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Identification of inhibitors of HSF1 functional activity by high-content target-based screening
Qingyan Au1, Yingjia Zhang, Jack R Barber
1Department of Biology, CytRx Corporation, San Diego, California 92109, USA.
Abstract:
Cancer cells are known to experience a high level of stress and may require constant repair for survival and proliferation. Recent studies showed that inhibition of heat shock factor 1 (HSF1), the key regulator for the stress-activated transcription of heat shock protein (HSP), can reduce the tumorigenic potential of cancer cells. Such a "nononcogene addiction" phenomenon makes HSF1 an attractive cancer drug target. Here, the authors report an image-based high-content screening (HCS) assay for HSF1 functional inhibitors. A heat shock-based methodology was used to stimulate the stress response followed by quantitative measurement of HSF1/HSP70 granules for compound-induced inhibitory effects. The authors discovered a small molecule from a compound library that inhibits HSF1 granule formation substantially in heat-shocked HeLa cells with IC(50) at 80 nM. Electorphoretic mobility shift of HSF1 by this compound suggested significant inhibition of HSF1 phosphorylation, accompanied by reduced expression levels of HSP70 and HSP90 after heat induction. Importantly, HeLa cells stably transfected with HSF1 shRNA were more resistant to the compound treatment under lethal temperature than cells containing HSF1, further validating an HSF1-dependent mechanism of action. The HCS assay the authors developed was robust with a Z' factor of 0.65 in a 384-well plate format, providing a valuable method for identifying small-molecule functional inhibitors of HSF1 for potential cancer treatment.
Insights
Researchers developed a high-content screening assay to find inhibitors of heat shock factor 1 (HSF1), a key protein in cancer cell survival. A novel compound was identified that significantly reduces HSF1 activity, offering potential for new cancer therapies.
Area of Science:
- Molecular Biology
- Cancer Research
- Drug Discovery
Background:
- Cancer cells exhibit high stress, relying on heat shock factor 1 (HSF1) for survival.
- HSF1 regulates heat shock protein (HSP) transcription, making it a potential cancer drug target.
- Inhibiting HSF1 can reduce cancer cell proliferation, a phenomenon known as 'nononcogene addiction'.
Purpose of the Study:
- To develop and validate an image-based high-content screening (HCS) assay for identifying HSF1 functional inhibitors.
- To discover small molecules that inhibit HSF1 activity and granule formation.
- To explore the potential of HSF1 inhibitors as a cancer treatment strategy.
Main Methods:
- Developed a heat shock-based HCS assay to measure HSF1/HSP70 granules.
- Screened a compound library to identify inhibitors of HSF1 granule formation.
- Utilized electrophoretic mobility shift assays and Western blotting to assess HSF1 phosphorylation and HSP expression.
- Validated the HSF1-dependent mechanism using HSF1 shRNA in HeLa cells.
Main Results:
- Identified a small molecule inhibitor of HSF1 granule formation with an IC(50) of 80 nM in heat-shocked HeLa cells.
- Demonstrated that the compound inhibits HSF1 phosphorylation, leading to reduced HSP70 and HSP90 expression.
- Confirmed an HSF1-dependent mechanism, as HSF1-depleted cells showed resistance to the compound.
- Validated the HCS assay's robustness with a Z' factor of 0.65.
Conclusions:
- The developed HCS assay is a reliable method for identifying functional HSF1 inhibitors.
- The identified small molecule demonstrates significant HSF1 inhibitory activity.
- HSF1 inhibition presents a promising therapeutic strategy for cancer treatment.
