A Semi-Quantitative Drug Affinity Responsive Target Stability (DARTS) assay for studying Rapamycin/mTOR interaction

Drug affinity responsive target stability, DARTS, is a robust method for detection of novel small-molecule protein targets. It can be used to verify known small-molecule protein interactions and to find potential protein targets for natural products. In this study, we further enhance the data analysis capabilities of the DARTS experiment by monitoring the changes in protein stability and estimating the affinity of protein-ligand interactions.

The protein-ligand interactions can be plotted against two curves, a proteolytic curve and a dose-dependence curve. We've used the mTOR-rapamycin interaction as an exemplary case for the establishment of our protocol. Grow 293T cells using DMEM with 10%fetal bovine serum, two-millimolar glutamine, and 1%antibiotics.

Incubate the cultures at 37 degrees Celsius under 5%carbon dioxide. When the culture reaches 80 to 90%confluence, wash the cells twice with cold PBS. Use a cell scraper to collect the cells into an appropriate amount of cold cell lysis buffer and transfer the lysing cells into a 1.5-millimeter tube.

Invert to mix the lysis buffer and lysing cells well and incubate the tube on ice for 10 minutes. Centrifuge the tube at 18, 000 times g for 10 minutes at four degrees Celsius. Transfer the supernatant into a new 1.5-milliliter tube and keep chilled on ice.

Perform BCA assay to approximate the protein concentration of lysates and calculate the dilution of the pronase based on the protein concentration. First, transfer the lysate into two 1.5-milliliter tubes, 99 microliters each. Add one microliter of small-molecule stock solution to each aliquot of lysate and incubate the two tubes for 30 to 60 minutes at room temperature with shaking.

On ice, establish serial dilutions of freshly thawed pronase solution in 1x TNC. Following the incubation with the small molecule, divide each aliquot into 20 microliters of samples in five tubes. To ensure the same digestion time, at specific intervals of every 30 seconds, add two microliters of the prepared pronase solutions to the samples accordingly.

For the one control group, add two microliters of TNC buffer. After five to 20 minutes, halt the digestion of the six tubes via the addition of two microliters of cold 20x protease inhibitor cocktail at 30-second intervals. Mix well and incubate on ice for 10 minutes.

Dilute each sample with six microliters of 5x SDS-PAGE loading buffer and boil the samples in a water bath at 70 degrees Celsius for 10 minutes. Now, to perform Western blot, load equal amounts of protein into the wells of an 8%SDS-PAGE gel, along with an appropriate molecular weight marker. Run the gel for 30 minutes at 80 volts, then adjust the voltage to 120 volts and continue running for one to two hours.

Verify that the small molecule can bind directly to the potential target proteins. In this protocol, incubation with the small molecule confirmed the protection against proteolysis. Three bands were found to be protected by incubation with rapamycin over vehicle control.

Western blotting illustrated the presence of mTOR protein at low pronase to protein ratios and its reduction and loss with increasing ratios. Proteolysis of mTOR by pronase was clearly inhibited by the presence of rapamycin and the addition of rapamycin generated an obvious shift in the proteolytic curve. Rapamycin dose-dependently enhanced the level of mTOR, suggesting the rising stability of mTOR with rapamycin treatment.

Quantification of the target protein band intensities suggested that mTOR is the target protein of rapamycin. In order to get the best stepwise effect, this experiment may have to be repeated several times to obtain a suitable range of pronase over protein ratios.