Assessing Cellular Target Engagement by SHP2 (PTPN11) Phosphatase Inhibitors

The tyrosine phosphatase SHP2, is a key mediator of cellular signaling, promoting cell survival and proliferation. My team is focused on finding small molecule SHP2 inhibitors for the treatment of cancer. Our technique provides a rapid and powerful method to establish cell-penetrants, as well as target engagement of small molecule SHP2 inhibitors, in cells.

This is assures, discovery resources are efficiently directed. SHP2 as an attractive target for cancer therapy. And several SHP2 inhibitors, are in clinical trials.

Our technique to facilitate the discovery of next generation inhibitors with improved efficacy profiles. Demonstrating to procedure would be Celeste Romero, a research assistant and Douglas Sheffler, a research assistant professor, from our laboratory. To begin detach HEK293 T-cells from the plates, using three milliliters of cell detachment reagent.

Dilute the cells with 12 milliliters of growth media, and collect them by centrifugation at 1, 400 times G, for four minutes. Resuspend the cell palate in 10 milliliters of growth media and measure the concentration and viability of the cells with trypan blue and a cell counter. Plate 700, 000 exponentially growing cells into each well of a six well culture plate and incubate them for 24 hours at 37 degrees Celsius, and 5%carbon dioxide.

On the next day, dilute two micrograms of plasma DNA into 200 microliters of transfection buffer. Vortex the plasma DNA for 10 seconds and centralfuge it at 1, 400 times G for four minutes. Add four microliters of transfection reagent to the diluted DNA.

Vortex it for 10 seconds, and repeat the centrifugation. Incubate the DNA at 23 degrees Celsius for 10 minutes, then add the transfection mix to the attached HEK293 T cells in the six well plate, and return the cells to the incubator for another 24 hours. To prepare assay plates, dilute inhibitor solutions in DMSO, for a stock concentration of 10 millimolar and dispense them into a 384 well low dead volume source plate, for immediate use.

Spot the desired volume of inhibitors or vehicle, using a liquid handler into 384 well real time PCR plates at a target final volume of less than 0.5%DMSO. Seal the plates using a plate sealer with inert gas purging. To prepare the transfected cells pre incubate growth media and sell detachment reagent in a 37 degrees Celsius water bath.

Remove the cells from the incubator and gently aspirate the media from the wells. Add 0.3 milliliters of the cell detachment reagent to each well, and gently rock the plate back and forth to thoroughly cover the surface of the plate bottom. Incubate the plate at 23 degrees Celsius for two minutes.

Add one milliliter of growth media to each well, and gently pipette the cells in the well. Then transfer them to a 15 milliliter Falcon centrifuge tube Centrifuge the cells at 1, 400 times G for four minutes to collect the cells. Gently aspirate the media, then resuspend the cell pellet in two milliliters of growth media.

Ensure the cell viability is greater than 90%using try pan blue and a cell counter. Dilute cells to a concentration of 125 cells per microliter, keeping the cells in suspension for no more than two hours for optimal viability. For incubation with SHP2 inhibitors, dispense the cells into a sterile single channel solution trough.

Centrifuge the previously prepared 384 well real-time PCR plate at 2, 500 times G for five minutes, then remove the seal and use a 125 microliter multi-channel pipette, to add five microliters of the diluted cells to the desired Wells. Centrifuge the plate at 42 times G for 30 seconds without a lid, then attach a lid and incubate the plate for one hour at 37 degrees Celsius and 5%carbon dioxide. Program the Thermo cycler, according to manuscript directions for either the thermal profile gradient or isothermal experiment.

Start the thermocycler heat pulse program, and place the assay plate on the thermal block. When the program is finished, remove the assay plate. Supplement each well of the assay plate with five microliters of licensed detection master mix.

Centrifuge the plate at 42 times G for 30 seconds, and store it at 23 degrees Celsius in darkness for 30 to 60 minutes. Measure chemiluminescence using a microplate reader with the optimized integration time. The thermal gradient experiment for wild type SHP2, resulted in a sigmoidal cellular thermal profile with a narrow melting transition that is typical for a folded protein.

Incubation of wild type SHP2 with the allosteric inhibitor SHP099, stabilized to the thermal profile to a significant and measurable degree. The stabilization of SHP2, also tracked with the potency of SHP099 like allosteric compounds. At 10 micromolar, the more potent RMC-4550, produced a greater degree of stabilization than SHP099 for wild type SHP2.

Due to their unique mechanism, SHP099 like allosteric inhibitors are less effective against many SHP2 oncogenic mutants, including SHP2-E76K. Consistent with these known properties only marginal thermal stabilization of SHP2-E76K by SHP099 is observed. The expression level of the EPL tag protein can dramatically influence the signal intensity.

Thermal profiles for transient and stably integrated wild type cells. under identical asset conditions are shown. Normalization of the disparate curves affords comparable thermal profiles, revealing the wide range of the EFC detection system.

Taking advantage of the thermal cycler ability to produce thermal gradients on the short axis of a 384 well plate. a series of isothermal titrations can be performed on a single plate. At an optimal temperature, an isothermal titration using a full 10 point Dose-Response, yielded a useful EC50, for RMC-4550.

The principles of this asset could be used to monitor the degradation of proteins in cells, induced by PROTAC compounds. We are using cellular Thelma shift technique to discover inhibitors of mutant SHP2 and leukemias, drugs specific against frequent SHP2 oncogenic mutants will have a significant impact for the treatment of these cancers.