Name: direct measurement of kdm1a target engagement using chemoprobe-based immunoassays
Uploaded: 2019-06-13T13:00:00
Description: Watch this Scientific Journal Video about Direct Measurement of KDM1A Target Engagement Using Chemoprobe-based Immunoassays at JoVE.com

This study was financed by Oryzon Genomics. S.A., Hoffman-La Roche, and partially supported by the CIIP-20152001 and RETOS collaboration program RTC-2015-3332-1.

Blood samples were obtained from the Instituto de Investigaci&#243;n Biom&#233;dica Sant Pau Biobank according to Spanish legislation (Real Decreto de Biobancos 1716/2011) and approval of the local ethics committees. Studies with animal tissues were performed in accordance with the institutional guidelines for the care and use of laboratory animals (European Communities Council Directive 86/609/EEC) established by the Ethical Committee for Animal Experimentation at the PRAAL-PCB.
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<ol>
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The protocol presented here was developed to directly measure KDM1A target engagement using a novel KDM1A chemoprobe capture based ELISA. The method has been validated on cultured human cell lines and ex vivo samples from human, rat and mouse and baboon (including PBMCs, lung, brain, skin, tumors), but can be readily applied to other species in which the KDM1A antibody target epitopes and catalytic center are conserved. As OG-881 is an activity based chemoprobe, the sample quality is important and proper manipulation and...

Lysine specific demethylase 1 (KDM1A)1 is a demethylase involved in the control of gene transcription. This protein has emerged as a candidate pharmacological target2 in oncology; including Acute Myeloid Leukemia3 (AML), Myelodysplasia Syndrome (MDS)4, Myelofibrosis (MF)5,6, Small Cell Lung Cancer (SCLC)7; in Sickle Cell Disease (SCD)8,9, and in central nervous system diseases including Alzheimer&#39;s Disease (AD), Multiple Sclerosis (MS); and in aggression10.
Most of the KDM1A inhibiting compounds in clinical development are cyclopropylamine derivatives and inhibit the protein by covalent binding to its flavin adenine dinucleotide (FAD) cofactor11. Inhibition of KDM1A induces gene expression changes, but these changes vary enormously across tissues, cell types, or disease cases. Inhibition of KDM1A also changes histone marks12, yet these changes are generally produced locally at a specific site in the genome, and are again, highly tissue and cell specific.
The protocol was developed to directly measure KDM1A target engagement in biological samples and has been optimized for the use with cyclopropylamine derived inhibitors. The assay is based on ELISA technology and analyzes, in parallel, Total and Free (i.e. unbound by inhibitor) KDM1A in a native protein extract from a biological sample in a solid phase assay. As a first step, the biological sample is lysed in the presence of the biotinylated KDM1A selective chemoprobe OG-88113,14, derived from the selective KDM1A inhibitor ORY-1001 (iadademstat), a potent inhibitor of KDM1A in clinical development for the treatment of oncological disease. The chemoprobe has an IC50 for KDM1A of 120 nM and includes a FAD binding moiety linked to a biotinylated polyethylene glycol (PEG)-tail. The chemoprobe exclusively binds to the free KDM1A, but not to the inhibitor-bound KDM1A in the sample. After the chemoprobe binding, the KDM1A containing complexes in the sample are captured on microtiter plates with streptavidin coated surface to determine free KDM1A, or on plates coated with a monoclonal anti-KDM1A capture antibody to determine total KDM1A. After washing, both plates are incubated with an anti-KDM1A detection antibody, washed again, and incubated with a secondary HRP-conjugated donkey anti-rabbit IgG antibody for detection using a luminescent substrate and quantification by measuring relative light units (RLU) in a luminometer (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59390/59390fig1.jpg" /> 
Figure 1. Schema of ELISA Enzyme linked chemoprobe immunoabsorbent assay for KDM1A target engagement: A) Determination of total KDM1A using sandwich ELISA and B) Determination of free KDM1A using chemoprobe ELISA. <a href="https://www.jove.com/files/ftp_upload/59390/59390fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
A standard curve is included in both ELISA plates to verify the linearity of each assay. The determination of KDM1A target engagement in each sample is then calculated as a relative value to the pre-dose or vehicle treated sample.

