We thank Barbara Goricnick and Nadja Hinterreiter for their help with cell culturing and preparation of experimental solutions. The authors gratefully acknowledge financial support by the Research Training Group 1910 &#34;Medicinal chemistry of selective GPCR ligands&#34; funded by the German Research Foundation (DFG) under grant number 222125149. JAS is particularly grateful for a scholarship granted by the Bavarian Gender Equality Program.

1. Cell seeding on electrode arrays
NOTE: Selection of electrode layout is a trade-off between sensitivity and number of cells under study. The smaller the electrode, the more sensitive is the measurement but the smaller is the number of cells under study. For cells showing strong impedance fluctuations over time under baseline conditions, bigger or interdigitated electrodes are preferable.
<ol>
	<li>Pre-warm all solutions needed for standard cell passaging and seeding in a 37 &#176;C water bath. For assays with human U-373 MG cells it takes: phosphate buffered saline (PBS) without calcium and magnesium, 0.05% (w/v) trypsin, cell culture medium (Eagle&#39;s Minimum Essential Medium (EMEM) supplemented with 5% (v/v) fetal calf serum (FCS), 2 mM L-glutamine and 100 &#181;g/mL penicillin/streptomycin).</li>
	<li>Rinse the cell layer of the stock culture grown on the bottom of conventional cell culture flasks or dishes with PBS twice.</li>
	<li>Remove the PBS, add the trypsin solution (1 mL for 25 cm2) and let the cells incubate at 37 &#176;C for 5 min (applies to U-373 MG cells).</li>
	<li>Control for complete detachment of the cells from the bottom of the growth substrate by microscopy.</li>
	<li>Stop the trypsin reaction as soon as the cells are completely detached by adding 9 mL of cell culture medium per 1 mL of trypsin to the cell suspension. Carefully rinse remaining cells from the bottom of the cell culture substrate by pipetting the suspension over the substrate.</li>
	<li>Collect the cell suspension with a pipette and transfer it to a centrifugation tube (15 mL or 50 mL tube).</li>
	<li>Spin down the cells by centrifugation at 110 x g for 10 min at room temperature.</li>
	<li>Carefully remove the supernatant and thoroughly re-suspend the cell pellet in culture medium before counting the cells (e.g. by using a hemacytometer for phase contrast microscopes and manual counting).</li>
	<li>Adjust the cell suspension to the desired cell density. For experiments with U-373 MG cells, use 100 000 cells/cm2 to grow a confluent cell layer within 48 h. This translates to a cell density of 200 000 cells/mL for 8-well electrode arrays with a growth area of ~0.8 cm2 and a well volume of 400 &#181;L. 
	NOTE: For reproducible GPCR activation experiments, cells should be grown to confluent monolayers on the electrode arrays. To ensure proper receptor expression on the cell surface, cells should be seeded at least 36 h before performing the experiment. Testing different cell densities for cell seeding is often meaningful to identify the best experimental conditions.</li>
	<li>Add the cell suspension to the wells of the electrode array and let the sells settle down at room temperature for 10 &#8211; 15 min in order to ensure homogenous distribution of cells on the bottom of the wells.</li>
	<li>Grow cells for at least 36 h in a standard cell culture incubator with 5% CO2 (dependent on medium type) at 37 &#176;C and humidified atmosphere. Change cell culture medium 24 h prior to the experiment.</li>
	<li>On the day of the experiment, inspect the cell layers on the electrode arrays by (phase contrast) microscopy to ensure complete coverage of electrodes with cells.</li>
</ol>
2. Equilibration of cells in serum-free medium
<ol>
	<li>Pre-warm serum-free medium, in this study: Leibovitz&#39;s L15 medium.</li>
	<li>Remove cell culture medium from cells grown on electrode arrays and replace by pre-warmed serum-free medium. Use 200 &#181;L for 8-well format electrode arrays, and 150 &#181;L for 96-well electrode arrays.</li>
	<li>Let the cells equilibrate in serum-free medium at 37 &#176;C for at least 2 h. The equilibration time strongly depends on the cell type. For example, U-373 MG cells need 2 h, CHO cells need 4 h, BAEC may require overnight equilibration in L-15 medium. 
