Name: using high content imaging to quantify target engagement in adherent cells
Uploaded: 2018-11-29T14:45:00
Description: Watch this Scientific Journal Video about Using High Content Imaging to Quantify Target Engagement in Adherent Cells at JoVE.com

The authors acknowledge infrastructure support from Science for Life Laboratory and Karolinska Institutet. The authors also acknowledge input and discussions with Michaela Vallin, Magdalena Otrocka and Thomas Lundb&#228;ck.

1. Seeding of Cells
NOTE: For a general overview of the workflow see Figure 1. A detailed list of materials and reagents are available in the Table of Materials.
<ol>
	<li>Prior to seeding of the cells, drill holes with a standard drill in the frame of black 384-well imaging assay plates to avoid air bubbles being trapped under the plate later during the heating step. To avoid plastic particles entering the wells during this ste...

<ol>
	<li>Morgan, P., 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=Impact+of+a+five-dimensional+framework+on+R&amp;D+productivity+at+AstraZeneca.">Impact of a five-dimensional framework on R&amp;D productivity at AstraZeneca.</a> Nature Reviews Drug Discovery. 17 (3), 167-181 (2018).</li><li>Freedman, L. P., Cockburn, I. M., Simcoe, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Economics+of+Reproducibility+in+Preclinical+Research.">The Economics of Reproducibility in Preclinical Research.</a> PLOS Biology. 13 (6), e1002165 (2015).</li><li>Waring, M. J., 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=An+analysis+of+the+attrition+of+drug+candidates+from+four+major+pharmaceutical+companies.">An analysis of the attrition of drug candidates from four major pharmaceutical companies.</a> Nature Reviews Drug Discovery. 14 (7), 475-486 (2015).</li><li>Morgan, P., 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=Can+the+flow+of+medicines+be+improved?+Fundamental+pharmacokinetic+and+pharmacological+principles+toward+improving+Phase+II+survival.">Can the flow of medicines be improved? Fundamental pharmacokinetic and pharmacological principles toward improving Phase II survival.</a> Drug Discovery Today. 17 (9), 419-424 (2012).</li><li>Bunnage, M. E., Chekler, E. L. P., Jones, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+validation+using+chemical+probes.">Target validation using chemical probes.</a> Nature Chemical Biology. 9 (4), 195-199 (2013).</li><li>Sch&#252;rmann, M., Janning, P., Ziegler, S., Waldmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-Molecule+Target+Engagement+in+Cells.">Small-Molecule Target Engagement in Cells.</a> Cell Chemical Biology. 23 (4), 435-441 (2016).</li><li>Robers, M. B., 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=Target+engagement+and+drug+residence+time+can+be+observed+in+living+cells+with+BRET.">Target engagement and drug residence time can be observed in living cells with BRET.</a> Nature Communications. 6, 10091 (2015).</li><li>Martinez Molina, D., 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=Monitoring+drug+target+engagement+in+cells+and+tissues+using+the+cellular+thermal+shift+assay.">Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay.</a> Science. 341 (6141), 84-87 (2013).</li><li>Martinez Molina, D., Nordlund, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cellular+Thermal+Shift+Assay:+A+Novel+Biophysical+Assay+for+In+situ+Drug+Target+Engagement+and+Mechanistic+Biomarker+Studies.">The Cellular Thermal Shift Assay: A Novel Biophysical Assay for In situ Drug Target Engagement and Mechanistic Biomarker Studies.</a> Annual Review of Pharmacology and Toxicology. 56, 141-161 (2016).</li><li>Jafari, R., 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+cellular+thermal+shift+assay+for+evaluating+drug+target+interactions+in+cells.">The cellular thermal shift assay for evaluating drug target interactions in cells.</a> Nature Protocols. 9 (9), 2100-2122 (2014).</li><li>Almqvist, H., 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=CETSA+screening+identifies+known+and+novel+thymidylate+synthase+inhibitors+and+slow+intracellular+activation+of+5-fluorouracil.">CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil.</a> Nature Communications. 7, 11040 (2016).</li><li>Axelsson, H., 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=In+situ+Target+Engagement+Studies+in+Adherent+Cells.">In situ Target Engagement Studies in Adherent Cells.</a> ACS Chemical Biology. 13 (4), 942-950 (2018).</li><li>Seashore-Ludlow, B., Perspective Lundb&#228;ck, T. E. a. r. l. y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microplate+Application+of+the+Cellular+Thermal+Shift+Assay+(CETSA).">Microplate Application of the Cellular Thermal Shift Assay (CETSA).</a> Journal of Biomolecular Screening. 21 (10), 1019-1033 (2016).</li><li>Axelsson, H., Almqvist, H., Seashore-Ludlow, B., Lundback, T., Sittampalam, G. S., 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=.">.</a> Assay Guidance Manual [Internet]. , (2016).</li><li>Mateus, 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=Prediction+of+intracellular+exposure+bridges+the+gap+between+target-+and+cell-based+drug+discovery.">Prediction of intracellular exposure bridges the gap between target- and cell-based drug discovery.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (30), E6231-E6239 (2017).</li></ol>

