Related Experiment Video
Updated: Jun 15, 2026
Malachite Green Assay for the Discovery of Heat-Shock Protein 90 Inhibitors
Published on: January 20, 2023
Mutations that increase both Hsp90 ATPase activity in vitro and Hsp90 drug resistance in vivo
Anna Zurawska1, Jakub Urbanski, Jurgita Matuliene
1International Institute of Molecular and Cell Biology, Warsaw, Poland.
Abstract:
Hsp90 inhibitors are currently tested in clinical trials as anticancer agents. We investigated whether inhibitor resistance may arise as a result of a point mutation in Hsp90. We used yeast cells that expressed human Hsp90beta to select inhibitor-resistant mutants from the randomly mutagenized library. Single amino acid substitution, I123T, in a selected mutant was sufficient to confer inhibitor resistance. Transfection of human cells with the HSP90beta I123T and the corresponding HSP90alpha I128T yielded cell lines resistant to inhibitors of the Hsp90 ATPase. Unexpectedly, mutations did not result in diminished inhibitor binding in vitro. Similarly resistant cells were obtained after transfection with previously described A116N and T31I mutants of HSP90beta that cause increase in ATPase activity in vitro. Inhibitor-resistant phenotypes of the I123T and A116N mutants depended on their increased affinity for Aha1, whereas T31I mutation did not result in increased Aha1 binding. These results show possible scenario by which resistance may arise in patients treated with Hsp90 inhibitors. Additionally, our results show that each isoform of Hsp90 can alone sustain cellular functions.
Insights
Heat shock protein 90 (Hsp90) inhibitors can lead to drug resistance through specific mutations. These Hsp90 mutations, like I123T, confer resistance by altering protein interactions, not necessarily inhibitor binding.
Area of Science:
- Molecular Biology
- Biochemistry
- Oncology
Background:
- Heat shock protein 90 (Hsp90) is a crucial molecular chaperone involved in cancer progression.
- Hsp90 inhibitors are under clinical investigation as anticancer therapeutics.
- Understanding mechanisms of drug resistance is vital for effective cancer treatment.
Purpose of the Study:
- To investigate if point mutations in Hsp90 can lead to resistance against Hsp90 inhibitors.
- To identify specific mutations conferring resistance and elucidate their underlying mechanisms.
Main Methods:
- Utilized yeast cells expressing human Hsp90beta to screen for inhibitor-resistant mutants.
- Introduced specific Hsp90 mutations (I123T, I128T, A116N, T31I) into human cells via transfection.
- Assessed inhibitor resistance, Hsp90 ATPase activity, and binding affinity to Aha1 in vitro.
Main Results:
- A single amino acid substitution (I123T in Hsp90beta) was sufficient to confer resistance to Hsp90 inhibitors.
- Mutations I123T and I128T in Hsp90alpha/beta isoforms conferred resistance without diminishing in vitro inhibitor binding.
- Resistance in I123T and A116N mutants was linked to increased affinity for Aha1, while T31I showed no such correlation.
- Both Hsp90 isoforms (alpha and beta) can independently support cellular functions.
Conclusions:
- Identified specific Hsp90 mutations as a potential mechanism for acquired resistance to Hsp90-targeted cancer therapies.
- Demonstrated that resistance can arise from altered protein-protein interactions (e.g., with Aha1) rather than solely reduced drug binding.
- Highlighted the potential for Hsp90 isoforms to sustain cellular functions, offering insights into therapeutic strategies.
More Related Videos
Related Concept Videos
02:56Treatment Resistant Cancers
01:29Pharmacogenetic Phenotypes: Alterations in Pharmacokinetics, Drug Targets and Biologic Milieu