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.

Add glutathione donor beads at 10 micrograms per milliliter concentration in PBS. Add GST-FKBP51 to a final concentration of 10 micrograms per milliliter, and incubate the reaction for 10 minutes at 25 degrees Celsius in the dark.

Make serial dilutions of the test compound in DMSO. Add 0.25 microliters of test compound dilutions in triplicate, in the corner of each well of a 384-well plate.

For negative control, add 0.25 microliters of DMSO, and for positive control, add 0.25 microliters of Hsp90 C-terminal peptide in the wells.

Add 22.5 microliters of the solution containing glutathione donor beads with GST-tagged proteins to each well. Shake the plate thoroughly with hands and incubate in the dark at 25 degrees Celsius for 15 minutes.

Dilute the acceptor beads with the attached Hsp90 C-terminal peptide to 100 micrograms per milliliter concentration in 0.5x PBS. Add 2.25 microliters of diluted acceptor beads to each well. Shake and incubate the plate in the dark for 15 minutes as demonstrated.

Turn on the plate reader instrument, measure the signal of the plate, and analyze the data as described in the text manuscript.