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

NOTE: An overview of this protocol is shown in Figure 1.

1. Expression and purification of GST-FKBP51 and GST-FKBP52 (Figure 1A)

1. Plasmids
   NOTE: Obtain cDNA clones for human FKBP51 (clone id: 5723416) and for human FKBP52 (clone id: 7474554) from IMAGE consortium.
   1. Amplify the human FKBP51 DNA by PCR with primers (forward; 5`GGATCCATGACTACTGATGAAGGT-3`, reverse; 5`-CTCGAGCTATGCTTCTGTCTCCAC-3`) containing BamHI and XhoI overhangs and clone into pGEX6-1 vector at BamHI / XhoI restriction sites.
   2. Amplify the human FKBP52 DNA by PCR with primers (forward; 5`-GAATTCATGACAGCCGAGGAGATG-3`, reverse; 5`-CTCGAGCTATGCTTCTGTCTCCAC-3`) containing EcoRI and XhoI overhangs and clone into pGEX6-2 vector at EcoRI / XhoI restriction sites.
      NOTE: PCR reaction set up and conditions are shown in Table 1 and Table 2.
   3. Verify the inserted sequence and transform the plasmids into the chemically competent E. coli according to the manufacture protocol.
2. Protein expression and purification
   1. Add 25 g of Luria broth (LB) base in 1 L of distilled water to make the LB solution. Autoclave it at 121 °C for 15 min. After cooling, add 50 µg/mL ampicillin.
   2. Take a colony of bacteria expressing GST-FKBP51 or GST-FKBP52 and mix with 500 µL of LB solution in a 1.5 mL tube. Vortex.
   3. Add the mixture of "1.2.2" into 1 L of LB solution in the Erlenmeyer flask covered with an aluminum foil. Incubate the Erlenmeyer flask in the shaker overnight at 37 °C.
   4. Induce protein expression by adding 1 mM isopropyl-β-D-thiogalactoside (IPTG) to the Erlenmeyer flask and continue the incubation for a further 2 h.
   5. To get cell pellets, centrifuge at 5,000 x g for 15 min. Remove the supernatant.
      NOTE: The cell pellets can be stored at -20 °C.
   6. Resuspend the cell pellets in 40 mL of PBS and sonicate 3 x 20 s on ice. Add 1 mM PMSF, 1 mM EDTA, and protease inhibitor cocktail (1 tablet) to prevent proteolysis.
   7. Centrifuge the suspension for 30 min 50,000 x g to remove cell debris and apply the supernatant onto 5 mL GST-trap column.
   8. After washing the column with 30 mL PBS, elute GST-FKBP51 and GST-FKBP52 with 5 mL of 10 mM glutathione in PBS.
   9. Concentrate proteins on 15 mL 10.000 MWCO centrifugation unit. To remove free glutathione, pass concentrates through PD-10 column equilibrated with 0.5x PBS and again concentrate on the filter centrifuge device.
   10. Collect protein-containing fractions. Verify the proteins in SDS-PAGE and adjust protein concentrations to 1 mg/mL.
       NOTE: Typical protein yield is 2-5 mg/L culture. The protein can be stored at -20 °C.

