Development of a high-content imaging assay for screening compound aggregation.

Area of Science:

• Biochemistry
• Chemical Biology
• Drug Discovery

Background:

• Chemical compounds can aggregate, leading to non-specific interactions with proteins.
• These interactions can cause false positives or negatives in drug screening assays, affecting target identification.
• Understanding compound aggregation is crucial for accurate assessment of drug candidates.

Purpose of the Study:

• To develop a high-content imaging assay for detecting compound-protein interactions.
• To identify and quantify compound aggregation events.
• To determine the critical aggregation concentration (CAC) of small molecules.

Main Methods:

• Utilized a high-content imaging assay in a 384-well format.
• Employed fluorescently labeled target proteins for detection.
• Used an Operetta cell imager for high-throughput screening.
• Quantified compound aggregation and interaction with target proteins.

Main Results:

• Successfully developed a high-throughput assay to screen for compound aggregates.
• The assay directly detects interactions between compound aggregators and target proteins.
• Enabled determination of the critical aggregation concentration (CAC) for promiscuous small molecules.
• Demonstrated the assay's capability to differentiate specific from non-specific interactions.

Conclusions:

• The developed high-content imaging assay is effective for identifying compound aggregation.
• This method allows for direct detection of compound-target interactions and CAC determination.
• The assay aids in mitigating false results in drug discovery screening.
• Provides a valuable tool for characterizing promiscuous small molecules in early drug development.