As discussed in the results section, there are several key steps to the procedure. First, it is important to identify a high-quality affinity reagent. We recommend screening a small library of antibodies for each desired target. After a primary antibody has been selected, it is also important to validate the system for a number of different binding sites of the protein target if appropriate. Counter-screening for compounds that interfere with the assay signal as shown in Figure 2C by omittin...

When developing new drugs or chemical probes it is essential to couple the observed pharmacological effect or functional readout to measurements of target occupancy or engagement in live cells1,2,3. These data are necessary both to ensure that the small molecule in fact reaches its desired target and to validate the biological hypothesis behind protein target selection4,5. Furthermore, during drug development, model systems of increasing complexity are used to select and corroborate a lead compound prior to clinical trials. To confirm translation of biology across these preclinical systems, methods for tracing drug-target engagement and accompanying biology throughout this development process are critical.
Drug-target engagement has traditionally been challenging to monitor in live cells with unfunctionalized small molecules and proteins, especially at the single-cell level with spatial resolution6,7. One recent method to observe the interaction between unmodified drugs and proteins in live cells is the cellular thermal shift assay (CETSA) in which ligand-induced stabilization of a native protein in response to a heat challenge is quantified8,9,10. This is accomplished by quantifying remaining soluble protein after exposure to a heat challenge. In the initial disclosure of CETSA, western blot was used for detection. To enable screening campaigns and hit triaging of larger compound collections, efforts to increase the throughput of CETSA experiments have lead to the development of several homogenous, microplate-based assays10,11. However, one limitation with these methods is that they are currently best suited to compound treatment in cell suspensions and the detection requires cell lysis, leading to loss of spatial information. CETSA can be applied experimentally either as a ligand-induced shift in thermal aggregation temperature (Tagg) at a single concentration of the small molecule or the ligand concentration necessary to stabilize the protein at a single temperature. The latter is termed isothermal dose response fingerprints (ITDRF) to signify the dependence of these measurements on the specific experimental conditions.
The goal of this protocol is to measure target engagement using CETSA in adherent cells by immunofluorescent (IF) antibody detection with high-content microscopy12. This procedure extends the original CETSA platform to allow for single-cell quantification of target engagement with conservation of subcellular localization. Notably, unlike many previous reports, in this procedure compound treatment is performed in live adherent cells without surface detachment or washing prior to the heat challenge, thus preserving the established binding equilibrium we aim to measure13. Currently, the method is validated for one target protein p38&#945; (MAPK14) in several cell lines, and we hope that by sharing this procedure the technique can be applied broadly across the melting proteome. We anticipate that this protocol can be adapted throughout the drug development pipeline from screening, hit triaging through to monitoring of target engagement in vivo.

