The regulation of the eIF4F complex signaling is associated with increased translation of mRNA subset that are involved in cancer proliferation and survival. Here we describe an eIF4E-eIF4G cell-based protein-protein interaction assays that enables us to assess drug-induced perturbation in eIF4F complex integrity in live cells. There is a growing interest in the development of modalities that efficiently target protein-protein interface.
We envisioned that our eIF4E-eIF4G PPI assays will help in fostering these strategies and validate them. This assay will provide an optimal primary scheme for it to lead optimization of eIF4E-eIF4G inhibitors. Seeding the cell correctly and performing the transfer transfection are the most challenging aspect of this product since they may reflect directly on the suppression of the PPI reporting system.
After thawing and counting cells, seed 6 well plates with HEK 293 cells, using 2 milliliters of standard growth medium per well. On the morning of day 2, for each planed transfection, dilute 9 microliters of liposome-based solution in a tube containing 125 microliters of reduced serum medium without phenol red. While the diluted liposomes incubate at room temperature for 5 minutes, prepare the master mix of DNA by diluting 3 micrograms of each plasmid in 125 microliters of reduced serum medium for each transfection tube.
Add 12 microliters of enhancer reagents to the DNA master mix 2, then mix well. Immediately add this mixture to each tube of diluted liposomes in a ratio of 1 to 1. After incubating this DNA lipid complex for 15 minutes at room temperature, add the complex to each well of the 6 well plates.
Incubate the cells at 37 degrees Celsius with 5%carbon dioxide for 24 hours. On the morning of day 3, rinse each well with 1 milliliter of PBS, and add 0.3 milliliters of trypsin. Incubate the plates at 37 degrees Celsius for 5 minutes.
After incubation, neutralize the trypsin by adding 2 milliliters to each well of the reduced serum medium without phenol red. Transfer the transfected cells into a 15 milliliter tube. Next, centrifuge the cells at 290x G for 5 minutes.
Aspirate the medium, and re-suspend the cell pellet in 2 milliliters of the reduced serum medium. Seed the transfected HEK 293 cells in 96-well opaque plates, at a density of 30, 000 cells per well in 90 microliters of medium. To obtain 3 technical replicates for 3 different compounds within the same experiment, seed 60 wells.
Exclude the wells on the edges. Immediately after seeding the transfected cells, add 10 microliters of 10%DMSO compound solution to each well. Prepare 1 millimolar compound stock solutions by dissolving each compound of interest in 100%DMSO.
In order to have 3 replicates for each compound titration, use 8 microliters of the 1 millimolar compound stock solution. Perform a two-fold serial dilution of each stock compound solution in 8 wells of a 96-well PCR plate by pipetting 4 microliters of the 1 millimolar stock into 4 microliters of 100%DMSO for each titration point. Discard the excess 4 microliters after the last point of the two-fold serial dilution.
Add 36 microliters of HPLC grade sterile water to each tube to prepare 40 microliters of 10x compound serial dilution solutions in 10%DMSO. Also, prepare a control. 10%DMS only stock solution in HPLC grade sterile water.
Add 10 microliters of 10x working solutions to the cells in the 96-well opaque plate in order to yield the intended final concentration, with a residual DMSO concentration of 1%Incubate the plate at 37 degrees Celsius with 5%carbon dioxide for 3 hours. After 3 hours, start preparing the luciferase substrate reagent by combining 1 volume of substrate with 19 volumes of the dilution reagent. Use a multi-channel pipette to immediately add 25 microliters of the substrate reagent to each well of the 96 well plate with the cells.
Shake the plate on an orbital shaker at 350 RPM, for 50 minutes at room temperature. To assess luminescence, place the plate on a plate reader. Set the mirror reader to luminescence and the emission filter to 455.
Use a measurement height of 6.5 millimeters with a measurement time of 1 second. To assess cell viability, add 33 microliters of viability assay reagent to each well. After 15 minutes at room temperature, assess luminescence with the plate reader by setting the mirror reader to luminescence and the emission filter to 600.
Use a measurement height of 6.5 millimeters and a measurement time of 1 second. Use the data from the plate reader to determine the IC 50 value of each compound, by curve-fitting the data to the 4-parameter fitting curve equation described in the manuscript. HEK 293 cells were transfected with the eIF4E-eIF4G complementation system, and then receded and treated with mTOR inhibitors.
When luminescence was assessed 4 hours after treatment, PP242 and rapamycin both produced a dose-dependent inhibition of the signal. Neither PP242 nor rapamycin produced a significant decrease in cell viability, indicating that the decrease in luminescence in the eIF4E-eIF4G complementation system is not due to non-specific cell death, but rather to disruption of the EIF4E-4G interaction. Western blot analysis, following m7GTP pull down experiment, showed that 4EBP1 mediated disruption of endogenous eIF4E-eIF4G interaction correlates with the measured eIF4E-eIF4G assay signal.
PP242 was a more potent inhibitor of total 4EBP1 phosphorylation than rapamycin. Both inhibitors showed an impact on mTOR signaling normally, with rapamycin being more active against mTORC1 substrates, and PP242 targeting both mTORC1 and mTORC2. The most critical aspect of this product are seeding the cells on the day of transaction, re-seeding the cells in a medium without phenol red, assessing the luminescence of the eIF4E-eIF4G complementation assay, and run in the viability assay on the same plate.
A secondary viability assay can be performed to assess any drug of target and known specific effects.