2. Coupling of Hsp90 C-terminal peptide to the acceptor beads (Figure 1B)

1. Hsp90 peptide preparation
   1. Synthesize ten amino acid peptide NH₂-EDASRMEEVD-COOH corresponding to amino acids 714-724 of human Hsp90 beta isoform (UniProt ID: P08238) by a peptide synthesis service.
   2. Dilute the Hsp90 peptide in PBS to 1 mg/mL concentration.
2. Acceptor beads preparation
   1. Dilute the unconjugated acceptor beads in PBS to 1 mg/mL concentration and transfer to a 1.5 mL tube.
   2. Perform the washing by centrifugation at 16,000 x g for 15 min. Carefully remove the supernatant.
3. Conjugation
   1. Set the ratio between beads and peptide as 10:1. In the 1.5 mL tube containing 1 mg of acceptor bead pellet (prepared as described above), add 1 mL of PBS (pH 7.4), 0.1 mg of diluted peptide, 1.25 µL of Tween-20, 10 µL of a 400 mM solution of sodium cyanoborohydride (NaBH₃CN) in water.
      CAUTION: NaBH₃CN is toxic; use a fume hood and gloves. NaBH₃CN solution should be freshly prepared.
   2. Incubate for 24 h at 37 °C with end-over-end agitation (10-20 rpm) on a rotary shaker.
4. Reaction quenching and beads washing
   1. Add 20 µL of 1 M Tris-HCl (pH 8.0) solution to the reaction to block unreacted sites. Incubate for 1 h at 37 °C.
   2. Centrifuge at 16,000 x g (or maximum speed) for 15 min at 4 °C. Remove the supernatant and resuspend the bead pellet in 1 mL of Tris-HCl solution (100 mM, pH 8.0).
   3. Repeat the washing step three times.
   4. After the last centrifugation, resuspend the beads at 1 mg/mL in storage buffer (1 mL of 0.5 × PBS with 0.01% sodium azide as a preservative). Store the conjugated acceptor bead solution at 4 °C light protected.
      CAUTION: Sodium azide is toxic; use a fume hood and gloves.

3. The assay probing the interaction between GST-FKBP51 or GST-FKBP52 and Hsp90 C-terminal peptide, and inhibition with small molecular mass compounds (Figure 1C)

1. GST-tagged proteins interacting with glutathione donor beads
   1. Set up the reactions in 384-well plates.
   2. Prepare the solution containing 10 µg/mL of the glutathione donor beads in 0.5x PBS, pH 7.4.
      NOTE: After prolonged storage, beads settle down and need to be vortexed.
   3. Add GST-FKBP51 or GST-FKBP52 to a final concentration of 10 µg/mL.
   4. Incubate in the dark at 25 °C for 10 min.
      NOTE: At this step, GST-tagged proteins will interact with glutathione attached to the beads. For each well, 22.5 µL of this mixture will be used. The concentration of the binding partners must be determined empirically. Titrate GST-FKBP51 and GST-FKBP52 and choose the concentration that gives the best signal.
2. Compound addition
   1. Make serial dilutions of test compounds in DMSO.
      NOTE: The concentrations used are typically 10, 30, 100, 300, 1,000, and 3,000 µM.
   2. Add 0.25 µL of DMSO (negative control) or Hsp90 C-terminal peptide (positive control, 30 µM) or compounds in DMSO to the corner of each well of the plate. Use triplicates for every compound concentration.
   3. Add 22.5 µL of the solution containing glutathione donor beads with GST-tagged proteins to each well.
   4. Shake the plate gently with hand but thoroughly. Incubate in the dark at 25 °C for 15 min.
      NOTE: During this time, compounds will interact with the TPR domain at the Hsp90 C-terminal peptide binding site.
3. Acceptor beads addition
   1. Dilute the acceptor beads with attached Hsp90 C-terminal peptide to 100 µg/mL in 0.5x PBS.
   2. Add 2.25 µL of diluted acceptor beads to each well.
   3. Mix gently but thoroughly. Incubate in the dark at 25 °C for 15 min.
      NOTE: At this step, donor and acceptor beads are brought into proximity by the protein-peptide interactions. The final volume of the reaction mixture is 25 µL. Therefore, the final concentrations of compounds are ranging from 0.1 to 30 µM.
4. Plate reading
   NOTE: Read the plate using a plate reader set in the relevant mode.
   1. Turn on the instrument and open the software
   2. Choose the relevant protocol.
   3. Click Edit plate map and select the well being used in the plate for measurement.
   4. Click Next to continue and Run the selected protocol.
   5. After measurement, click Show Results to view results.
   6. Export the data.

Table 1: PCR reaction set up for human FKBP51 and FKBP52 DNA amplification.

Table 2: Thermocyler conditions for human FKBP51 and FKBP52 DNA amplification.