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.

<ol>
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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Hypomethylation+Signature+Predicts+Antitumor+Activity+of+LSD1+Inhibitors+in+SCLC.">DNA Hypomethylation Signature Predicts Antitumor Activity of LSD1 Inhibitors in SCLC.</a> Cancer Cell. 28 (1), 57-69 (2015).</li><li>Rivers, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RN-1,+a+potent+and+selective+lysine-specific+demethylase+1+inhibitor,+increases+&#947;-globin+expression,+F+reticulocytes,+and+F+cells+in+a+sickle+cell+disease+mouse+model.">RN-1, a potent and selective lysine-specific demethylase 1 inhibitor, increases &#947;-globin expression, F reticulocytes, and F cells in a sickle cell disease mouse model.</a> Experimental Hematology. 43 (7), 546-553 (2015).</li><li>Rivers, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+administration+of+the+LSD1+inhibitor+ORY-3001+increases+fetal+hemoglobin+in+sickle+cell+mice+and+baboons.">Oral administration of the LSD1 inhibitor ORY-3001 increases fetal hemoglobin in sickle cell mice and baboons.</a> Experimental Hematology. 67, 60-64 (2018).</li><li>Buesa, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dual+LSD1/MAO-B+inhibitor+ORY-2001+prevents+the+development+of+the+memory+deficit+in+samp8+mice+through+induction+of+neuronal+plasticity+and+reduction+of+neuroinflammation.">The dual LSD1/MAO-B inhibitor ORY-2001 prevents the development of the memory deficit in samp8 mice through induction of neuronal plasticity and reduction of neuroinflammation.</a> Alzheimer&#8217;s &amp; Dementia. 11 (7), P905 (2015).</li><li>Schmidt, D. M., McCafferty, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-2-Phenylcyclopropylamine+is+a+mechanism-based+inactivator+of+the+histone+demethylase+LSD1.">Trans-2-Phenylcyclopropylamine is a mechanism-based inactivator of the histone demethylase LSD1.</a> Biochemistry. 46 (14), 4408-4416 (2007).</li><li>Forneris, F., Binda, C., Vanoni, M. A., Battaglioli, E., Mattevi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+histone+demethylase+LSD1+reads+the+histone+code.">Human histone demethylase LSD1 reads the histone code.</a> Journal of Biological Chemistry. 280 (50), 41360-41365 (2005).</li><li>Gonz#225;lez, E. C., Maes, T., Crusat, C. M., Mu#241;oz, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oryzon+Genomics,+Methods+to+determine+kdm1a+target+engagement+and+chemoprobes+useful+therefor.">Oryzon Genomics, Methods to determine kdm1a target engagement and chemoprobes useful therefor.</a> , (2016).</li><li>Mascaró, C., Ortega, A., Carceller, E., Rruiz Rodriguez, R., Cicero, F., Lunardi, S., Yu, L., Hilbert, M., Maes, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoprobe-based+assays+of+histone+lysine+demethylase+1A+target+occupation+enable+in+vivo+pharmacokinetics+and+-dynamics+studies+of+KDM1A+inhibitors.">Chemoprobe-based assays of histone lysine demethylase 1A target occupation enable in vivo pharmacokinetics and -dynamics studies of KDM1A inhibitors.</a> Journal of Biological Chemistry. , (2019).</li><li>Rodriguez-Suarez, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+Homogeneous+Nonradioactive+Methyltransferase+and+Demethylase+Assays+Targeting+Histone+H3+Lysine+4.">Development of Homogeneous Nonradioactive Methyltransferase and Demethylase Assays Targeting Histone H3 Lysine 4.</a> Journal of Biomolecular Screening. 17 (1), 49-58 (2011).</li><li>Lynch, J. T., Cockerill, M. J., Hitchin, J. R., Wiseman, D. H., Somervaille, T. 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The author Tamara Maes is an executive director and shareholder, and the authors Cristina Mascar&#243; and Raquel Ruiz Rodriguez are employees of Oryzon Genomics S.A. Oryzon Genomics S.A. develops KDM1A inhibitors and holds patents covering compounds and methods used in this Article.