	NOTE: L-15 medium is CO2 independent and requires a CO2-free atmosphere. For equilibration in L-15 set the incubator to 0 % CO2. Equilibration can be monitored by impedance readings, which we recommend to do for the first experiments.</li>
</ol>
3. Monitoring cell equilibration with impedance readings
<ol>
	<li>Place the electrode array into the connecting array holder of the impedance analyzer.</li>
	<li>Ensure proper low impedance contact between electrodes and impedance analyzer. This check is individually different for different instruments. 
	NOTE: If the instrument fails to make connection to the electrodes, open the contact clamp again, readjust the electrode array for proper positioning inside the holder and retry.</li>
	<li>Select electrode type and/or multi-well format from the user interface of the software.</li>
	<li>Set up the measurement parameters. Different options are available. 
	NOTE: To select the most sensitive AC frequency, we refer to the literature and instrument manuals as it depends on electrode layout and cell type under study. Typically, the sensing frequency is in the range of 4 kHz &#8211; 50 kHz. Here, U-373 MG cells were grown on circular working electrodes of 250 &#181;m diameter and they were monitored at an AC frequency of 12 kHz.
	<ol>
		<li>If single and multiple frequency data acquisition modes are available, select the single frequency mode to ensure maximum time resolution. The measurement will be performed at this single frequency. For the most widely spread instruments, there is a number of pre-set frequencies along the available frequency window.</li>
		<li>If the number of wells under study is low or time resolution is not critical, select multiple frequency recordings instead. Impedance readings at a specified number of frequencies will be recorded for every well for later in-depth analysis. 
		NOTE: The time resolution decreases with increasing number of frequencies recorded per well and increasing number of wells. The options for frequency and data acquisition mode selection vary with instrument type and version.</li>
	</ol>
	</li>
	<li>Start acquisition of time course data.</li>
	<li>Follow the cell layer impedance (at least 2 h) until the impedance has stabilized. In the meantime, prepare the agonist solutions.</li>
	<li>When the cell layers have reached a stable impedance level, either (i) proceed to the serial agonist addition within the same experiment or (ii) terminate data acquisition and start a new dataset for monitoring agonist-induced receptor activation.</li>
</ol>
4. Preparation of agonist solutions for experiments in agonist mode
<ol>
	<li>Calculate the concentration of agonist solutions as needed for each step of serial dosing according to equation (1). n ranges from 1 to the total number of serial additions i. x denotes concentration and volume in the well at step n. y denotes concentration and volume of the &#34;solution-to-be-added&#34; in step n.</li>
</ol>
<img alt="Equation 1" src="/files/ftp_upload/60686/60686equ01.jpg" />
NOTE: Consider the number of replicates and calculate the total volume of &#34;solution-to-be-added&#34; for each concentration step. Results of a typical calculation are given in Tables 1-4. It takes a general idea about the agonist concentration range to be studied as the range defines the concentrations and number of portions to be administered during serial addition. Using the serial agonist addition protocol the agonist concentration is increased stepwise. Therefore, the amount of agonist already in the well when the next dose is added has to be taken into account. When the number of agonist molecules already present in the well is nx = cx &#8729;Vx (with the current concentration cx and volume Vx) and the number of molecules in the well after the next addition is nx+y, the number of molecules to be added ny is determined by the concentration cy and volume Vy of the solution to be applied to the well (ny = cy &#8729; Vy). After adding a portion of agonist, the new amount of agonist molecules in the well is: cx+y &#8729; Vx+y = cx &#8729; Vx + cy &#8729; Vy. This calculation applies for each subsequent step. Because of the interdependence of agonist concentration in the well and the amount of agonist in the portions to be added with each step, it is important to define final concentrations after each step in advance.
Mode 1: The volume in the well will increase with each step as liquid is continuously added. 
Using this mode and an 8-well format, use Vx1 = 200 &#181;L and Vy1 &#8230;. Vyi = 30 &#181;L.
Mode 2: The volume in the well is constant as the volume added with each step is removed just before the subsequent addition. 
