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Updated: May 19, 2026
Malachite Green Assay for the Discovery of Heat-Shock Protein 90 Inhibitors
Published on: January 20, 2023
Discovery and validation of small-molecule heat-shock protein 90 inhibitors through multimodality molecular imaging
Carmel T Chan1, Robert E Reeves, Ron Geller
1Department of Radiology, Molecular Imaging Program at Stanford (MIPS), Stanford University School of Medicine, Stanford, CA 94305, USA.
Abstract:
Up-regulation of the folding machinery of the heat-shock protein 90 (Hsp90) chaperone protein is crucial for cancer progression. The two Hsp90 isoforms (α and β) play different roles in response to chemotherapy. To identify isoform-selective inhibitors of Hsp90(α/β)/cochaperone p23 interactions, we developed a dual-luciferase (Renilla and Firefly) reporter system for high-throughput screening (HTS) and monitoring the efficacy of Hsp90 inhibitors in cell culture and live mice. HTS of a 30,176 small-molecule chemical library in cell culture identified a compound, N-(5-methylisoxazol-3-yl)-2-[4-(thiophen-2-yl)-6-(trifluoromethyl)pyrimidin-2-ylthio]acetamide (CP9), that binds to Hsp90(α/β) and displays characteristics of Hsp90 inhibitors, i.e., degradation of Hsp90 client proteins and inhibition of cell proliferation, glucose metabolism, and thymidine kinase activity, in multiple cancer cell lines. The efficacy of CP9 in disrupting Hsp90(α/β)/p23 interactions and cell proliferation in tumor xenografts was evaluated by non-invasive, repetitive Renilla luciferase and Firefly luciferase imaging, respectively. At 38 h posttreatment (80 mg/kg × 3, i.p.), CP9 led to selective disruption of Hsp90α/p23 as compared with Hsp90β/p23 interactions. Small-animal PET/CT in the same cohort of mice showed that CP9 treatment (43 h) led to a 40% decrease in (18)F-fluorodeoxyglucose uptake in tumors relative to carrier control-treated mice. However, CP9 did not lead to significant degradation of Hsp90 client proteins in tumors. We performed a structural activity relationship study with 62 analogs of CP9 and identified A17 as the lead compound that outperformed CP9 in inhibiting Hsp90(α/β)/p23 interactions in cell culture. Our efforts demonstrated the power of coupling of HTS with multimodality molecular imaging and led to identification of Hsp90 inhibitors.
Insights
Researchers developed a novel reporter system to discover Hsp90 inhibitors, identifying CP9 which selectively disrupts Hsp90α/p23 interactions and shows anti-cancer effects in mice. Further optimization yielded lead compound A17.
Area of Science:
- Oncology
- Molecular Biology
- Pharmacology
Background:
- Heat-shock protein 90 (Hsp90) chaperone protein up-regulation is vital for cancer progression.
- Hsp90 isoforms (α and β) exhibit distinct roles in chemotherapy response.
- Targeting Hsp90/cochaperone interactions offers a therapeutic strategy for cancer treatment.
Purpose of the Study:
- To develop a high-throughput screening (HTS) system for identifying isoform-selective Hsp90 inhibitors.
- To discover novel small molecules that disrupt Hsp90(α/β)/p23 interactions.
- To evaluate the in vivo efficacy of identified Hsp90 inhibitors using molecular imaging.
Main Methods:
- A dual-luciferase reporter system was established for HTS of a chemical library.
- Compound CP9 was identified and characterized for its Hsp90 inhibitory properties.
- In vivo efficacy of CP9 was assessed using non-invasive imaging in tumor xenografts.
- Small-animal PET/CT was employed to monitor metabolic changes in tumors.
Main Results:
- HTS identified CP9, which inhibits Hsp90(α/β) activity, cell proliferation, and glucose metabolism in cancer cell lines.
- CP9 selectively disrupted Hsp90α/p23 interactions in vivo.
- CP9 treatment reduced tumor glucose uptake by 40% in mice.
- Structural activity relationship studies led to the identification of A17 as a superior Hsp90 inhibitor.
Conclusions:
- The developed reporter system effectively facilitates the discovery of Hsp90 inhibitors.
- CP9 demonstrates potential as an anti-cancer agent by selectively targeting Hsp90α.
- Multimodality molecular imaging is a powerful tool for monitoring Hsp90 inhibitor efficacy in vivo.
- Lead compound A17 warrants further investigation for its therapeutic potential.