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 conservation of samples should be pursued especially during the initial steps of the protocol, to ensure the KDM1A activity is conserved.
The current experimental protocol was optimized to analyze KDM1A target engagement by covalent FAD targeting inhibitors. It can also be used with reversible inhibitors that block the access to the FAD cofactor of KDM1A. Potent reversible inhibitors with long residence times may employ the unmodified protocol.
The OG-881 chemoprobe may not be suitable for low potency reversible inhibitors with high off-rates. The particular chemoprobe used in this manuscript is not cell penetrant and therefore analyses are performed ex vivo on lysed samples.
The method can be run on instruments that are broadly available in research and analytical laboratories; it does not require genetic modifications to be introduced into cells, and it can easily be applied to different sample types. Another advantage is that it can be used on samples derived from different species that are frequently used in the preclinical proof of concept studies and in toxicology models and that it has successfully been translated to analyze clinical samples.
Other methods have been used for analysis of KDM1A target engagement. Many of these methods use proxy markers like changes of the H3K4me2 histone mark, using AlphaLisa15; or induction of expression markers using qRT-PCR or FACS analysis16. However, in cells or tissues, the histone marks are controlled by multiple factors, and assays that measure changes in the histone mark do not always provide a good dynamic range for analysis. KDM1A inhibition can induce potent changes in gene and protein expression, but the response tends to very heterogeneous and highly cell context dependent, which can complicate analyses of dose response3,7.
Direct assessment of the occupation of the target is, therefore, the best option to measure target engagement. One assay that has been proposed for this is the cellular thermal shift assay (CETSA), based on the increase of thermal stability of target proteins upon binding of inhibitors. This method may, in principle, be applied to unmodified cells and different tissue types and has recently been used to assess the cellular activity of KDM1A inhibitors in cultivated cells17. However, this technology has rarely been used for in vivo pharmacodynamics studies18&#160;and to the best of our knowledge,&#160;its use has not been reported in clinical trials.
The protocol provided here describes a fully validated chemoprobe based method which has been used to determine KDM1A target engagement in cells and tissue samples. The method has been successfully translated to analyze samples of human subjects treated with a KDM1A inhibitor19&#160;and will be of great use to model PK/PD responses in clinical trials.

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.bdigitaluss.remotexs.co/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><tr><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. The RLU values of Total and Free KDM1A detected in human PBMCs from the blood of 3 independent volunteers are superposed on the Standard curve in Figure 2C and 2D. 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.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59390/59390fig2.jpg" /> 
Figure 2. Determination of Total and Free rKDM1A in PBMCs of healthy volunteers. RLU values of Total rKDM1A assessed by ELISA (A) and of Free rKDM1A assessed by chemoprobe capture ELISA (B). Data were obtained from 3 replicate experiments, each analyzed in triplicate (N = 3; n = 3). RLU values of Total KDM1A assessed by ELISA (C) and of Free KDM1A (D) for the PBMCs of 3 independent untreated volunteers (red, blue and green squares) superposed on Standard Curve. Data were obtained from one experiment analyzed in triplicate (N = 1; n = 3). Values represented are the means &#177; SD. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59390/59390fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Analysis of KDM1A target engagement in cells 
AML cells were cultured following provider recommendations. Cells were treated with vehicle or ORY-1001 at different concentrations (0.25; 0.5; 1; 5 and 25 nM) (Figure 3). The native protein extracts were obtained in presence of 25 nM OG-881 chemoprobe. 0.5 &#181;g of total protein was used to perform the target engagement analysis as described previously. Total and free KDM1A were determined, and the percentage of target engagement of ORY-1001 to KDM1A was calculated relative to the vehicle as described.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59390/59390fig3.jpg" /> 
Figure 3. Dose-response of KDM1A Target Engagement in a human AML cell line. Cells were treated with vehicle or ORY-1001 at different concentrations (0.25; 0.5; 1; 5 and 25 nM) and used for determination of target engagement as described. Data were obtained from 3 replicate experiments, each analyzed in triplicate (N = 3, n = 3). Values represented are the means &#177; SD. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59390/59390fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Analysis of in vivo KDM1A target engagement
The objective of this experiment was to characterize the target engagement of ORY-1001 in different rat tissues, in function of the dose level. To achieve this goal, 15 Sprague-Dawley rats (200-250 g) were housed in a cytostatic security room to avoid potential contamination by the tested compound. A maximum of 3 rats/cage were randomly assigned to 5 study groups. The 5 different study groups received, respectively, vehicle; 1; 3; 10 or 30 &#181;g / kg of ORY-1001 by oral administration for 4 consecutive days. Compound stock solutions were prepared daily. Animals were weighed before each administration to adjust the required volume.&#160;All animals were housed at constant room temperature (20 - 24 &#186;C) and relative humidity (45 - 65 %) under a 12 h light-dark cycle (lights on at 6:00 AM). Food and water were available ad libitum. Blood samples were collected 2 h after last administration in K2EDTA tubes and PBMCs were isolated according to the procedure described previously in step 1.2.2. and preserved at -80 &#176;C until native protein extraction. Lung samples were also collected 2 h after the last drug administration, frozen immediately in liquid nitrogen, and stored at -80 &#176;C. Studies 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.
After pulverization, the native protein extracts from lung were obtained as described and quantified. 5 &#181;g of total protein from pooled PBMCs or 7.5 &#181;g of total protein from lung from 3 animals were used per dose group to run the KDM1A target engagement assay.
The dose-response of KDM1A target engagement in PBMCs and in lung treatment of rats with ORY-1001 by oral gavage, calculated relative to the vehicle group is shown in Figure 4A and 4B. As can see in Figure 4C, the ex vivo incubation with 25 nM 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 4 days with 30 &#181;g/kg ORY-1001, confirming KDM1A was already fully inhibited in vivo.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59390/59390fig4.jpg" /> 
Figure 4. In vivo and ex vivo native KDM1A target engagement. Dose-response of KDM1A target engagement in PBMCs (A) and lung samples (B) from rats treated with ORY-1001 for 4 consecutive days (p.o). Data were obtained from pooled PBMCs extracts from 3 animals per cohort, analyzed in duplicate (N = 1, n = 2) or from the lungs from 3 individual animals per cohort, analyzed in triplicate (N = 3, n = 3). C. Comparison of TE in pooled lung protein extracts of rats treated with vehicle (left) or 30 &#181;g/kg ORY-1001; after 1 h ex vivo incubation of the extracts without (grey bars) or with 25 nM ORY-1001 (blue bars) (N = 3, n = 3). All data are represented as means &#177; SD. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59390/59390fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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7.2K Views.  Oryzon Genomics. 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.