Using this mode and a 96-well format, use Vx1 = 150 &#181;L and Vy1 &#8230;. Vyi = 75 &#181;L.
<ol start="2">
	<li>Print the data sheet with the total volume per concentration and detailed instructions for pipetting.</li>
	<li>Prepare all solutions in the required amount. Make all agonist solutions in the same serum-free medium as used for equilibration of the cells. 
	CAUTION: Histamine dihydrochloride is considered hazardous by the 2012 OSHA Hazard Communication Standard (29 CFR 1910.1200). Histamine causes skin irritation, serious eye irritation, may cause an allergic skin reaction, allergy, asthma symptoms or breathing difficulties if inhaled and may cause respiratory irritation. Please consider the safety data sheet. 
	NOTE: Make agonist solutions as fresh as possible. Stability of agonists in solution may vary considerably. Store solutions at 4 &#176;C or below until use in the experiment. For some molecules additional stabilizing additives, like BSA when using peptide-based or lipid-based molecules, may be considered to prevent adsorption to the walls of wells and tubes.</li>
	<li>If performing the experiment in 96-well format, transfer solutions to a conventional 96-well plate (no electrodes) and use an 8- (or 12-) channel pipet for quick liquid transfer to the electrode array.</li>
</ol>
5. Preparation of agonist solutions for experiment in antagonist mode
NOTE: Prepare the antagonist solution(s) with the concentration to be applied throughout. Volume and concentration of antagonist solution depend on the mode of agonist addition (1 or 2). Examples for experiments in 8-well or 96-well format in addition mode 2: (A) 8-well format (Vx1 = 200 &#181;L, VAntagonist = 200 &#181;L); (B) 96-well format (Vx1 = 150 &#181;L, VAntagonist = 75 &#181;L).
<ol>
	<li>Calculate the concentration of each agonist solution needed for each step of serial dosing as described in step 4.1.</li>
	<li>Make all agonist solutions in the same serum-free medium as used for equilibration of the cells and add the antagonist at the same final concentration as planned for the respective wells in the experiment. 
	NOTE: In this case the histamine stock solution (10 mM) is prepared in L-15 medium. When agonists are dissolved in other solvents (e.g., dimethyl sulfoxid (DMSO), ethanol) a solvent control should be included to account for the increasing solvent load with each step of addition.</li>
</ol>
6. Performing the serial addition protocol in agonist mode
<ol>
	<li>Start data acquisition as described in steps 3.1 &#8211; 3.5.</li>
	<li>Pre-warm the agonist solutions prior to use by placing them in the incubator about 10 &#8211; 15 min before addition. 
	NOTE: When using thermo-labile substances, the solutions should not be kept at 37 &#176;C for too long. If pre-warming for 10 &#8211; 15 min is considered critical, bring solutions to 37 &#176;C shortly before addition in a water bath.</li>
	<li>Run the agonist serial dosing dependent on the selected addition mode. Following mode 1 the total volume in the well increases with each addition of the next dose. In mode 2 the same volume which is added with each step is also removed again just before adding the next higher dose. 
	NOTE: Time needed for equilibration of the cell layer in between two subsequent agonist doses depends on the cells&#39; response time. An initial experiment in parallel mode (one well &#8211; one concentration) reveals (i) cell response times for different agonist concentrations and (ii) the most sensitive curve parameter (e.g., impedance maximum, impedance after time x).</li>
</ol>
A: Mode 1 / 8-well format
<ol>
	<li>Add 30 &#181;L of the first solution with the lowest concentration of agonist to the cells that were equilibrated in 200 &#181;L of serum-free medium.</li>
	<li>Let the cells respond and equilibrate for the pre-defined period of time (e.g., 15 min).</li>
	<li>Add 30 &#181;L of the second solution with the next higher concentration.</li>
	<li>Repeat steps 6.3.1-6.3.3 with the third, fourth, and so on, agonist solution. 