<table>
	<tbody>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Medicago</td>
			<td>09-9400-100</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express</td>
			<td>ThermoFisher Scientific</td>
			<td>12604013</td>
			<td>for detaching cells and subculturing</td>
		</tr>
		<tr>
			<td>16% paraformaldehyde (PFA)</td>
			<td>ThermoFisher Scientific</td>
			<td>28908</td>
			<td>fixative</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG (H+L), Alexa Fluor 488 conjugated antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>A11008</td>
			<td>secondary antibody</td>
		</tr>
		<tr>
			<td>HCS CellMask Red stain</td>
			<td>ThermoFisher Scientific</td>
			<td>H32712</td>
			<td>Cytoplasm stain</td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Sigma-Aldrich</td>
			<td>56741</td>
			<td>for permeabilization</td>
		</tr>
		<tr>
			<td>Hoechst stain 33342</td>
			<td>Sigma-Aldrich</td>
			<td>B2261</td>
			<td>nuclear stain</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) - high Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>6429</td>
			<td>cell culture media component</td>
		</tr>
		<tr>
			<td>Heat-inactivated fetal bovine serum (FBS)</td>
			<td>Sigma-Aldrich</td>
			<td>F9665</td>
			<td>cell culture media component</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td>cell culture media component</td>
		</tr>
		<tr>
			<td>Corning, breathable plate seal</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3345</td>
			<td>for copound incubation step</td>
		</tr>
		<tr>
			<td>Rabbit anti-p38 antibody [E229]</td>
			<td>Abcam</td>
			<td>ab170099</td>
			<td>primary antibody, LOT:GR305364-16</td>
		</tr>
		<tr>
			<td>Falcon, Black 384-well clear bottom imaging plates</td>
			<td>VWR</td>
			<td>736-2044</td>
			<td>imaging plates</td>
		</tr>
		<tr>
			<td>Greiner, 384-well low volume polypropylene plates</td>
			<td>VWR</td>
			<td>784201</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive aluminum foil</td>
			<td>VWR</td>
			<td>30127790</td>
			<td></td>
		</tr>
		<tr>
			<td>Peelable aluminium seal</td>
			<td>Agilent</td>
			<td>24210-001</td>
			<td>for PlateLoc</td>
		</tr>
		<tr>
			<td>LY2228820</td>
			<td>Selleckchem</td>
			<td>S1494</td>
			<td>p38&#945; inhibitor</td>
		</tr>
		<tr>
			<td>PH797804</td>
			<td>Selleckchem</td>
			<td>PH797804</td>
			<td>p38&#945; inhibitor</td>
		</tr>
		<tr>
			<td>BIRB796</td>
			<td>Selleckchem</td>
			<td>S1574</td>
			<td>p38&#945; inhibitor</td>
		</tr>
		<tr>
			<td>SB203580</td>
			<td>Tocris</td>
			<td>1202</td>
			<td>p38&#945; inhibitor</td>
		</tr>
		<tr>
			<td>AMG 548</td>
			<td>Tocris</td>
			<td>3920</td>
			<td>p38&#945; inhibitor</td>
		</tr>
		<tr>
			<td>RWJ 67657</td>
			<td>Tocris</td>
			<td>2999</td>
			<td>p38&#945; inhibitor</td>
		</tr>
		<tr>
			<td>L-Skepinone</td>
			<td>CBCS compound collection</td>
			<td></td>
			<td>p38&#945; inhibitor</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A7030</td>
			<td>blocking agent</td>
		</tr>
		<tr>
			<td>SDS (sodium dodecyl sulfate)</td>
			<td>BDH</td>
			<td>44244</td>
			<td>used in antigen retrieval</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G8898</td>
			<td>used in antigen retrieval</td>
		</tr>
		<tr>
			<td>A-431 cells</td>
			<td>ATCC</td>
			<td>ATC-CRL-1555</td>
			<td></td>
		</tr>
		<tr>
			<td>Echo 550</td>
			<td>Labcyte</td>
			<td></td>
			<td>For preparation of compound plates</td>
		</tr>
		<tr>
			<td>Plate sealer</td>
			<td>Agilent</td>
			<td></td>
			<td>PlateLoc</td>
		</tr>
		<tr>
			<td>Bulk reagent dispenser</td>
			<td>Thermo Scientific</td>
			<td>5840300</td>
			<td>Multidrop Combi</td>
		</tr>
		<tr>
			<td>Automated liquid handling</td>
			<td>Agilent</td>
			<td></td>
			<td>Bravo liquid handling platform; used for compound plate preparation</td>
		</tr>
		<tr>
			<td>Plate washer</td>
			<td>Tecan</td>
			<td></td>
			<td>Hydrospeed</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Julabo</td>
			<td>TW12</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocouple</td>
			<td>VWR</td>
			<td></td>
			<td>Thermocouple traceable lab thermometer</td>
		</tr>
		<tr>
			<td>High content imager</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>ImageXpress Micro XLS Widefield High-Content Analysis System</td>
		</tr>
	</tbody>
</table>

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.

The protocol outlined in Figure 1 describes the basic workflow for running CETSA assays on adherent cells with detection of remaining soluble protein by high content imaging. This workflow can be easily adapted to all stages of assay development by modifying the plate layout of the compounds or reagents14. We detail expected results for several anticipated use cases below.
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Watch this Scientific Journal Video about Using High Content Imaging to Quantify Target Engagement in Adherent Cells at JoVE.com

Using High Content Imaging to Quantify Target Engagement in Adherent 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 ...

