Using High Content Imaging to Quantify Target Engagement in Adherent Cells

Summary

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).

Abstract

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.

Introduction

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 cells^1,2,3. 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 selection^4,5. 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 resolution^6,7. 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 quantified^8,9,10. 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 assays^10,11. 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 (T_agg) 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 p38α (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.

Protocol

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.

1. 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 step and to maintain sterile conditions, seal the plates with an adhesive aluminum foil or cover the plate prior to drilling in a tissue culture hood. Typically, 3 holes with a diameter of 3.5 mm on each of side of the plate (short edge) suffice.
2. Prepare a laminar flow bench by cleaning with 70% ethanol. Following standard aseptic tissue culture techniques, remove media from cell flask or dish. Wash cells with 5-10 mL of phosphate-buffered saline (PBS) and then add 2 mL trypsin to the flask. Incubate the flask at 37 °C until the A-431 cells detach. Count the cells either using a haemocytometer or cell counter. Prepare a cell suspension of 50,000 cells/mL in culture medium.
3. Dispense 40 µL of cell suspension (giving a final cell density of 2,000 cells per well) into each well of an assay plate using a bulk reagent dispenser or a multichannel pipet, depending on the scale of the experiment. Briefly move the plate from side-to-side to disperse cells evenly on the bottom of the plate.
4. To minimize plate-edge effects, allow the cells to settle at the bottom of the assay plate for 20 minutes at room temperature in the back of the laminar flow hood. Then, place the plate in a plastic container with damp paper towels to ensure a humid atmosphere. Prior to use, wipe the plastic container with 70% ethanol.
5. Incubate the box with the assay plate for 2-3 days at 37 °C and 5% CO₂ in a conventional humidified incubator. Monitor confluency of the cells until 50-75%, as assessed by visual inspection with a light microscope.

2. Compound Treatment

1. On the day of the experiment, aspirate the medium from each well using a plate washer. Place the plate on the plate washer and select the aspiration program. If a plate washer is not available, then the liquid can be removed by inverting the plate with a rapid hand twist over a waste tray or sink. Complete removal of liquid is essential for good performance. Any excess liquid is then removed by dabbing with paper towels.
2. Add 30 µL of compounds diluted to the appropriated concentration in cell culture medium using automated dispensing or a multichannel pipette depending on the scale of the experiment. Ensure to add a negative (DMSO) and positive (known ligand) control to several wells on each assay plate. Since this is a thermal shift assay, it is necessary that the compound concentration exceeds the dissociation constant to observe protein stabilization. Thus, a rough guideline for compound concentration is 50-100 times the IC₅₀, but more detail descriptions for compound concentrations are found in the Discussion section.
   NOTE: DMSO tolerability of the cell lines should be tested prior to the experiment.
3. Seal the compound-treated assay plate with a breathable plate seal and incubate at 37 °C and 5% CO₂ in a humidified incubator for 30 minutes.

3. Heat Challenge

1. First, set the water bath to the desired temperature. Note that the final temperature that is reached inside the wells of the assay plate can be different from the final temperature in the water bath. Investigate the offset beforehand with a dummy plate and thermocouple thermometer. It typically takes 30 minutes for the bath to stabilize at the desired temperature.
2. To verify that the desired temperature is reached in the wells of the assay plate during the heating step, prepare an unsealed dummy plate containing the same volume of medium as the assay plate.
3. Remove the assay plates from the incubator. Take off the breathable seal and re-seal the assay plate containing the compound-treated cells with a tight adhesive aluminum foil to ensure that no water will leak into the wells during the subsequent heating in the water bath. Ensure that the drilled holes in plate frame are accessible.
4. Place the assay plate and the dummy plate in the water bath with the bottom of the plate angled towards the water surface to force any remaining air out from under the plates.
5. Monitor the temperature inside the wells of a new dummy plate using a thermocouple thermometer.
6. Heat the assay plate in the water bath for 3 minutes. Immediately transfer the assay and dummy plate to another water bath with room-tempered water to cool down for 5 minutes. The assay plate is now ready for further processing.

4. Fixation

1. Dispense 10 µL 16% (w/v) paraformaldehyde (PFA) directly to the assay plate using a bulk reagent dispenser or a multichannel pipet. Incubate at room temperature for 20 minutes.
   Note: Some fixatives are classified as carcinogenic and institutional safety regulations should be followed.
2. Aspirate the PFA solution and wash the cells with 300 µL PBS using a plate washer. Place the plate on the plate washer and select the aspiration program.
   NOTE: This procedure has been optimized using an overflow protocol on the plate washer in which liquid is simultaneously dispensed and removed. If a plate washer or similar procedure is not available, the washing step can alternatively be done manually.
3. Manual washing procedure: Remove the liquid from the wells by inverting the plate with a rapid hand twist over a waste tray or sink. Complete removal of liquid is essential for good performance. Add 80 µL of PBS with a multichannel pipette and invert the plate again as described above, repeat the washing procedure two times. After the last wash, blot the plate against clean paper towels to remove any excess liquid.

5. Permeabilization

1. Add 20 µL of 0.1% (v/v) NP-40 to the wells with a multichannel pipet and incubate at room temperature for 10 minutes. Wash the cells using the same procedure as described above (step 4.2).
2. Alternatively, when appropriate, apply an antigen retrieval protocol, e.g.:
   1. Add 20 µL of 1% SDS to the wells with a multichannel pipet. Incubate at room temperature for 5 minutes and wash according to the same procedure described above (step 4.2).
   2. Add 80 μL of 10 mM glycine at pH 7.2 and incubate for 10 minutes at room temperature. Aspirate the glycine solution using a plate washer by placing on the plate washer and selecting the aspiration protocol. See notes in 2.1 and 4.2 if a plate washer is not available.

