Development of a Fluorescence Probe for High-Throughput Screening of Allosteric Inhibitors Targeting TRAP1

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.