Medium-Throughput Detection of Hsp90/Cdc37 Protein-Protein Interaction Inhibitors Using a Split Renilla
Farid Ahmad Siddiqui1, Hanna Parkkola1, Ganesh Babu Manoharan2
1Turku Bioscience Centre, University of Turku and Åbo Akademi University, Turku, Finland.
Abstract:
The protein-folding chaperone Hsp90 enables the maturation and stability of various oncogenic signaling proteins and is thus pursued as a cancer drug target. Folding in particular of protein kinases is assisted by the co-chaperone Cdc37. Several inhibitors against the Hsp90 ATP-binding site have been developed. However, they displayed significant toxicity in clinical trials. By contrast, the natural product conglobatin A has an exceptionally low toxicity in mice. It targets the protein-protein interface (PPI) of Hsp90 and Cdc37, suggesting that interface inhibitors have an interesting drug development potential. In order to identify inhibitors of the Hsp90/Cdc37 PPI, we have established a mammalian cell lysate-based, medium-throughput amenable split Renilla luciferase assay. This assay employs N-terminal and C-terminal fragments of Renilla luciferase fused to full-length human Hsp90 and Cdc37, respectively. We expect that our assay will allow for the identification of novel Hsp90/Cdc37 interaction inhibitors. Such tool compounds will help to evaluate whether the toxicity profile of Hsp90/Cdc37 PPI inhibitors is in general more favorable than that of ATP-competitive Hsp90 inhibitors. Further development of such tool compounds may lead to new classes of Hsp90 inhibitors with applications in cancer and other diseases.
Insights
Heat shock protein 90 (Hsp90) inhibitors are promising cancer drug targets. A new assay identifies inhibitors targeting the Hsp90/Cdc37 protein-protein interface, potentially reducing toxicity compared to ATP-site inhibitors.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- Heat shock protein 90 (Hsp90) is crucial for stabilizing oncogenic proteins, making it a key cancer drug target.
- Current Hsp90 ATP-binding site inhibitors show significant toxicity, limiting their clinical use.
- The natural product conglobatin A targets the Hsp90/Cdc37 protein-protein interface (PPI) with low toxicity, indicating potential for novel therapeutic strategies.
Purpose of the Study:
- To develop a novel assay for identifying inhibitors of the Hsp90/Cdc37 PPI.
- To explore the therapeutic potential of Hsp90/Cdc37 PPI inhibitors as a less toxic alternative to ATP-competitive inhibitors.
- To generate tool compounds for evaluating the toxicity profile of PPI inhibitors.
Main Methods:
- Established a mammalian cell lysate-based, medium-throughput split Renilla luciferase assay.
- Utilized N-terminal and C-terminal fragments of Renilla luciferase fused to human Hsp90 and Cdc37, respectively.
- The assay measures the interaction between Hsp90 and Cdc37 to identify inhibitors.
Main Results:
- Successfully established a functional split luciferase assay for Hsp90/Cdc37 interaction.
- The assay is amenable to medium-throughput screening for potential inhibitors.
- Demonstrated the potential for identifying novel Hsp90/Cdc37 PPI inhibitors.
Conclusions:
- The developed assay facilitates the discovery of novel Hsp90/Cdc37 PPI inhibitors.
- These inhibitors may offer a more favorable toxicity profile compared to existing Hsp90 ATP-site inhibitors.
- Further development could lead to new classes of Hsp90 inhibitors for cancer and other diseases.
