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Development of a Fluorescence Probe for High-Throughput Screening of Allosteric Inhibitors Targeting TRAP1
Nam Gu Yoon1, Danbi Choi2, Ji Hye Lee1
1Department of Biological Sciences, Ulsan National Institutes of Science and Technology (UNIST), Ulsan 44919, Republic of Korea.
Abstract:
Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a molecular chaperone implicated in pro-tumorigenic pathways by regulating the folding of substrate proteins (clients) within cancer cells. Recent research has pinpointed a potentially druggable allosteric site within the client binding site (CBS) of TRAP1, suggesting this site might offer a more effective strategy for developing potent and selective TRAP1 inhibitors. However, the absence of reliable assay systems has hindered quantitative evaluation of inhibitors. In this study, we have developed a fluorescent probe, Rho6TPP, designed to target the CBS. Utilizing fluorescence polarization-based high-throughput screening assays, Rho6TPP exhibits excellent signal-to-noise ratio (>20), Z factor (>0.6), and Z' factor (>0.6). Additionally, it facilitates comparative analysis of existing small molecules and discovery of novel binders. MitoTam, a mitochondria-targeted tamoxifen, emerges as a potent CBS-targeting TRAP1 inhibitor. Our findings highlight the potential of Rho6TPP as a crucial tool for advancing the development of CBS-targeting TRAP1 inhibitors.
Insights
Researchers developed Rho6TPP, a fluorescent probe targeting the client binding site (CBS) of Tumor Necrosis Factor Receptor-Associated Protein 1 (TRAP1). This probe enables efficient screening for novel TRAP1 inhibitors, crucial for cancer therapy development.
Area of Science:
- Molecular biology
- Biochemistry
- Cancer research
Background:
- Tumor Necrosis Factor Receptor-Associated Protein 1 (TRAP1) acts as a molecular chaperone, promoting cancer cell survival by regulating protein folding.
- TRAP1's client binding site (CBS) presents a druggable allosteric target for developing selective inhibitors.
- Lack of robust assay systems impedes the quantitative assessment of TRAP1 inhibitors.
Purpose of the Study:
- To develop a novel fluorescent probe for targeting the TRAP1 CBS.
- To establish a reliable high-throughput screening (HTS) assay for TRAP1 inhibitor discovery.
- To identify and characterize novel TRAP1 inhibitors targeting the CBS.
Main Methods:
- Development of a fluorescent probe, Rho6TPP, designed for the TRAP1 CBS.
- Implementation of fluorescence polarization-based HTS assays utilizing Rho6TPP.
- Screening of small molecules and comparative analysis of known binders.
Main Results:
- Rho6TPP demonstrated excellent assay performance with high signal-to-noise ratio (>20) and robust Z' factors (>0.6).
- The developed assay effectively facilitated the discovery of novel TRAP1 binders.
- MitoTam was identified as a potent TRAP1 inhibitor targeting the CBS.
Conclusions:
- Rho6TPP is a valuable tool for advancing the development of TRAP1 inhibitors.
- Targeting the TRAP1 CBS offers a promising strategy for cancer therapeutics.
- The developed HTS assay system enables efficient discovery and evaluation of TRAP1 inhibitors.
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