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Malachite Green Assay for the Discovery of Heat-Shock Protein 90 Inhibitors
Published on: January 20, 2023
5'-N-ethylcarboxamidoadenosine is not a paralog-specific Hsp90 inhibitor
Shanshan Liu1, Timothy O Street1
1Department of Biochemistry, Brandeis University, Waltham, MA, 02454.
Abstract:
The molecular chaperone Hsp90 facilitates the folding and modulates activation of diverse substrate proteins. Unlike other heat shock proteins such as Hsp60 and Hsp70, Hsp90 plays critical regulatory roles by maintaining active states of kinases, many of which are overactive in cancer cells. Four Hsp90 paralogs are expressed in eukaryotic cells: Hsp90α/β (in the cytosol), Grp94 (in the endoplasmic reticulum), Trap1 (in mitochondria). Although numerous Hsp90 inhibitors are being tested in cancer clinical trials, little is known about why different Hsp90 inhibitors show specificity among Hsp90 paralogs. The paralog specificity of Hsp90 inhibitors is likely fundamental to inhibitor efficacy and side effects. In hopes of gaining insight into this issue we examined NECA (5'-N-ethylcarboxamidoadenosine), which has been claimed to be an example of a highly specific ligand that binds to one paralog, Grp94, but not cytosolic Hsp90. To our surprise we find that NECA inhibits many different Hsp90 proteins (Grp94, Hsp90α, Trap1, yeast Hsp82, bacterial HtpG). NMR experiments demonstrate that NECA can bind to the N-terminal domains of Grp94 and Hsp82. We use ATPase competition experiments to quantify the inhibitory power of NECA for different Hsp90 proteins. This scale: Hsp82 > Hsp90α > HtpG ≈ Grp94 > Trap1, ranks Grp94 as less sensitive to NECA inhibition. Because NECA is primarily used as an adenosine receptor agonist, our results also suggest that cell biological experiments utilizing NECA may have confounding effects from cytosolic Hsp90 inhibition.
Insights
NECA, a potential Hsp90 inhibitor, unexpectedly targets multiple Hsp90 proteins, not just Grp94. This challenges its specificity and suggests broader implications for cancer research and drug development.
Area of Science:
- Molecular Biology
- Biochemistry
- Cancer Research
Background:
- Heat shock protein 90 (Hsp90) is crucial for protein folding and kinase regulation, with overactive kinases implicated in cancer.
- Four Hsp90 paralogs exist: Hsp90α/β (cytosolic), Grp94 (ER), and Trap1 (mitochondria).
- Hsp90 inhibitors are in clinical trials, but their paralog specificity and impact on efficacy/side effects are poorly understood.
Purpose of the Study:
- To investigate the paralog specificity of NECA (5'-N-ethylcarboxamidoadenosine), a compound previously claimed to be a Grp94-specific ligand.
- To determine if NECA inhibits other Hsp90 paralogs, particularly cytosolic Hsp90, and to quantify its inhibitory potency across different Hsp90 family members.
Main Methods:
- Nuclear Magnetic Resonance (NMR) spectroscopy to identify NECA binding sites on Hsp90 proteins.
- ATPase competition assays to quantify the inhibitory activity of NECA against various Hsp90 paralogs.
Main Results:
- NECA demonstrated inhibitory activity against multiple Hsp90 proteins, including Grp94, Hsp90α, Trap1, yeast Hsp82, and bacterial HtpG.
- NMR experiments confirmed NECA binding to the N-terminal domains of Grp94 and Hsp82.
- ATPase assays revealed an inhibitory potency scale: Hsp82 > Hsp90α > HtpG ≈ Grp94 > Trap1, indicating Grp94 is less sensitive to NECA.
Conclusions:
- NECA is not a specific Grp94 inhibitor and broadly inhibits various Hsp90 family members.
- The findings challenge the assumed specificity of NECA and suggest potential confounding effects in cell biological studies using NECA as an adenosine receptor agonist.
- Understanding Hsp90 paralog specificity is critical for developing effective Hsp90-targeted cancer therapies.
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