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 molecu