	NOTE: Working with 10 concentration steps will result in a total volume of 500 &#181;L in the end of the experiment, which is just below the maximum applicable volume of these wells of ~ 550 &#181;L.</li>
</ol>
B: Mode 2 / 96-well format
NOTE: Pause data acquisition during each liquid handling step (addition/removal) via the impedance instrument&#39;s software when running experiments in 96-well format. The more elaborate liquid handling may interfere with data acquisition. Use a multi-channel pipette.
<ol>
	<li>Pause data acquisition.</li>
	<li>Add 75 &#181;L of the first solution with the lowest concentration of agonist to the cells that were equilibrated in 150 &#181;L of serum-free medium.</li>
	<li>Resume data acquisition.</li>
	<li>Let the cells respond and equilibrate for the pre-defined period of time (e.g., 15 min).</li>
	<li>About 1 &#8211; 2 min before the regular equilibration time ends, pause the measurement and remove 75 &#181;L from each well. 
	NOTE: The time point at which solution should be removed depends on the number of wells monitored in parallel and the pipetting speed. Time needed for removal of the solutions should not exceed the time between subsequent steps.</li>
	<li>Add 75 &#181;L of the second solution with the next higher concentration and resume the measurement.</li>
	<li>Repeat steps 6.3.8-6.3.10 with the third, fourth, and so on, agonist solution.</li>
</ol>
7. Performing the serial addition protocol in antagonist mode
<ol>
	<li>Start the measurement as described in steps 3.1 &#8211; 3.5.</li>
	<li>During equilibration of the cell layers, prepare antagonist solution (e.g., 200 &#181;L of 1.5 &#181;M diphenhydramine hydrochloride in L15 medium). 
	CAUTION: Diphenhydramine hydrochloride has potential acute health effects. It is harmful if swallowed or inhaled, may cause eye and skin irritation. It may cause respiratory and digestive tract irritation. Please consider the safety data sheet.</li>
	<li>Pre-warm the antagonist and agonist solutions by placing them in the incubator about 10 &#8211; 15 min before addition to the cell cultures (cf. 6.2). If antagonist-free wells are included in the assay as well, also pre-warm serum-free medium.</li>
	<li>Add the antagonist solution to the designated wells. Let the cells equilibrate with antagonist for 15 &#8211; 20 min. If antagonist-free wells are included, add the same volume of serum-free medium to these wells.</li>
	<li>According to antagonist addition in mode 2, remove solution from the wells 
	(A) 8-well format (200 &#181;L) 
	(B) 96-well format (75 &#181;L)</li>
	<li>Run the agonist addition sequence as described in step 6.3.</li>
</ol>
8. Data export and analysis
<ol>
	<li>Export data using the impedance instrument&#39;s software in order to convert all recorded data from proprietary into common data formats (e.g., csv). This step allows for data re-organization and presentation with other software packages.</li>
	<li>Load the csv-formatted data to scientific data analysis software.</li>
	<li>Normalize impedance values by subtracting the impedance of the last data point before the first addition of agonist solution and by setting the time of addition to t = 0. Plot the time course of normalized impedance.</li>
	<li>Plot the individual time courses and identify the maxima in impedance after each addition step. Compose a data sheet with these values.</li>
	<li>Plot the values of maximum (or minimum, if applicable) impedance changes as a function of agonist concentration. This can be done for individual wells or for the averages (mean &#177; SD).</li>
	<li>Use a data fitting routine to determine half-maximal effective concentrations (EC50) and maximum response (EMax) by using the four parameter logistic model (equation 2):</li>
</ol>
<img alt="Equation 2" src="/files/ftp_upload/60686/60686equ02.jpg" />
NOTE: c indicates the agonist concentration, A1 is the minimum and A2 is the maximum asymptote of the sigmoidal dose-response curve (A2 = EMax). EC50 is the concentration at the inflection point of the curve, and n corresponds to the Hill slope.

