3.5. Fluorescence polarization assay (FP)

FP is an intrinsically powerful technique for the rapid and homogeneous analysis of molecular interactions in biological/chemical systems. The basic principle of FP is shown in Fig. 9. A fluorescent probe is excited with polarized light, and emission intensity is measured as FP. An unbound fluorescent inhibitor (red) shows a fast tumbling, resulting in a higher depolarized emission signal. The fluorescent probe binds to a larger protein, resulting in a slow tumbling and a higher polarized emission signal128. This technique has been successfully used in monitoring enzyme kinetics, protein–protein interactions, DNA diagnostics, biological interactions and high throughput screening in drug discovery.

The basic principle of the fluorescence polarization.

Cornish's group129 modified this method and constructed a broadly applicable high-throughput screening for small-molecule targets. The assay requires a target receptor and a reporter molecule, which can couple the target molecule with a fluorophore. The target molecules compete with the reporter for binding to the common receptor. Thus, the target concentration is inversely proportional to the ratio of bound to unbound reporter molecules, which can be measured by FP. They used the receptor FKBP12 and reporter molecule FK506-fluorescein to construct FP assay for screening FK506 (tacrolimus) from Streptomyces tsukubaensisin in 384-well, round, black-bottom plates. Berg's group130 used the STAT5a FP–based binding assay for HTS of chemical libraries comprising a total of 3289 natural products and/or known bioactive substances. NF023 and NF449 display improved selectivity for the SH2 domains of STATa/b and NF449 was the most active candidate among the STATa/b SH2 domain inhibitors. Dysregulation of MLL1 catalytic function is relevant to mixed-lineage leukemia and targeting WDR5–MLL1 interaction could be a promising therapeutic strategy for leukemia harboring MLL1 fusion proteins. Ye et al.131 discovered several small-molecule inhibitors with potent inhibitory activities in vitro against WDR5–MLL1 interaction through FP-based high throughput screening.