Imaging-Based High-Content Screening with Clickable Probes Identifies XPB Inhibitors

Area of Science:

• Chemical Biology
• Drug Discovery
• Molecular Imaging

Background:

• High-content screening (HCS) is vital in drug discovery but limited by indirect readouts for drug-protein interactions.
• Assessing drug interactions at endogenous levels and within subcellular compartments remains challenging.
• Existing methods struggle to directly measure drug occupancy at specific protein sites in live cells.

Purpose of the Study:

• To develop a novel assay combining HCS with bio-orthogonal labeling for direct drug-protein interaction assessment.
• To validate the approach by identifying ligands for xeroderma pigmentosum type B (XPB), a key DNA repair protein.
• To demonstrate a new framework for drug discovery targeting difficult protein systems.

Main Methods:

• Synthesized a clickable probe (Triptolide-alkyne, TL-alk) for specific labeling of XPB.
• Combined confocal imaging-based HCS with bio-orthogonal chemistry for visualizing drug-target engagement.
• Developed a colorimetric assay to quantify drug occupancy at nuclear XPB in live cells.

Main Results:

• The developed HCS platform enabled direct and precise measurement of drug occupancy in nuclear XPB.
• Screening 1874 FDA-approved drugs identified pelitinib as a novel XPB ligand.
• Pelitinib covalently bound XPB at Cys342, inhibited ATPase activity, impaired nucleotide excision repair (NER), and enhanced chemotherapy.

Conclusions:

• The integrated HCS and bio-orthogonal labeling approach overcomes limitations of traditional HCS methods.
• This platform provides a powerful tool for direct assessment of drug-target interactions in live cells.
• The study offers a transformative framework for drug discovery, particularly for challenging protein targets like XPB.