<ol>
	<li>Rosenbaum, D. M., Rasmussen, S. G., Kobilka, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+and+function+of+G-protein-coupled+receptors.">The structure and function of G-protein-coupled receptors.</a> Nature. 459, 356-363 (2009).</li><li>Sriram, K., Insel, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G+Protein-Coupled+Receptors+as+Targets+for+Approved+Drugs:+How+Many+Targets+and+How+Many+Drugs.">G Protein-Coupled Receptors as Targets for Approved Drugs: How Many Targets and How Many Drugs.</a> Molecular Pharmacology. 93, 251-258 (2018).</li><li>Kaitin, K. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deconstructing+the+drug+development+process:+The+new+face+of+innovation.">Deconstructing the drug development process: The new face of innovation.</a> Clinical Pharmacoly and Therapeutics. 87, 6 (2010).</li><li>Kenakin, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+assays+as+portals+to+seven-transmembrane+receptor-based+drug+discovery.">Cellular assays as portals to seven-transmembrane receptor-based drug discovery.</a> Nature reviews. Drug discovery. 8, 617-626 (2009).</li><li>Zhang, R., Xie, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+for+GPCR+drug+discovery.">Tools for GPCR drug discovery.</a> Acta Pharmacologica Sinica. 33, 372-384 (2012).</li><li>Scott, C. W., Peters, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+whole-cell+assays:+expanding+the+scope+of+GPCR+screening.">Label-free whole-cell assays: expanding the scope of GPCR screening.</a> Drug Discovery Today. 15, 704-716 (2010).</li><li>Giaever, I., Keese, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+fibroblast+behavior+in+tissue+culture+with+an+applied+electric+field.">Monitoring fibroblast behavior in tissue culture with an applied electric field.</a> Proceedings of the National Academy of Sciences of the United States of America. 81, 3761-3764 (1984).</li><li>Giaever, I., Keese, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+morphological+biosensor+for+mammalian+cells.">A morphological biosensor for mammalian cells.</a> Nature. 366, 591-592 (1993).</li><li>Fang, Y., Ferrie, A. M., Fontaine, N. H., Mauro, J., Balakrishnan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resonant+waveguide+grating+biosensor+for+living+cell+sensing.">Resonant waveguide grating biosensor for living cell sensing.</a> Biophysical Journal. 91, 16 (2006).</li><li>Stolwijk, J. A., Matrougui, K., Renken, C. W., Trebak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impedance+analysis+of+GPCR-mediated+changes+in+endothelial+barrier+function:+overview+and+fundamental+considerations+for+stable+and+reproducible+measurements.">Impedance analysis of GPCR-mediated changes in endothelial barrier function: overview and fundamental considerations for stable and reproducible measurements.</a> Pflugers Archiv : European Journal of Physiology. 467, 2193-2218 (2015).</li><li>Lieb, 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=Label-free+versus+conventional+cellular+assays:+Functional+investigations+on+the+human+histamine+H1+receptor.">Label-free versus conventional cellular assays: Functional investigations on the human histamine H1 receptor.</a> Pharmacological Research. 114, 13-26 (2016).</li><li>Stolwijk, J. 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=Increasing+the+throughput+of+label-free+cell+assays+to+study+the+activation+of+G-protein-coupled+receptors+by+using+a+serial+agonist+exposure+protocol.">Increasing the throughput of label-free cell assays to study the activation of G-protein-coupled receptors by using a serial agonist exposure protocol.</a> Integrative Biology. 11, 10 (2019).</li></ol>

The authors have nothing to disclose.

This protocol describes a method for label-free impedance measurements to determine the dose-response relationship of agonist-induced GPCR activation in absence or presence of specific antagonists for the same receptor. The proof of concept of this method was presented in a recent publication12. To our knowledge it is the first study describing the establishment of a full dose-response curve of agonist-mediated GPCR activation using a single cell layer in vitro. The approach inevitably requires th...

This report presents a detailed description of a serial addition protocol developed for the quantification of ligand-induced G protein-coupled receptor (GPCR) activation in adherently grown cells by label-free impedance measurements. G protein-coupled receptors (GPCRs) are involved in a multitude of physiological functions and human diseases1. Because of this and their good accessibility at the cell surface, GPCRs are one of the most important drug targets. This assessment is reflected in an estimated number of ~700 approved drugs targeting GPCRs, equivalent to a ~35% share on all marketed drugs2.