Video: Direct Measurement of KDM1A Target Engagement Using Chemoprobe-based Immunoassays

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. Therefore, DNA-protein interactions regulate multiple physiological, pathophysiological, and biological functions, such as cell differentiation, cell proliferation, cell cycle control, chromosome stability, epigenetic gene regulation, and cell transformation. In eukaryotic cells, the DNA interacts with histone and nonhistone proteins and is condensed into chromatin. Several technical tools can be used to analyze DNA-protein interactions, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting. However, these techniques analyze the protein-DNA interaction in vitro, not within the cellular context. Chromatin immunoprecipitation (ChIP) is a technique that captures proteins at their specific DNA binding sites, thereby allowing for the identification of DNA-protein interactions within their chromatin context. It is done by fixation&#160;of the DNA-protein interaction, followed by&#160;immunoprecipitation of the protein of interest. Subsequently, the genomic site that the protein was bound to is characterized. Here, we describe and discuss ChIP and demonstrate its analytical value for the identification of the Transforming Growth Factor-&#946; (TGF-&#946;)-induced binding of the transcription factor SMAD2 to SMAD Binding Elements (SBE) within the promoter region of the tyrosine-protein kinase Kit (c-KIT) receptor ligand Stem Cell Factor (SCF).

DNA-protein interactions are essential for multiple biological processes. During the evaluation of cellular functions, the analysis of DNA-protein interactions is indispensable for understanding gene regulation. Chromatin immunoprecipitation (ChIP) is a powerful tool to analyze such interactions in vivo.

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

Genetics

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Cancer Research

Using High Content Imaging to Quantify Target Engagement in Adherent Cells

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe validation. Methods that measure this phenomenon without modification of the protein target or small molecule are particularly valuable though technically challenging. The cellular thermal shift assay (CETSA) is one technique to monitor target engagement in living cells. Here, we describe an adaptation of the original CETSA protocol, which allows for high throughput measurements while retaining subcellular localization at the single cell level. We believe this protocol offers important advances to the application of CETSA for in-depth characterization of compound-target interaction, especially in heterogeneous populations of cells.

Measurements of drug target engagement are central to effective drug development and chemical probe validation. Here, we detail a protocol for measuring drug-target engagement using high content imaging in a microplate-compatible adaption of the cellular thermal shift assay (CETSA).

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe ...

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death.
In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by &#947;[32P] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.

We characterized a new kinase protein using robust biochemical approaches: Western Blot analysis with a dedicated specific antibody on different cell lines and tissues, interactions by coimmunoprecipitation experiments, kinase activity detected by Western Blot using a phospho-specific antibody and by &#947;[32P] ATP labeling.