6. Blocking

1. Add 15 µL of 1% (w/v) bovine serum albumin (BSA) in PBS to the wells using a bulk reagent dispenser or a multichannel pipet. Incubate the plate at room temperature for 1 hour or overnight at 4 °C with an aluminum foil plate seal.

7. Primary Antibody

1. Aspirate the blocking solution using a plate washer by placing on the plate washer and selecting the aspiration protocol. See notes in 2.1 and 4.2 if a plate washer is not available.
2. Add 10 µL of primary antibody diluted accordingly in 1% (w/v) BSA in PBS to the wells using a multichannel pipet. Incubate the plate at room temperature for 1 hour or overnight at 4 °C with an aluminum foil plate seal.

8. Secondary Antibody

1. Aspirate the primary antibody solution and wash the wells according to same procedure as described above (step 4.2).
2. Add 10 µL of Alexa 488 secondary antibody diluted accordingly in 1% (w/v) BSA in PBS. Incubate at room temperature for 1 hour. Seal the plate with an adhesive aluminum foil seal to protect from light.
   NOTE: Protect the plate from light during this and subsequent steps.

9. Nuclear Staining and Cell Mask

1. Add 10 µL of nuclear dye diluted to 0.05 mg/mL in PBS to the wells using a multichannel pipet. Incubate at room temperature for additional 10 minutes.
2. Aspirate the secondary antibody and Hoechst solution and wash the wells using the same procedure as described above (step 4.2).
3. Add 10 µL of cell mask diluted to 200 ng/mL in PBS to the wells using a multichannel pipet. Incubate at room temperature for 30 minutes.
4. Aspirate the cell mask solution and wash the wells using the same procedure as described above (step 4.2).
5. Dispense 60 µL of PBS to all wells using the plate washer, a bulk reagent dispenser, or a multichannel pipet, and seal the plates with an adhesive aluminum foil.

10. Image Acquisition and Analysis

1. Capture images on a high content imager using 3 fluorescent channels: DAPI (387/447), GFP (472/520), and TexasRed (562/624). Acquire 4 images per well using 10X objective. Use automated laser autofocus and apply binning 2 during acquisition. Store images as 16 bit, gray scale tiff files along with metadata.
2. Analyze images using available software. Identify cell boundaries using a Cell Scoring algorithm with DAPI (nucleus) and TexasRed (cytoplasm).
3. Extract average intensity for all acquired wavelengths for further data analysis.
4. Calculate the Z-factor to ensure the robustness of the assay.
5. Calculate % stabilization using the following formula: 100*(1-(well intensity – average well intensity negative control)/(average intensity positive control-average intensity negative control)). Here negative control is DMSO and positive control is the reference substance. Since the maximum stabilization between compounds can vary and in fact be greater than the positive control for ITDRF curves, the maximum and minimum stabilization intensities for each compound are sometimes used in place of the intensity values of the control wells.

Results

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.

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Phosphate-buffered saline (PBS)	Medicago	09-9400-100
TrypLE Express	ThermoFisher Scientific	12604013	for detaching cells and subculturing
16% paraformaldehyde (PFA)	ThermoFisher Scientific	28908	fixative
Goat anti-rabbit IgG (H+L), Alexa Fluor 488 conjugated antibody	ThermoFisher Scientific	A11008	secondary antibody
HCS CellMask Red stain	ThermoFisher Scientific	H32712	Cytoplasm stain
NP-40	Sigma-Aldrich	56741	for permeabilization
Hoechst stain 33342	Sigma-Aldrich	B2261	nuclear stain
Dulbecco’s modified Eagle’s medium (DMEM) - high Glucose	Sigma-Aldrich	6429	cell culture media component
Heat-inactivated fetal bovine serum (FBS)	Sigma-Aldrich	F9665	cell culture media component
Penicillin-Streptomycin	Sigma-Aldrich	P4333	cell culture media component
Corning, breathable plate seal	Sigma-Aldrich	CLS3345	for copound incubation step
Rabbit anti-p38 antibody [E229]	Abcam	ab170099	primary antibody, LOT:GR305364-16
Falcon, Black 384-well clear bottom imaging plates	VWR	736-2044	imaging plates
Greiner, 384-well low volume polypropylene plates	VWR	784201
Adhesive aluminum foil	VWR	30127790
Peelable aluminium seal	Agilent	24210-001	for PlateLoc
LY2228820	Selleckchem	S1494	p38α inhibitor
PH797804	Selleckchem	PH797804	p38α inhibitor
BIRB796	Selleckchem	S1574	p38α inhibitor
SB203580	Tocris	1202	p38α inhibitor
AMG 548	Tocris	3920	p38α inhibitor
RWJ 67657	Tocris	2999	p38α inhibitor
L-Skepinone	CBCS compound collection		p38α inhibitor
Bovine serum albumin (BSA)	Sigma-Aldrich	A7030	blocking agent
SDS (sodium dodecyl sulfate)	BDH	44244	used in antigen retrieval
Glycine	Sigma-Aldrich	G8898	used in antigen retrieval
A-431 cells	ATCC	ATC-CRL-1555
Echo 550	Labcyte		For preparation of compound plates
Plate sealer	Agilent		PlateLoc
Bulk reagent dispenser	Thermo Scientific	5840300	Multidrop Combi
Automated liquid handling	Agilent		Bravo liquid handling platform; used for compound plate preparation
Plate washer	Tecan		Hydrospeed
Water bath	Julabo	TW12
Thermocouple	VWR		Thermocouple traceable lab thermometer
High content imager	Molecular Devices		ImageXpress Micro XLS Widefield High-Content Analysis System