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><li>Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histone+demethylation+mediated+by+the+nuclear+amine+oxidase+homolog+LSD1%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Histone demethylation mediated by the nuclear amine oxidase homolog LSD1.</a> Cell. 119 (7), 941-953 (2004).</li>
<li>Maiques-Diaz, A., Somervaille, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((LSD1%3A+biologic+roles+and+therapeutic+targeting%5BTitle%5D)+AND+%22Epigenomics%22%5BJournal%5D)">LSD1: biologic roles and therapeutic targeting.</a> Epigenomics. 8 (8), 1103-1116 (2016).</li>
<li>Maes, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ORY-1001%2C+a+Potent+and+Selective+Covalent+KDM1A+Inhibitor%2C+for+the+Treatment+of+Acute+Leukemia%5BTitle%5D)+AND+%22Cancer+Cell%22%5BJournal%5D)">ORY-1001, a Potent and Selective Covalent KDM1A Inhibitor, for the Treatment of Acute Leukemia.</a> Cancer Cell. 33 (3), 495-511 (2018).</li>
<li>Sugino, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+LSD1+inhibitor+NCD38+ameliorates+MDS-related+leukemia+with+complex+karyotype+by+attenuating+leukemia+programs+via+activating+super-enhancers%5BTitle%5D)+AND+%22Leukemia%22%5BJournal%5D)">A novel LSD1 inhibitor NCD38 ameliorates MDS-related leukemia with complex karyotype by attenuating leukemia programs via activating super-enhancers.</a> Leukemia. 31 (11), 2303-2314 (2017).</li>
<li>Kleppe, M., Shank, K., Efthymia, P., Riehnhoff, H., Levine, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lysine-Specific+Histone+Demethylase%2C+LSD1%2C+%28KDM1A%29+As+a+Novel+Therapeutic+Target+in+Myeloproliferative+Neoplasms%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Lysine-Specific Histone Demethylase, LSD1, (KDM1A) As a Novel Therapeutic Target in Myeloproliferative Neoplasms.</a> Blood. 126, 601(2015).</li>
<li>Jutzi, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((LSD1+Inhibition+Prolongs+Survival+in+Mouse+Models+of+MPN+by+Selectively+Targeting+the+Disease+Clone%5BTitle%5D)+AND+%22HemaSphere%22%5BJournal%5D)">LSD1 Inhibition Prolongs Survival in Mouse Models of MPN by Selectively Targeting the Disease Clone.</a> HemaSphere. 2 (3), 54(2018).</li>
<li>Mohammad, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+Hypomethylation+Signature+Predicts+Antitumor+Activity+of+LSD1+Inhibitors+in+SCLC%5BTitle%5D)+AND+%22Cancer+Cell%22%5BJournal%5D)">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/pubmed?term=((RN-1%2C+a+potent+and+selective+lysine-specific+demethylase+1+inhibitor%2C+increases+%CE%B3-globin+expression%2C+F+reticulocytes%2C+and+F+cells+in+a+sickle+cell+disease+mouse+model%5BTitle%5D)+AND+%22Experimental+Hematology%22%5BJournal%5D)">RN-1, a potent and selective lysine-specific demethylase 1 inhibitor, increases γ-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/pubmed?term=((Oral+administration+of+the+LSD1+inhibitor+ORY-3001+increases+fetal+hemoglobin+in+sickle+cell+mice+and+baboons%5BTitle%5D)+AND+%22Experimental+Hematology%22%5BJournal%5D)">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/pubmed?term=((The+dual+LSD1%2FMAO-B+inhibitor+ORY-2001+prevents+the+development+of+the+memory+deficit+in+samp8+mice+through+induction+of+neuronal+plasticity+and+reduction+of+neuroinflammation%5BTitle%5D)+AND+%22Alzheimer%E2%80%99s+%26+Dementia%22%5BJournal%5D)">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’s & Dementia. 11 (7), P905(2015).</li>
<li>Schmidt, D. M., McCafferty, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Trans-2-Phenylcyclopropylamine+is+a+mechanism-based+inactivator+of+the+histone+demethylase+LSD1%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">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/pubmed?term=((Human+histone+demethylase+LSD1+reads+the+histone+code%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Human histone demethylase LSD1 reads the histone code.</a> Journal of Biological Chemistry. 280 (50), 41360-41365 (2005).</li>