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using three-dimensional (3D) cell-based invasion and migration assays. In this study, we have optimized these 3D assays for live-cell immunocytochemistry. An Antibody Labeling Reagent was used to detect in real-time the expression of the adhesion molecule CD44, on the plasma membrane of migrating and invading cells of a DMG H3K27M primary patient-derived cell line. CD44 is associated with cancer stem cell phenotype and tumor cell migration and invasion and is involved in the direct interactions with the central nervous system (CNS) extracellular matrix. Neurospheres (NS) from the DMG H3K27M cell line were embedded into the basal membrane matrix (BMM) or placed onto a thin coating layer of BMM, in the presence of an anti-CD44 antibody in conjunction with the antibody labeling reagent (ALR). The live-3D-cell immunocytochemistry image analysis was performed on a live-cell analysis instrument to quantitatively measure the overall CD44 expression, specifically on the migrating and invading cells. The method also allows visualizing in real-time the intermittent expression of CD44 on the plasma membrane of migrating and invading cells. Moreover, the assay also provided new insights into the potential role of CD44 in the mesenchymal to amoeboid transition in DMG H3K27M cells.

This study presents a protocol of live-3D-cell immunocytochemistry applied to a pediatric diffuse midline glioma cell line, useful to study in real-time the expression of proteins on the plasma membrane during dynamic processes like 3D cell invasion and migration.

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

Biology

Measuring Ubiquitylated, SUMOylated, and ADP-Ribosylated DNA-Protein Crosslinks

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, methyltransferases, etc.) malfunctioning, or exogenous agents such as chemotherapeutics and crosslinking agents. Once DPCs are induced, several types of post-translational modifications (PTMs) are promptly conjugated to them as early response mechanisms. It has been shown that DPCs can be modified by ubiquitin, small ubiquitin-like modifier (SUMO), and poly-ADP-ribose, which prime the substrates to signal their respective designated repair enzymes and, in some cases, coordinate the repair in sequential manners. As PTMs transpire quickly and are highly reversible, it has been challenging to isolate and detect PTM-conjugated DPCs that usually remain at low levels. Presented here is an immunoassay to purify and quantitatively detect ubiquitylated, SUMOylated, and ADP-ribosylated DPCs (drug-induced topoisomerase DPCs and aldehyde-induced non-specific DPCs) in vivo. This assay is derived from the RADAR (rapid approach to DNA adduct recovery) assay that is used for the isolation of genomic DNA containing DPCs by ethanol precipitation. Following normalization and nuclease digestion, PTMs of DPCs, including ubiquitylation, SUMOylation, and ADP-ribosylation, are detected by immunoblotting using their corresponding antibodies. This robust assay can be utilized to identify and characterize novel molecular mechanisms that repair enzymatic and non-enzymatic DPCs&#160;and has the potential to discover small molecule inhibitors targeting specific factors that regulate PTMs to repair DPCs.

Author Spotlight: Quantitative Detection of DNA Protein Crosslinks and Their Post-Translational Modifications  

The present protocol highlights a modified method to detect and quantify DNA-protein crosslinks (DPCs) and their post-translational modifications (PTMs), including ubiquitylation, SUMOylation, and ADP-ribosylation induced by topoisomerase inhibitors and by formaldehyde, thereby allowing the study of the formation and repair of DPCs and their PTMs.

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, ...

Identifying PD-1/PD-L1 Inhibitors with Surface Plasmon Resonance Technology

The disruption of the PD-1/PD-L1 interaction is a promising strategy for cancer immunotherapy. Reliable screening platforms are essential for evaluating the efficacy of PD-1/PD-L1 inhibitors. A previously established human PD-1/PD-L1 blockade assay utilizing Surface Plasmon Resonance (SPR) technology (first-generation PD-1/PD-L1 inhibitor SPR screening platform) demonstrated results comparable to those obtained through Homogeneous Time-Resolved Fluorescence (HTRF) and cell-based assays, with potential for large-scale screening. Herein, an optimized version of this assay (second-generation PD-1/PD-L1 inhibitor SPR screening platform) is presented, featuring a dual-step coupling process that combines amine and bio-streptavidin coupling to enhance PD-1 orientation control on the chip and reduce PD-1 protein consumption. The updated platform was successfully validated using the PD-1/PD-L1 inhibitor BMS-1166, showing blockade effects comparable to the previous SPR-based method and other established techniques such as ELISA. These results confirm the reliability of the approach. This optimized SPR screening platform offers a high-throughput and reliable tool for identifying novel PD-1/PD-L1 inhibitors, advancing cancer immunotherapy research, and highlighting the potential of SPR in immune checkpoint inhibitor screening.

This protocol describes a blockade assay for PD-1/PD-L1 inhibitors using surface plasmon resonance technology. It employs a dual-step immobilization strategy and a tailored buffer system to accurately measure response units, facilitating the assessment of blockade rates for compounds or biologics. Additionally, it supports the high-throughput identification of PD-1/PD-L1 inhibitors.

The disruption of the PD-1/PD-L1 interaction is a promising strategy for cancer immunotherapy. Reliable screening platforms are essential for evaluating ...