology. Finding specific inhibitors of human Heat Shock Factor 1 (hHSF1) has proven to be a challenging task, while screening for inhibitors of human Heat Shock Factor 2 (hHSF2) has never been described. We report the development of a novel system based on an in vivo cell growth restoration assay designed to identify specific inhibitors of human HSF2 in a high-throughput format. This system utilizes a humanized yeast strain in which the master regulator of molecular chaperone genes, yeast HSF, has been replaced with hHSF2 with no detrimental effect on cell growth. This replacement preserves the general regulatory patterns of genes encoding major molecular chaperones including Hsp70 and Hsp90. The controlled overexpression of hHSF2 creates a slow-growth phenotype, which is the basis of the growth restoration assay used for high-throughput screening. The phenotype is most robust when cells are cultured at 25 °C, while incubation at temperatures greater than 30 °C leads to compensation of the phenotype. Overexpression of hHSF2 causes overexpression of molecular chaperones which is a likely cause of the slowed growth. Our assay is characterized by two unique advantages. First, screening takes place in physiologically relevant, in vivo conditions. Second, hits in our screen will be of medically relevant potency, as compounds that completely inhibit hHSF2 function will further inhibit cell growth and therefore will not be scored as hits. This caveat biases our screening system for compounds capable of restoring hHSF2 activity to a physiologically normal level without completely inhibiting this essential system. |
| Databáze: | OpenAIRE |
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