<li>Oryzon Genomics, Methods to determine kdm1a target engagement and chemoprobes useful therefor. , WO2017158136 (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/pubmed?term=((Chemoprobe-based+assays+of+histone+lysine+demethylase+1A+target+occupation+enable+in+vivo+pharmacokinetics+and+-dynamics+studies+of+KDM1A+inhibitors%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">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. , In Press (2019).</li>
<li>Rodriguez-Suarez, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+Homogeneous+Nonradioactive+Methyltransferase+and+Demethylase+Assays+Targeting+Histone+H3+Lysine+4%5BTitle%5D)+AND+%22Journal+of+Biomolecular+Screening%22%5BJournal%5D)">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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD86+expression+as+a+surrogate+cellular+biomarker+for+pharmacological+inhibition+of+the+histone+demethylase+lysine-specific+demethylase+1%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">CD86 expression as a surrogate cellular biomarker for pharmacological inhibition of the histone demethylase lysine-specific demethylase 1.</a> Analytical Biochemistry. 442 (1), 104-106 (2013).</li>
<li>Schulz-Fincke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure-activity+studies+on+N-Substituted+tranylcypromine+derivatives+lead+to+selective+inhibitors+of+lysine+specific+demethylase+1+%28LSD1%29+and+potent+inducers+of+leukemic+cell+differentiation%5BTitle%5D)+AND+%22European+Journal+of+Medicinal+Chemistry%22%5BJournal%5D)">Structure-activity studies on N-Substituted tranylcypromine derivatives lead to selective inhibitors of lysine specific demethylase 1 (LSD1) and potent inducers of leukemic cell differentiation.</a> European Journal of Medicinal Chemistry. 144, 52-67 (2018).</li>
<li>Ishii, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CETSA+quantitatively+verifies+in+vivo+target+engagement+of+novel+RIPK1+inhibitors+in+various+biospecimens%5BTitle%5D)+AND+%22Scientific+Report%22%5BJournal%5D)">CETSA quantitatively verifies in vivo target engagement of novel RIPK1 inhibitors in various biospecimens.</a> Scientific Report. 7, 13000(2017).</li>
<li>Maes, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ORY-2001%3A+An+Epigenetic+drug+for+the+treatment+of+cognition+defects+in+Alzheimer%E2%80%99s+disease+and+other+neurodegenerative+disorders%5BTitle%5D)+AND+%22Alzheimer%E2%80%99s+%26+Dementia%22%5BJournal%5D)">ORY-2001: An Epigenetic drug for the treatment of cognition defects in Alzheimer’s disease and other neurodegenerative disorders.</a> Alzheimer’s & Dementia. 12 (7), P1192(2017).</li>
</ol>

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...

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><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. ...

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 ...

direct-measurement-kdm1a-target-engagement-using-chemoprobe-based

Research

JoVE Journal

Biochemistry

7.3K Views.  Oryzon Genomics. De beoordeling van de doelbetrokkenheid, dat wil zeggen de interactie van een geneesmiddel met het eiwit waarvoor het is ontworpen, is een basisvereiste voor de interpretatie van de biologische activiteit van een verbinding in de ontwikkeling van geneesmiddelen, of in basisonderzoeksprojecten. Het belangrijkste voordeel van deze techniek is dat het een directe meting van het dosis-effect en de dynamiek van de KDM1A-doelbetrokkenheid mogelijk maakt, in plaats van metingen van downstream-effect...

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 ...

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 ...

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

Bioengineering

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 ...

Direct Measurement of KDM1A Target Engagement Using Chemoprobe-based Immunoassays

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