Detection of Small-Molecule Aggregation with High-Throughput Microplate Biophysical Methods

Area of Science:

• Drug discovery and development
• Biophysical chemistry
• Analytical chemistry

Background:

• Small-molecule drug discovery is frequently impeded by compound aggregation.
• Aggregates can lead to false positives in high-throughput screening (HTS).
• These aggregates complicate structure-activity relationship (SAR) studies during optimization.

Purpose of the Study:

• To present microplate-based protocols for identifying compound aggregation.
• To establish resonant waveguide grating (RWG) and dynamic light scattering (DLS) as reliable methods for detecting aggregation.
• To validate the equivalence of RWG and DLS for aggregation assessment.

Main Methods:

• Development of Basic Protocol 1: Resonant waveguide grating (RWG) assay in 384-well or 1536-well plate formats.
• Development of Basic Protocol 2: Dynamic light scattering (DLS) assay in a 384-well plate format.
• Utilizing Bland-Altman analysis to compare results from RWG and DLS.

Main Results:

• Both RWG and DLS assays effectively identify compound aggregation in microplate formats.
• RWG and DLS yield equivalent results when assessing a compound test set.
• Bland-Altman analysis confirms the agreement between the two methods.

Conclusions:

• RWG and DLS are validated as robust, high-throughput methods for detecting compound aggregation.
• These assays can mitigate false positives in drug discovery screening campaigns.
• The described protocols facilitate reliable aggregation assessment, aiding compound optimization.