Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

NAPPA is a powerful protein microarray platform that can be used for the study of protein activity and function in an unbiased high throughput manner. The proteins displayed on NAPPA are produced in a human based expression system using human derived ribosomes and chaperones in order to improve the likelihood of natural folding and activity. The use of NAPPA arrays for the screening of kinase inhibitors provide a new platform for the test of new drugs and clinically relevant kinase mutations.

Protein microarrays generated by NAPPA methodology can be used for many distinct applications, including biomarker discovery, protein-protein interactions, substrate identification, and drug screening. Perform quality control steps along the way. Test the quality of your DNA preparation, the DNA microarray, and the protein microarray.

If in any step the quality's not good, just start over. Demonstrating the procedure will be Lisa Miller, a technician in my laboratory. To prepare bacterial growth for in house high throughput mini-prep, first inoculate the bacteria in the auger plate in a 96 well format, and let it grow for 16 hours.

The next day, fill each well of the deep well plate with 1.5 milliliters of terrific broth medium, supplemented with antibody ampicillin. The antibody depends on the selection marker on the plasmid. Then, sterilize a 96-pin device with 80%ethanol and flame.

Use the sterile 96-pin device to pick colonies from the overnight incubated agar plate and submerge into the wells of the deep well plate to inoculate the culture. Cover the block with a gas-permeable seal and incubate on a shaker for 22 to 24 hours at 37 degrees Celsius, and 300 to 800 rpm. After that, pellet cultures and resuspend the cultures according to the manuscript.

Lyse bacteria by adding 200 microliters of solution two into each well, seal the plate with an aluminum seal, and invert five times. Incubate the cells for exactly five minutes. To neutralize the solution, add 200 microliters of solution three, seal the plate with an aluminum seal, and invert five times.

Then, spin the plate at 3800 G, four degrees Celsius, for 30 minutes to clear the lysate supernatant. To prepare anion exchange resin plates, first, mix the anion exchange slurry, and pour the slurry into a glass trough. Stack filter plates on top of a deep well plate to act as a waste collection vessel.

Then, using wide board P1000 tips, mix the slurry, and transfer 450 microliters of the slurry into each well of the filter plates. Centrifuge and discard the flow-through. Now, transfer the lysate supernatants to the stack of resin plate and deep well block.

Spin the stacked plates for five minutes at 30 times G with slowest ramp up speed and discard the flow-through. To wash the column, add 400 microliters of solution N3 wash buffer to each well. Transfer the resin plate to vacuum manifold to remove the wash buffer.

Repeat the washing step three times and then remove any remaining washer buffer with a brief five minute spin at 30 times G.To elute the DNA, place the resin plate onto a clean 800 microliter collection plate. Add 300 microliters of solution N5 to each well and let it sit at room temperature for ten minutes. Then, spin the stacked plates for five minutes at 20 times G with slow ramp up speed to 233 times G for one minute.

Store plates at negative 20 degrees Celsius prior to use. First, place the slides on a rack and submerge the slides in glass reservoir containing the coating solution of 2%aminosilane reagent in acetone. Rock the slides for 15 minutes.

Then, rinse the slides in acetone followed by a final rinse in water. Next, dry the slides using pressured air. Blowing on them from all angles for about three minutes until all water droplets have been removed.

Store the coated slides at room temperature on a metal rack inside a tightly sealed box. Now, proceed to precipitate the DNA as described in the manuscript. Then, resuspend the DNA pellet in 20 microliters of ultrapure water and shake at 1000 rpm for two hours.

To prepare array sample, add 10 microliters of freshly prepared printing mix which contains the antibody, crosslinker, and polylysine to each DNA sample. Seal plates with aluminum foil and shake at room temperature for 90 minutes at 1000 rpm. Store the plates overnight at four degrees Celsius.

On the printing day, briefly vortex and spin the plates. Transfer 28 microliters of each sample to a 384 well plate. Place the aminosilane coated slides and the 384 well plate, on the arrayer deck, and start the microarray printing program.

When the printing is done, label the microarrays. The microarrays can be stored for months until further use. To measure the levels of DNA, place the slides in a pipette box and block them with 50 milliliters of blocking buffer.

Incubate the slides on a rocking shaker at room temperature for one hour. Then, discard the solution and add 20 milliliters of blocking buffer and 33 microliters of fluorescent DNA intercalating dye. Incubate for 15 minutes with agitation.