<table>
	<tbody>
		<tr>
			<td>0,05% Trypsin-EDTA (1X)</td>
			<td>Thermo Scientific</td>
			<td>#25300-062</td>
			<td></td>
		</tr>
		<tr>
			<td>10 X Protease Inhibitor Tablets</td>
			<td>Roche</td>
			<td>#11836153001</td>
			<td></td>
		</tr>
		<tr>
			<td>96 deep well storage block</td>
			<td>VWR</td>
			<td>#734-1679</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well ELISA plates</td>
			<td>Nunc</td>
			<td>#436110</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive black Film</td>
			<td>Perkin Elmer</td>
			<td>#6050173</td>
			<td></td>
		</tr>
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			<td>Adhesive transparent Film</td>
			<td>VWR</td>
			<td>#60941-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated KDM1A probe OG-881</td>
			<td>Oryzon Genomics S.A.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td># 3117057001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin Standard</td>
			<td>Thermo Scientific</td>
			<td>#23208)</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford Protein Assay</td>
			<td>BioRad</td>
			<td>#500-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell lysis buffer 10X</td>
			<td>Cell Signaling</td>
			<td>#9803</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge for 96- well plates</td>
			<td>Hettich</td>
			<td>Rotina 420R</td>
			<td></td>
		</tr>
		<tr>
			<td>Flask</td>
			<td>Thermo Scientific</td>
			<td>#156499</td>
			<td></td>
		</tr>
		<tr>
			<td>Full length, enzymatically active human Recombinant LSD1 / KDM1A</td>
			<td>Active Motif</td>
			<td>#31426</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphpad Prism 5 Project</td>
			<td>GraphPad Software</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminol-Enhacer and Peroxide Solution (Chemiluminescent Substrate)</td>
			<td>Thermo Scientific</td>
			<td>#37074</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Centrifuge</td>
			<td>Eppendorf</td>
			<td>5415 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader Infinite 200-Tecan</td>
			<td>Tecan</td>
			<td>Infinite 200</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse monoclonal capture antibody Anti-KDM1A (N-terminal epitope)</td>
			<td>Abcam</td>
			<td>#ab53269</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle G18 gauge blunt</td>
			<td>BD</td>
			<td>#303129</td>
			<td></td>
		</tr>
		<tr>
			<td>ORY-1001 (iadademstat)</td>
			<td>Oryzon Genomics S.A.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>PBMC separation tubes 10 ml</td>
			<td>Greiner bio-one</td>
			<td>#163288</td>
			<td></td>
		</tr>
		<tr>
			<td>PBMC separation tubes 50 ml</td>
			<td>Greiner bio-one</td>
			<td>#227288</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1x</td>
			<td>Sigma</td>
			<td>#D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate shaker</td>
			<td>Heidolph Instruments</td>
			<td>Rotamax 120</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysorbate 20</td>
			<td>Sigma</td>
			<td>#P7949</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit monoclonal detection antibody Anti-KDM1A (C-terminal epitope)</td>
			<td>Cell Signaling</td>
			<td>#672184BF-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Secondary antibody Peroxidase-conjugated Donkey Anti-rabbit IgG</td>
			<td>Thermo Scientific</td>
			<td>#31458</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer cuvette 1.5</td>
			<td>Deltalab</td>
			<td>#302100</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer for cuvette</td>
			<td>GE Healthcare</td>
			<td>GeneQuant 1300</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin</td>
			<td>Promega</td>
			<td>#Z704A</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>BD</td>
			<td>#303172</td>
			<td></td>
		</tr>
		<tr>
			<td>Type 1 ultrapure water</td>
			<td>Millipore</td>
			<td>Milli-Q Advantage A10</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner</td>
			<td>VWR</td>
			<td>USC200T</td>
			<td></td>
		</tr>
	</tbody>
</table>

direct measurement of kdm1a target engagement using chemoprobe-based immunoassays

The assessment of the target engagement, defined as 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. In epigenetics, target engagement is most often assessed by the analysis of proxy markers instead of measuring the union of the compound to the target. Downstream biological readouts that have been analyzed include the histone mark modulation or gene expression changes. KDM1A is a lysine demethylase that removes methyl groups from mono- and dimethylated H3K4, a modification associated with the silencing of gene expression. Modulation of the proxy markers is dependent on the cell type and function of the genetic make-up of the cells investigated, which can make interpretation and cross-case comparison quite difficult. To circumvent these problems, a versatile protocol is presented to assess the dose effects and dynamics of the direct KDM1A target engagement. The assay described makes use of a KDM1A chemoprobe to capture and quantify uninhibited enzyme, can be broadly applied to cells or tissue samples without the need for genetic modification, has an excellent window of detection, and can be used both for basic research and analysis of clinical samples.

The Linearity of Total and Free KDM1A determination.
A Standard Series was prepared as described in step 5.3.2., using 0 to 2500 pg of full-length human recombinant KDM1A enzyme. The RLU values of Total and Free rKDM1A were assessed to verify the linearity (Figure 2A and 2B). Data are represented as mean from 3 experiments with 3 technical replicates (n) &#177; SD. ...

Watch this Scientific Journal Video about Direct Measurement of KDM1A Target Engagement Using Chemoprobe-based Immunoassays at JoVE.com

Direct Measurement of KDM1A Target Engagement Using Chemoprobe-based Immunoassays

Here, we present a protocol to measure KDM1A target engagement in a human or animal cell, tissue or blood samples treated with KDM1A inhibitors. The protocol employs chemoprobe tagging of the free KDM1A enzyme and direct quantification of the target occupation using chemoprobe-based immunoassays and can be used in preclinical and clinical studies.

The assessment of the target engagement, defined as the interaction of a drug with the protein it was designed for, is a basic requirement for the ...

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.

Research

JoVE Journal

Biochemistry

Covalent Binding of Antibodies to Cellulose Paper Discs and Their Applications in Naked-eye Colorimetric Immunoassays

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Cortisol Measurement in Koala (Phascolarctos cinereus) Fur

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Fixed Target Serial Data Collection at Diamond Light Source

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