Amplified Luminescent Proximity Homogeneous Assay: A Bead-Based Proximity Assay to Screen Small Molecules Inhibiting Protein-Protein Interactions

In biological systems, a protein's proximity to its specific partner is a crucial determinant of successful protein-protein interaction. With small inhibitory molecules present, these proteins cannot move near each other, disrupting their interactions.

To screen inhibitory molecules for disrupting interactions between two proteins - a chaperone and co-chaperone - take a multi-well plate containing various small molecules. Supplement the wells with donor beads having co-chaperones attached to their surfaces. These beads contain photosensitizers for chemiluminescence assay.

Add a slurry of acceptor beads conjugated to the chaperone-derived peptide sequences that bind specifically to the co-chaperone. The beads contain a thioxene-based dye and fluorophores essential for visualization.

In wells with non-inhibitory molecules, high affinities between the chaperone-binding peptides and co-chaperones attract the acceptor and donor beads. While the beads remain distant when inhibitors are present.

Using a chemiluminescence reader, study the interaction. Upon illumination at a specific wavelength, the donor bead photosensitizers emit highly reactive singlet oxygens.

With no inhibitory molecules, the emitted species reach near proximally-located acceptor beads and react with dye molecules, causing chemiluminescence. This energy activates the acceptor beads' fluorophores, causing light emission.

In contrast, in the wells with inhibitory molecules, singlet oxygens undergo decay, leading to an absence of light emission, indicating successful inhibition of protein-protein interactions.