Direct Measurement of KDM1A Target Engagement Using Chemoprobe-based Immunoassays

The assessment of the target engagement, that is, the interaction of a drug with the protein it was designed for, is a basic requirement for the interpretation of the biological activity of any compound in drug development, or in basic research projects. The main advantage of this technique is that it permits direct measurement of dose effect and dynamics of KDM1A target engagement, rather than measurements of downstream effects such as histone marks and expression. This technique can be applied in basic research and also in clinical trials, in oncological CNS or other diseases subject to treatment with KDM1A inhibitor to study pharmacodynamics.

This technique is based on a chemoprobe developed to study ORY-1001, iadademstat, but can be really used for other KDM1A inhibitors and the strategy can be applied to other targets. The procedure will be demonstrated by Raquel Ruiz, operation manager of the PK/PD discovery platform from my lab. During cell treatment with ORY-1001 the compound binds to the KDM1A protein present in the nucleus.

To begin this procedure, retrieve a 10 microliter single-use aliquot of 20 millimolar biotinylated probe OG-881 stock solution from the refrigerator and let it warm up at room temperature for 10 minutes. Using a micropipet with filter tips, serially dilute the stock solution to prepare the two micromolar working solution. Next, dissolve one tablet of protease inhibitor in one milliliter of PBS in a microcentrifuge tube.

For each milliliter of the desired volume of 1x cell lysis buffer with the chemoprobe, mix 100 microliters of commercial obtained 10x cell lysis buffer, 150 microliters of the 10x protease inhibitor, 12.5 microliters of the two micromolar OG-881 working solution and 737.5 microliters of type-1 double distilled water. Cells are lysed in 1x lysis buffer in presence of the chemoprobe that binds to any free KDM1A protein. To prepare from tissues, use a mortar and pestle that are chilled on dry ice to pulverize and homogenize a one cubic centimeter piece of frozen tissue.

Aliquot the tissue into single-use vials, making sure to transfer approximately 40 milligrams of tissue powder into each vial, while avoiding thawing at all times. Store the samples at 80 degrees Celsius until ready to process. To prepare from cell pellets, resuspend the pellet of approximately 10 million cells in 200 microliters of 1x cell lysis buffer containing 25 nanomolar OG-881.

Vortex each sample briefly and keep them on ice for five minutes. Using a sonicator, sonicate the samples with three pulses at 45 kilohertz lasting for 20 seconds each. Place the samples on ice for 20 seconds between pulses.

Keep the samples on ice for an additional five minutes. Then, vortex briefly and centrifuge the samples at 14, 000 times G for 10 minutes in a centrifuge that is pre-chilled at four degrees Celsius. Using a one milliliter micropipet transfer each supernatant into a separate 1.5 milliliter microcentrifuge tube.

Leave the tubes on ice for two hours before processing. Then, quantify the native protein using the Bradford assay as outlined in the text protocol. A total plate is coated with anti-KDM1A antibody.

Free plate is coated with streptavidin. Cell lysate is then added to both plates and KDM1A complex is captured directly or through the chemoprobe. Plates are washed and incubated with the anti-KDM1A detection antibody and a secondary antibody coupled to horseradish peroxidase.

Finally, luminescent substrate is added and signal generated. For total KDM1A ELISA, prepare 10 milliliters of KDM1A capture antibody in PBS to a final concentration of two micrograms per milliliter for each plate. Transfer 100 microliters into each well of the plate.

For free KDM1A ELISA, prepare 10 milliliters of streptavidin in PBS as a concentration of 10 micrograms per milliliter for each plate. Transfer 100 microliters into each well of the plate. Then, top seal each plate with adhesive film and incubate overnight at four degrees Celsius.

The next day, remove the plates from the refrigerator and let the them equilibrate at room temperature for approximately 45 minutes. Wash each plate three times with wash buffer, making sure to tap the plate on paper towels after each washing step to remove any residual solution. Add 200 microliters of blocking buffer to each well of both plates.

