1 University of Cambridge, UK
AbstractIn a screening campaign, some small molecule hits can interact with the intended molecular target in a non-specific manner, for example, through compound aggregation. These compound aggregates will lead to the discovery of false positives and hinder the drug discovery process. As such, it is beneficial to identify compound aggregators and their critical aggregation concentration (CAC) at the early stage of hit discovery, especially after a high-throughput screening campaign. Various techniques have been developed to identify compound aggregators, for example, dynamic light scattering (DLS), confocal static light scattering (cSLS), nuclear magnetic resonance (NMR) and β-lactamase assay. Here a fluorescence polarisation (FP) assay has been developed to identify compound aggregators using 5-dodecanoylaminofluorescein (5-DAF) as a lipophilic probe. 5-DAF was described previously as a probe to determine critical micelle concentration (CMC). We postulate that the ability of 5-DAF to insert its lipophilic tail into detergent micelles can apply to compound aggregates as well. Several known aggregators and non-aggregators alongside other compounds with unknown aggregation potential were screened with 5-DAF as an FP probe. The FP assay correctly identified the non-aggregators and aggregators except indirubin and rottlerin. It is hypothesised that the insolubility of high concentrations of indirubin and rottlerin in an aqueous buffer prevents the probe from working in a homogenous state. While the DLS technique allows direct visualisation of compound aggregation, the FP technique enables a cost-effective and rapid method to identify compound aggregators in a high-throughput manner using standard laboratory equipment.