This method can help answer key questions in chemical biology and drug discovery. Such as, does your compound bind to the target protein? The main advantages for this technique are that there's no requirement of detachment of the adherent cells.And spatial localization can be determined by imaging. Though we've applied this method in cell lines, it's also possible to apply this method to other cell systems such as primary cultures and cocultures. Before seeding the cells, place black 384-well imaging assay plates in a tissue culture hood, and cover the plates with aluminum foil before using a standard drill to make three 3.5-millimeter-diameter holes on both ends of each plate.When the plates are ready, use aseptic technique to wash an A-431 cell culture with 5 to 10 milliliters of PBS. And incubate the cells with two milliliters of trypsin at 37 degrees Celsius until the cells detach. After counting, dilute the cells to 5 times 10 to the 4th cells per milliliter in culture medium, and seed 40 microliters of cells to each well of each assay plate.Gently shaking each plate from side to side after seeding to ensure a homogenous distribution of the cells across the well bottoms. To minimize plate edge effects, allow the cells to settle at the bottom of the assay plates for 20 minutes at room temperature in the back of the laminar flow hood before placing the plates in a custom humidified chamber for two to three days at 37 degrees Celsius and 5%carbon dioxide. On the day of the experiment, use a plate washer to aspirate the medium from each well.And add 30 microliters of the compound of interest at the appropriate experimental concentration to the appropriate experimental wells. Seal the compound-treated assay plates with breathable plate seals. And return the plates to the cell culture incubator for 30 minutes.To expose the cells to a heat challenge, first set the water bath to the appropriate experimental temperature, and place an unsealed dummy plate containing the same volume of medium per well as the assay plate into the bath, along with a thermocouple thermometer for monitoring the temperature within the wells. When the appropriate experimental temperature has been reached, remove the breathable seal from each assay plate and reseal the plates with a tight adhesive aluminum foil to ensure that no water leaks into the wells during their subsequent heating in the water bath. Keeping the drilled holes within the plate frame accessible.Place the assay plates and a new dummy plate into the water bath, with the bottoms of the plates angled toward the water surface to force any remaining air from under the plates, and begin monitoring the temperature of the new dummy plate. An even heating of the plate is critical for assay performance. Take care to place the plate in the water bath such that there are no air bubbles trapped underneath.After three minutes, immediately cool the plates in a second water bath of room-temperature water for five minutes before their analysis. To image the cells, first add 10 microliters of 16%paraformaldehyde directly to the assay plates for a 20-minute incubation at room temperature. At the end of the fixation period, wash the cells with 300 microliters of PBS on the plate washer, and add 20 microliters of 0.1%NP40 for a 10-minute incubation at room temperature.At the end of the incubation, wash the cells as demonstrated. And block the cells with 15 microliters of 1%bovine serum albumin, or BSA, for one hour at room temperature. Next, use the plate washer to aspirate the blocking serum, and add 10 microliters of the primary antibody of interest to the appropriate wells for a one-hour incubation at room temperature.At the end of the incubation, wash each well with 300 microliters of PBS, and label the cells with 10 microliters of the appropriate secondary antibody for one hour at room temperature protected from light. For nuclear staining of the cells, add 10 microliters of an appropriate nuclear dye to each well for 10 minutes at room temperature. And wash the cells in PBS as demonstrated.Label the cells with 10 microliters of cell mask per well for 30 minutes at room temperature. Followed by a last wash with PBS. Then dispense 60 microliters of fresh PBS to each well, and seal the plates with aluminum foil until imaging.To image the cells, capture four images per well on a high-content imager using the appropriate fluorescent channels under the 10-times objective using automated laser autofocus and apply binning to during the acquisition. Then store the images as 16-bit grayscale dot-tiff files along with the metadata. Antibody recognition should not be disrupted by conformational changes in the target protein that may be induced by ligand binding.For example, BIRB796 has a long off rate, and quantification of the target engagement is only possible by applying an antigen retrieval step. This representative thermal aggregation curve experiment for cells treated with positive and negative control compounds illustrates typical protein stabilization ranges after treatment of the cells with the compounds at various temperatures. Here a typical isothermal dose-response fingerprint experiment is shown to demonstrate representative ranges of protein stabilization in response to different doses of experimental compound.The application of isothermal heat challenges for compound screening to identify novel binders of the target protein allows the analysis of a large number of compounds at a single concentration at one time, before isothermal dose-response fingerprinting of the hits to confirm target protein stabilization. While attempting this procedure, it's important to remember that the exact experimental conditions, such as the length and temperature of the heat challenge impact the observed potency of the compounds. Don't forget that working with paraformaldehyde can be extremely hazardous.And precautions, such as following institutional safety guidelines, should always be taken while performing this procedure.

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