Top seal the plates with adhesive film and incubate at room temperature for two hours. Meanwhile, dilute the previously obtained native protein extracts with PBS to the appropriate concentration and lay out the plate to prepare a standard curve using human rKDM1A as outlined in the text protocol. After the two hour incubation is complete, discard the blocking buffer and wash the plates with wash buffer, making sure to tap the plate on paper towels after each washing step to remove any residual solution.

Transfer the appropriate diluted samples to a refrigerated 96 deep well storage block following the plate distribution found in table two of the text protocol. Keep this block on ice while pipetting 100 microliters per well into the total and free ELISA plates following the plate distribution found in table three of the text protocol. Incubate at room temperature for one hour, then discard the samples and wash the plates five times with wash buffer.

Prepare 20 milliliters of rabbit anti-KDM1A detection antibody in blocking buffer at a concentration of 0.125 micrograms per milliliter. Add 100 microliters of the detection antibody solution to each well of both plates, except those corresponding to the negative controls. Top seal the plates with adhesive film and incubate at room temperature for one hour.

After this, discard the detection antibody solution and wash the plates six times with wash buffer. Prepare 25 milliliters of secondary goat anti-rabbit antibody, HRP, to a dilution of one to 5, 000 in blocking buffer. Add 100 microliters of the secondary antibody solution to each well of the microtiter plates and incubate at room temperature for one hour.

30 minutes before the end of this incubation, mix equal parts of luminol enhancer and peroxide solution in an amber bottle under soft light conditions. Leave this mixture at room temperature. When the one hour incubation is complete, discard the secondary antibody solution and wash the plates six times with wash buffer.

Next, pipet 100 microliters of the luminol working solution to each well of the plates, making sure to pipet very slowly to avoid bubble formation. Use a timer to control the time between the addition of the solution and the luminescence measurement of the plates and keep this time constant to achieve a good inter-assay reproducibility. Top seal the plates with adhesive film and centrifuge at 500 times G and at room temperature for 45 seconds to eliminate any remaining bubbles.

Incubate the plates on a plate shaker at 100 rpm for one minute. Then, remove the adhesive film and insert the plate into a microplate reader and leave it for three minutes to stabilize the temperature at 25 degrees Celsius. Read the relative luminescence units of each ELISA plate assay.

Save and copy the raw RLU values from the raw data spreadsheet files for further analysis. In this study, a novel KDM1A chemoprobe capture based ELISA is used to directly measure KDM1A target engagement in cells and tissue samples. The RLU values of total and free rKDM1A are assessed to verify the linearity.

The RLU values of total and free KDM1A detected in human PBMCs from three independent volunteers are then superimposed on the standard curve. AML cells are cultured following provider recommendations and are treated with either vehicle or ORY-1001 at different concentrations. The native protein extract are obtained in the presence of 25 millimolar OG-881 chemoprobe and 0.5 micrograms of total protein is used to perform the target engagement's analysis.

Total and free KDM1A are then determined and the percentages of target engagement of ORY-1001 to KDM1A is calculated relative to the vehicle. The dose-responsive KDM1A target engagement in PBMCs and in lung treatment of rats with ORY-1001 by oral gavage, calculated relative the vehicle group is shown here. The ex vivo incubation with 25 nanomolar ORY-1001 of lung protein extracts from the vehicle treated animals, yields full TE, yet, but does not further increase TE in samples from rats treated for four days with 30 micrograms per kilogram ORY-1001, confirming KDM1A was already fully inhibited in vivo.

This protocol assess KDM1A target engagement by measuring free and total KDM1A. Remember it requires native protein extraction. This technique can be used on samples from different species to assess a specific inhibitor, also to screen for new inhibitors.

The chemoprobe used in this study can also be applied for chemoproteomics to study the KDM1A interactum. Remember to work safely and respect at all times preventative measures when handling biological samples and bioactive compounds like KDM1A inhibitor.