The development of new drugs comprises two central processes: (i) the identification and functional characterization of biological target molecules, and (ii) the discovery of new lead substances and their development into administrable drugs. In both processes, efficient methods are required to quantitatively evaluate drug-target interactions and the subsequent biological downstream response. Different stages of the pre-clinical drug development process make use of different methods of analysis ranging from biomolecular interaction studies between drug and target, over functional studies on cells in culture, to experiments on excised organ material or whole animals. Both, physiological significance and biological complexity increase from the former to the latter3. Although it is the overall goal to minimize animal experiments, pharmacological studies using isolated organs from laboratory animals or even whole animals are regarded inevitable to comprehensively characterize new drug candidates. In terms of analytical readout, organ pharmacology studies provide a distal, integrative &#34;holistic&#34; functional response of highest physiological relevance. A drawback of such experiments is that they are not compatible with high throughput screening for technical as well as ethical reasons and have been largely substituted by studies based on in vitro cell culture4.
Methods to quantify GPCR activation in cell cultures include different label-based chemical assays, which specifically detect second messengers, phosphorylation state of downstream signaling proteins, transcriptional activation via certain transcription factors or ligand-induced intracellular receptor trafficking4,5. A drawback of such label-based assays is the necessity to label the cells with potentially harmful dyes or radioactive markers. This often requires running the assay as an endpoint-determination for an exposure time that has to be specified a priori. Using label-based endpoint assays suffers from this very limited and biased timing of the experiment and the risk that chemical labels and probes may interfere with the regular cell physiology &#8211; potentially unnoticed by the experimenter.
In recent years, label-free assays for monitoring GPCR activation have emerged, like impedance-based techniques or optical methods applying resonant waveguide gratings5,6. Labelling of the cells is experimentally not required with these approaches. As these physical readouts operate on low amplitude signals, such methods are considered non-invasive, they allow for continuous cell monitoring potentially in real time and the observation time is only limited by the cell culture not by the readout. Similar to readouts from whole organs, label-free approaches commonly report on holistic cell responses, far downstream of receptor activation when integration along the entire signaling network leads to time-dependent but rather unspecific changes in cell morphology or mass redistribution. Whereas impedance-based assays measure the dielectric signature of changes in cell shape7,8, measurements using resonant waveguide gratings are sensitive to changes in refractive index at the cell-substrate interface arising from dynamic mass redistribution (DMR)9. The integrative character makes label-free methods extremely sensitive to receptor-mediated events regardless of the type(s) of G protein (Gq, Gi/0, Gs, G12/13) or &#946;-arrestin involved in the signaling cascade6 and well-suited for endogenous expression levels of the receptor.
In a standard label-free impedance-based assay the cells are adherently grown in multi-well plates with coplanar gold-film electrodes deposited on the bottom of each well10. These electrode arrays are connected to an impedance analyzer and the cell responses to an experimental stimulus are recorded from individual wells by time-resolved impedance readings. In a typical GPCR assay a ligand is added in individually different concentrations to each individual well. The ligand-induced changes in the impedance time course are then analyzed with respect to characteristic curve features such as maximum signal change, area under the curve, signal change within a given time interval or slope of the curve at a specified time point, in order to quantify the ligand&#39;s potency and efficacy11.
The cost of the electrode arrays may limit the application of this technique in high throughput screening (HTS) campaigns. Moreover, with increasing numbers of samples to be followed in parallel, the number of individual measurements increases and thereby reduces the available time resolution for each well gradually - even for state of the art multichannel recordings. Under such conditions fast and transient cell responses may escape the measurement. In addition, the conventional one well &#8211; one concentration approach imposes a significant time and cost factor on perfused organ-on-chip or body-on-chip developments with respect to their suitability in ligand-GPCR interaction analysis.
For this reason, we developed a stepwise dosing protocol that enables recording of full dose-response curves of ligand-induced GPCR activation in cultured cell monolayers by continuously monitoring the impedance of a single well while the agonist concentration is increased stepwise. The serial agonist addition protocol significantly increases throughput per well from one concentration to 10 or more concentrations, as shown on the current example of human U-373 MG cells, which endogenously express the histamine 1 receptor (H1R). Thus, the method has the potential to significantly improve throughput in label-free dose-response studies, while the time resolution is retained at the instrumental maximum.

