We built a homogeneous bead-based assay to study chaperone-cochaperone interactions. Using this technique, we screen small molecules inhibiting interactions between Hsp90 and FKDP51 or FKBP52 and identify potent and selective inhibitors. This high throughput screening technique is robust and reliable.
Results can be obtained within one hour, and it only requires small amounts of beads protein and compounds. Hsp90 interaction with this cochaperones was implicated in human disorders, such as Alzheimer's disease, Cancer, Autoimmune disease. This technique provides the opportunity to screen chaperone-cochaperone inhibitors with high medical importance.
Here is the basic principle of this assay. When the interaction between Hsp90C-Terminal peptide and TPR domain of cochaperones brings the donor and acceptor beads into proximity, a highly amplified signal is generated. When the interaction inhibitors are added, the donor and acceptor beads cannot reach proximity, and no detectable signal is produced.
Begin by diluting the Hsp90 peptide in PBS at one milligram per milliliter concentration. Dilute the unconjugated acceptor beads in PBS, and transfer the beads into a 1.5 milliliter tube. Centrifuge the diluted beads for washing, and then carefully remove the supernatant.
For conjugating, set the acceptor beads in peptides at a ratio of 10 to 1. To the paletide beads at PBS diluted peptide, Tween 20 and Sodium cyanaboroughhydride solution, and incubate with end over end agitation on a rotary shaker. To block the unreacted sites, add 20 microliters of one molar Tris hydrochloride solution of pH 8.0 to the beads, and quench the reaction by incubating for one hour at 37 degrees Celsius.
At the end of the incubation, wash the beads by centrifugation and re-suspend the bead pellet in one milliliter of Tris hydrochloride solution. Repeat the washing two more times. After the last washing, re-suspend the beads at one milligram per milliliter concentration in storage buffer and store the conjugated acceptor bead solution at 4 degrees Celsius protected from light.
Add glutathione donor beads at 10 micrograms per milliliter concentration in PBS. Add GSTFKBP51 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 and 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 Hsp90C-Terminal peptide to 100 micrograms per milliliter concentration in 0.5 times 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. Create an X-Y data table in the welcome dialog, select X numbers, Y and enter three replicate values in side-by-side columns.
Import the concentration values to the X column and the signal values to the Y column. Click analyze, select transform concentration X, and then select transform to logarithms. Click analyze and choose nonlinear regression under X-Y analysis.
Open the dose response inhibition option and choose log versus response variable slope equation. Click okay to view the results containing IC50 value and graphs. This assay was used for screening small molecules, inhibiting interactions between Hsp90 and FKBP51 or FKBP52.
The Z score of more than 0.8 and signal to background ratio of 13.35, demonstrate the robustness and reliability for high throughput screening. The effect of small molecular weight test compound D10 on inhibition of chaperone-cochaperone interactions was assessed and IC50 values were calculated, where D10 showed dose dependent inhibition. The IC50 value for Hsp90 GSTFKBP51 interactions was 65 nanomolar.
Whereas for Hsp90, GSTFKBP52 interactions, complete inhibition was not achieved with the highest compound concentration indicates that selective inhibition of protein-protein interaction with small molecules can be achieved. The critical step is the order of addition. We first form a complex between a small molecule and its TPR target.
Otherwise longer incubation and higher compound concentrations are required. We can use this technique to screen for small molecules disrupting the interaction between Hsp90 and FKB51 or Hsp90 and FKB52. It can also be extended to any interaction between Hsp90, Hsp70 and a TPR motif containing cochaperone screening selective inhibitors.
