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Updated: May 6, 2026
Intracellular Refolding Assay
Published on: January 24, 2012
Development of a high-throughput screening cancer cell-based luciferase refolding assay for identifying Hsp90
Takrima Sadikot1, Megan Swink, Jeffery D Eskew
11 University of Kansas Cancer Center, University of Kansas Medical Center , Kansas City, Kansas.
Abstract:
The 90 kDa heat-shock protein (Hsp90) and other cochaperones allow for proper folding of nascent or misfolded polypeptides. Cancer cells exploit these chaperones by maintaining the stability of mutated and misfolded oncoproteins and allowing them to evade proteosomal degradation. Inhibiting Hsp90 is an attractive strategy for cancer therapy, as the concomitant degradation of multiple oncoproteins may lead to effective anti-neoplastic agents. Unfortunately, early clinical trials have been disappointing with N-terminal Hsp90 inhibitors, as it is unclear whether the problems that plague current Hsp90 inhibitors in clinical trials are related to on-target or off-target activity. One approach to overcome these pitfalls is to identify structurally diverse scaffolds that improve Hsp90 inhibitory activity in the cancer cell milieu. Utilizing a panel of cancer cell lines that express luciferase, we have designed an in-cell Hsp90-dependent luciferase refolding assay. The assay was optimized using previously identified Hsp90 inhibitors and experimental novobiocin analogues against prostate, colon, and lung cancer cell lines. This assay exhibits good interplate precision (% CV), a signal-to-noise ratio (S/N) of ≥7, and an approximate Z-factor ranging from 0.5 to 0.7. Novobiocin analogues that revealed activity in this assay were examined via western blot experiments for client protein degradation, a hallmark of Hsp90 inhibition. Subsequently, a pilot screen was conducted using the Prestwick library, and two compounds, biperiden and ethoxyquin, revealed significant activity. Here, we report the development of an in-cell Hsp90-dependent luciferase refolding assay that is amenable across cancer cell lines for the screening of inhibitors in their specific milieu.
Insights
We developed a new assay to screen for heat-shock protein 90 (Hsp90) inhibitors in cancer cells. This method identifies novel compounds like biperiden and ethoxyquin for potential anti-cancer therapies.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Heat-shock protein 90 (Hsp90) is crucial for cancer cell survival by stabilizing oncoproteins.
- Hsp90 inhibition is a promising cancer therapy strategy, but early clinical trials faced challenges.
- Identifying novel Hsp90 inhibitors effective within the cancer cell environment is critical.
Purpose of the Study:
- To develop and optimize an in-cell assay for screening Hsp90 inhibitors.
- To identify novel Hsp90 inhibitors using a luciferase refolding assay in various cancer cell lines.
- To validate Hsp90 inhibition by assessing client protein degradation.
Main Methods:
- Designed an Hsp90-dependent luciferase refolding assay using cancer cell lines.
- Optimized the assay with known inhibitors and novobiocin analogues.
- Screened the Prestwick library and confirmed hits via Western blot analysis for client protein degradation.
Main Results:
- The developed assay demonstrated good precision (CV), signal-to-noise ratio (≥7), and Z-factor (0.5–0.7).
- Novobiocin analogues showed activity, leading to client protein degradation.
- Pilot screening identified biperiden and ethoxyquin as active compounds.
Conclusions:
- An effective in-cell Hsp90-dependent luciferase refolding assay was established.
- The assay is suitable for screening inhibitors across diverse cancer cell lines.
- This platform facilitates the discovery of novel Hsp90-targeting anti-cancer agents.