<table><tbody><tr><td>B&amp;uuml;rker counting chamber</td><td>Marienfeld (Lauda-K&amp;ouml;nigshofen, Germany)</td><td>640210</td><td></td></tr><tr><td>cell culture flasks 25 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>Greiner bio-one (Frickenhausen, Germany)</td><td>690175</td><td></td></tr><tr><td>Cell incubator (Heraeus Function Line BB15)</td><td>Thermo Scientific (Darmstadt, Germany)</td><td>\</td><td></td></tr><tr><td>Centrifuge (Heraeus 1S-R)</td><td>Thermo Scientific (Darmstadt, Germany)</td><td>\</td><td></td></tr><tr><td>Diphenhydramine hydrochloride</td><td>Sigma Aldrich (Taufkirchen, Germany)</td><td>D3630</td><td></td></tr><tr><td>Eagle&#039;s Minimum Essential Medium with 4.5 g/L D-Glucose and 2.2 g/L NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma Aldrich (Taufkirchen, Germany)</td><td>D5671</td><td></td></tr><tr><td>Impedance Instrument (ECIS Z&amp;theta;)</td><td>Applied BioPhysics Inc. (Troy, NY, USA)</td><td>\</td><td></td></tr><tr><td>8-well electrode Arrays (8W1E PET)</td><td>Applied BioPhysics Inc. (Troy, NY, USA)</td><td>\</td><td>PET base with 0.049 mm&lt;sup&gt;2&lt;/sup&gt; working electrode and ~50 mm&lt;sup&gt;2&lt;/sup&gt; counter electrode (gold)</td></tr><tr><td>96-well electrode arrays (96W1E+ PET)</td><td>Applied BioPhysics Inc. (Troy, NY, USA)</td><td>\</td><td>PET base with two electrodes (gold) with 0.256 mm&lt;sup&gt;2&lt;/sup&gt; total electrode area</td></tr><tr><td>Fetal calf serum (FCS)</td><td>Biochrom (Berlin, Germany)</td><td>S0615</td><td></td></tr><tr><td>Histamine dihydrochloride</td><td>Carl Roth (Karlsruhe, Germany)</td><td>4017.1</td><td></td></tr><tr><td>Laminar flow hood (Herasafe, KS 12)</td><td>Thermo Scientific (Darmstadt, Germany)</td><td>51022515</td><td>class II safety cabinet</td></tr><tr><td>Leibovitz&#039; L-15 medium</td><td>Thermo Scientific (Darmstadt, Germany)</td><td>21083-027</td><td></td></tr><tr><td>L-glutamine</td><td>Sigma Aldrich (Taufkirchen, Germany)</td><td>G7513</td><td></td></tr><tr><td>Micropipette large (100 - 1000 &amp;micro;L)</td><td>Brandt (Wertheim, Germany)</td><td>704780</td><td></td></tr><tr><td>Micropipette large (20 - 200 &amp;micro;L)</td><td>Brandt (Wertheim, Germany)</td><td>704778</td><td></td></tr><tr><td>Microscope (phase contrast, Nikon Diaphot)</td><td>Nikon (D&amp;uuml;sseldorf, Germany)</td><td></td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Sigma Aldrich (Taufkirchen, Germany)</td><td>P0781</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma Aldrich (Taufkirchen, Germany)</td><td>D8537</td><td></td></tr><tr><td>Pipette, serological</td><td>Greiner bio-one (Frickenhausen, Germany)</td><td>607 180</td><td></td></tr><tr><td>Pipettor (accu-jet pro)</td><td>Brandt (Wertheim, Germany)</td><td>26300</td><td></td></tr><tr><td>Trypsin</td><td>Sigma Aldrich (Taufkirchen, Germany)</td><td>T4174</td><td>in PBS with 1 mM EDTA</td></tr><tr><td>Tube, 15 mL</td><td>Greiner bio-one (Frickenhausen, Germany)</td><td>188 271</td><td></td></tr><tr><td>Tube, 50 mL</td><td>Greiner bio-one (Frickenhausen, Germany)</td><td>210 261</td><td></td></tr><tr><td>U-373 MG cells</td><td>ATCC (Rockville, MD, USA)</td><td>ATCC HTB-17</td><td></td></tr><tr><td>water bath (TW21)</td><td>Julabo (Seelbach, Germany)</td><td>\</td><td></td></tr></tbody></table>

stepwise dosing protocol for increased throughput in label-free impedance-based gpcr assays

