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Updated: Sep 21, 2025
Malachite Green Assay for the Discovery of Heat-Shock Protein 90 Inhibitors
Published on: January 20, 2023
Development of a First-in-Class Small-Molecule Inhibitor of the C-Terminal Hsp90 Dimerization
Sanil Bhatia1, Lukas Spanier2, David Bickel2
1Department of Pediatric Oncology, Hematology and Clinical Immunology, Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf 40225, Germany.
Abstract:
Heat shock proteins 90 (Hsp90) are promising therapeutic targets due to their involvement in stabilizing several aberrantly expressed oncoproteins. In cancerous cells, Hsp90 expression is elevated, thereby exerting antiapoptotic effects, which is essential for the malignant transformation and tumor progression. Most of the Hsp90 inhibitors (Hsp90i) under investigation target the ATP binding site in the N-terminal domain of Hsp90. However, adverse effects, including induction of the prosurvival resistance mechanism (heat shock response or HSR) and associated dose-limiting toxicity, have so far precluded their clinical approval. In contrast, modulators that interfere with the C-terminal domain (CTD) of Hsp90 do not inflict HSR. Since the CTD dimerization of Hsp90 is essential for its chaperone activity, interfering with the dimerization process by small-molecule protein-protein interaction inhibitors is a promising strategy for anticancer drug research. We have developed a first-in-class small-molecule inhibitor (5b) targeting the Hsp90 CTD dimerization interface, based on a tripyrimidonamide scaffold through structure-based molecular design, chemical synthesis, binding mode model prediction, assessment of the biochemical affinity, and efficacy against therapy-resistant leukemia cells. 5b reduces xenotransplantation of leukemia cells in zebrafish models and induces apoptosis in BCR-ABL1+ (T315I) tyrosine kinase inhibitor-resistant leukemia cells, without inducing HSR.
Insights
A novel small-molecule inhibitor, 5b, targets the C-terminal domain of Heat shock protein 90 (Hsp90) to treat resistant leukemia. This approach avoids the heat shock response (HSR) seen with other Hsp90 inhibitors.
Area of Science:
- Oncology
- Molecular Biology
- Drug Discovery
Background:
- Heat shock proteins 90 (Hsp90) are crucial for stabilizing oncoproteins in cancer, promoting malignant transformation and tumor progression.
- Current Hsp90 inhibitors targeting the N-terminal ATP-binding site induce the heat shock response (HSR), leading to toxicity and limiting clinical application.
- Modulators targeting the Hsp90 C-terminal domain (CTD) offer an alternative therapeutic strategy by avoiding HSR.
Purpose of the Study:
- To develop a novel small-molecule inhibitor targeting the Hsp90 CTD dimerization interface.
- To evaluate the efficacy of this inhibitor against therapy-resistant leukemia cells.
- To assess whether the inhibitor induces the heat shock response (HSR).
Main Methods:
- Structure-based molecular design and chemical synthesis of a tripyrimidonamide scaffold.
- Prediction of binding mode and assessment of biochemical affinity.
- Evaluation of efficacy in zebrafish xenotransplantation models and apoptosis induction in BCR-ABL1+(T315I) leukemia cells.
Main Results:
- A first-in-class small-molecule inhibitor, designated 5b, was successfully developed.
- Compound 5b demonstrated efficacy in reducing leukemia cell xenotransplantation in zebrafish.
- 5b induced apoptosis in TKI-resistant leukemia cells without triggering the HSR.
Conclusions:
- Targeting Hsp90 CTD dimerization with small-molecule inhibitors is a viable anticancer strategy.
- Compound 5b represents a promising therapeutic candidate for treating resistant leukemia.
- Inhibiting Hsp90 CTD dimerization offers a potential advantage over N-terminal inhibitors by avoiding HSR and associated toxicities.
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