Pharmacy, Health Sciences & Exercise Science
Event Title
The Search for Small-Molecule Inhibitors of HSF: A Novel Cell-Based Assay for Use in High-Throughput Screens
Document Type
Oral Presentation
Location
Indianapolis, IN
Subject Area
Pharmacy, Health Sciences & Exercise Science
Start Date
11-4-2014 8:30 AM
End Date
11-4-2014 10:00 AM
Sponsor
Andrew Erkine (Butler University)
Description
Background: The most basic unit of "life" is the cell. All cells constantly encounter stressors that threaten their ability to survive, such as temperature changes, pathogens, and oxidative stress. Heat shock factor (HSF) is a transcription factor that regulates the cellular response to stress, the accordingly named "heat shock response." Under normal growth conditions in metazoans, HSF1 activity is repressed and exists either in the cytosol or nucleus in an inert monomeric state.The mechanism by which HSF mediates cytoprotection is through the expression of heat shock proteins (HSPs), which serve as molecular chaperones, molecules that repair aberrantly folded and damaged proteins. Because this function is essential to cell survival, it is conserved amongst all eukaryotes, to such an extent that human HSF (isoforms 1 and 2) is sufficient to sustain growth when expressed in Saccharomyces cerevisiae, the most basic of eukaryotes. Recent research has shown increased HSF expression in tumors and implicated HSF in tumorigenesis, putatively by way of increased expression of HSPs, which refold misfolded proteins that otherwise would induce cell-death pathways and alert the immune system to their presence; by this mechanism, increased HSF expression may allow cancer cells to survive where they otherwise would not.
Study Objective: We set out to develop a cell-based assay using Saccharomyces cerevisiae to search for inhibitors of HSF, contributing to future anticancer therapies.
Methods: A strain of Saccharomyces cerevisiae was developed, whose sole source of HSF was the human HSF2 isoform expressed at basal levels, which is sufficient to support growth. Additionally, a plasmid was constructed placing human HSF2 under an inducible Gal1 promoter, allowing overexpression of HSF2. Because overexpression of human HSF2 proved cytotoxic and significantly slowed growth, we developed an assay in which inhibitors of HSF2 would ameliorate this cytotoxicity and allow our strain to grow normally. Such an assay is a novel method for drug discovery, and thus required development and optimization, including statistical validation by a simple calculation, Z-factor.
Results: Replication and support of assay validity was ultimately achieved in October 2013, when our assay was validated on a larger scale by the National Institutes of Health in Bethesda, Maryland. Future work will involve use of our assay to screen a library of 400,000 small molecule compounds for inhibitors of human HSF2.
Conclusions: This work pioneered a new method for drug discovery, and currently a screen is underway, searching for small molecule compounds which may be important for future anticancer therapies. Additionally, this assay is applicable to any polypeptide drug target, whose overexpression in Saccharomyces cerevisiae diminishes growth.
The Search for Small-Molecule Inhibitors of HSF: A Novel Cell-Based Assay for Use in High-Throughput Screens
Indianapolis, IN
Background: The most basic unit of "life" is the cell. All cells constantly encounter stressors that threaten their ability to survive, such as temperature changes, pathogens, and oxidative stress. Heat shock factor (HSF) is a transcription factor that regulates the cellular response to stress, the accordingly named "heat shock response." Under normal growth conditions in metazoans, HSF1 activity is repressed and exists either in the cytosol or nucleus in an inert monomeric state.The mechanism by which HSF mediates cytoprotection is through the expression of heat shock proteins (HSPs), which serve as molecular chaperones, molecules that repair aberrantly folded and damaged proteins. Because this function is essential to cell survival, it is conserved amongst all eukaryotes, to such an extent that human HSF (isoforms 1 and 2) is sufficient to sustain growth when expressed in Saccharomyces cerevisiae, the most basic of eukaryotes. Recent research has shown increased HSF expression in tumors and implicated HSF in tumorigenesis, putatively by way of increased expression of HSPs, which refold misfolded proteins that otherwise would induce cell-death pathways and alert the immune system to their presence; by this mechanism, increased HSF expression may allow cancer cells to survive where they otherwise would not.
Study Objective: We set out to develop a cell-based assay using Saccharomyces cerevisiae to search for inhibitors of HSF, contributing to future anticancer therapies.
Methods: A strain of Saccharomyces cerevisiae was developed, whose sole source of HSF was the human HSF2 isoform expressed at basal levels, which is sufficient to support growth. Additionally, a plasmid was constructed placing human HSF2 under an inducible Gal1 promoter, allowing overexpression of HSF2. Because overexpression of human HSF2 proved cytotoxic and significantly slowed growth, we developed an assay in which inhibitors of HSF2 would ameliorate this cytotoxicity and allow our strain to grow normally. Such an assay is a novel method for drug discovery, and thus required development and optimization, including statistical validation by a simple calculation, Z-factor.
Results: Replication and support of assay validity was ultimately achieved in October 2013, when our assay was validated on a larger scale by the National Institutes of Health in Bethesda, Maryland. Future work will involve use of our assay to screen a library of 400,000 small molecule compounds for inhibitors of human HSF2.
Conclusions: This work pioneered a new method for drug discovery, and currently a screen is underway, searching for small molecule compounds which may be important for future anticancer therapies. Additionally, this assay is applicable to any polypeptide drug target, whose overexpression in Saccharomyces cerevisiae diminishes growth.