After the incubation is over, quickly rinse the slides with ultra pure water and dry with pressured air. The microarrays are ready to be scanned. To express the microarrays, block the slides as previously shown and then rinse them with ultra pure water, and dry with filtered compressed air.

Attach a sealing gasket to each slide. Add 130 milliliters of in vitro transcription and translation mix onto the slides, and gently massage the sealing gaskets so that the in vitro transcription and translation mix spreads out and covers the whole area of the array without any bubbles. Apply the small round port seals to both ports.

Incubate for 90 minutes at 30 degrees Celsius for protein expression, followed by 30 minutes at 15 degrees Celsius for immobilization of the query protein. Then, remove the gasket, wash the slides three times for five minutes each in 15 milliliters TBST with milk, and block in the same solution for one hour. For the detection of the proteins on the array, remove the TBST with milk, place the slides on a grid plate, and apply 600 microliters of primary antibody, mouse anti-Flag, diluted one to two hundred, and 1x TBST supplemented with 5%milk.

Incubate for one hour at room temperature. Wash the slides with 50 milliliters of 1x TBST and 5%milk on the rocking shaker three times for five minutes each. Repeat the procedure for the secondary antibody.

After the one hour incubation is over, wash the slides with 1x TBST on the rocking shaker three times for five minutes each. Then, quickly rinse the slides with ultra pure water, and dry using pressured air. The slides are ready to be scanned.

To perform the phosphatase and DNase treatment, wash and block the expressed slides with TBST supplemented with 3%BSA as described in the manuscript. Then, place the slides on a grid plate and apply 200 microliters of phosphatase DNase solution. Place a microarray cover slip on top to avoid evaporation.

Incubate at 30 degrees Celsius for one hour and 30 minutes in the oven. Then, wash slides with 1x TBST and 0.2 molar sodium chloride on the rocking shaker three times for five minutes each. To perform the drug treatment and kinase reaction, place the slides on the grid plate and apply 200 microliters of drug kinase solution.

Place a cover slip on top to avoid evaporation. Incubate for one hour at 30 degrees Celsius in the oven. The key to reproducible data is consistent incubation time across slides.

This especially important during the kinase reaction. The time required to process each slide should be accounted for. After that, wash slides with 1x TBST and 0.2 molar sodium chloride on the rocking shaker three times for five minutes each.

Repeat protein detection using as primary antibody phosphotyrosine antibody diluted one to 100 in TBST, supplemented with 3%bovine serum albumin. Use 1x TBST and 3%bovine serum albumin for washing after the incubation with primary antibody, and TBST for washes after incubations with secondary antibody. Wash and dry as previously.

For image acquisition, load the microarrays into the slide holder magazine. Load the magazine into the microarray scanner. Scan all microarrays with the optimized settings and remember to turn the auto gain off when comparing images across experiments.

Transfer the images to a quantification software, align the grid with the spots, and quantify the signal intensity of each feature on the microarray. Proceed with statistical analysis. In this study, the microarray showed the majority of spots containing complementary DNA successfully displayed detectable levels of protein.

NAPPA kinase microarrays showed good reproducibility among slides, with the correlation of the levels of protein display among distinct printing batches higher than 0.88. Representative results of kinase activity in NAPPA kinase arrays showed high levels of protein phosphorylation after expression. The comparison between microarrays in which the phosphorylation levels were measured right after phosphatase treatment, and after 60 minutes of autophosphorylation reaction, suggests the presence of active protein kinases on the array.

On NAPPA kinase arrays, imatinib showed a significant reduction in ABL1 and BCR-ABL1 activity, whereas other kinases remained mostly unaffected. The kinase activity normalized against the dephosphorylated array and represented as a percentage of the positive control microarray showed selective inhibition of imatinib towards ABL1 and BCR-ABL1. Typical results obtained for the ibrutinib screening showed the kinase activity of ABL1 non-relevant kinase is unaffected.

BTK canonical target, and ERBB4 potential new target, Showed reduced activity in the presence of ibrutinib. The data suggests ERBB4 can be inhibited by ibrutinib in a dose-specific fashion. The success of protein microarray screenings are highly dependent on the quality of the microarray itself.

Several positive and negative control features should be used to allow proper data analysis. NAPPA kinase assay is a screening platform, and as such, the data obtained should be validated in followup assays, which may include in vitro kinase assays, or cell based assays. Since our protocol starts with cDNA, any kinase mutation or variation can be easily incorporated in the microarray and studied in high throughput.

During the preparation of the anion exchange resin slurry and plates, it is recommended the use of masks to prevent possible inhalation of the resin particles.