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach provides real-time cell monitoring with a device-dependent time resolution down to several tens of milliseconds and it is highly automated. However, when sample numbers get high (e.g., dose-response studies for various different ligands), the cost for the disposable electrode arrays as well as the available time resolution for sequential well-by-well recordings may become limiting. Therefore, we here present a serial agonist addition protocol which has the potential to significantly increase the output of label-free GPCR assays. Using the serial agonist addition protocol, a GPCR agonist is added sequentially in increasing concentrations to a single cell layer while continuously monitoring the sample&#39;s impedance (agonist mode). With this serial approach, it is now possible to establish a full dose-response curve for a GPCR agonist from just one single cell layer. The serial agonist addition protocol is applicable to different GPCR coupling types, Gq Gi/0 or Gs and it is compatible with recombinant and endogenous expression levels of the receptor under study. Receptor blocking by GPCR antagonists is assessable as well (antagonist mode).

A typical scheme for preparing the various agonist solutions is shown for an experiment using 8-well electrode arrays with histamine as agonist in Tables 1-4. Table 1 and Table 2 present volumes and concentrations for an experiment using addition mode 1 (cf., Figure 1), while Table 3 and Table 4 present volumes and concentrations for an experiment following addition mode 2 (...

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Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays

This protocol demonstrates real-time recording of full dose-response relationships for agonist-induced GPCR activation from a single cell layer grown on a single microelectrode using label-free impedance measurements. The new dosing scheme significantly increases throughput without loss in time resolution.

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach ...

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6.5K Views.  University of Regensburg. Dit protocol maakt het mogelijk om een volledige functionele concentratieresponsrelatie van GPCR-agonist uit één celpopulatie op te nemen en biedt gedetailleerde agonistische karakterisering, zelfs voor lage celmonsternummers. Het belangrijkste voordeel van seriële dosering is de toename van de doorvoer, omdat een enkele put voldoende is voor een volledige concentratieresponsanalyse. Deze techniek is zeer geschikt om enkele transductie te bestuderen in zeldzame en complexe biologische mode...

Video: Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists1. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the use of four routinely used biosensor platforms in our laboratory to evaluate the binding affinity and kinetics of ten high-affinity monoclonal antibodies (mAbs) against human proprotein convertase subtilisin kexin type 9 (PCSK9). While both Biacore T100 and ProteOn XPR36 are derived from the well-established Surface Plasmon Resonance (SPR) technology, the former has four flow cells connected by serial flow configuration, whereas the latter presents 36 reaction spots in parallel through an improvised 6 x 6 crisscross microfluidic channel configuration. The IBIS MX96 also operates based on the SPR sensor technology, with an additional imaging feature that provides detection in spatial orientation. This detection technique coupled with the Continuous Flow Microspotter (CFM) expands the throughput significantly by enabling multiplex array printing and detection of 96 reaction sports simultaneously. In contrast, the Octet RED384 is based on the BioLayer Interferometry (BLI) optical principle, with fiber-optic probes acting as the biosensor to detect interference pattern changes upon binding interactions at the tip surface. Unlike the SPR-based platforms, the BLI system does not rely on continuous flow fluidics; instead, the sensor tips collect readings while they are immersed in analyte solutions of a 384-well microplate during orbital agitation.
Each of these biosensor platforms has its own advantages and disadvantages. To provide a direct comparison of these instruments&#39; ability to provide quality kinetic data, the described protocols illustrate experiments that use the same assay format and the same high-quality reagents to characterize antibody-antigen kinetics that fit the simple 1:1 molecular interaction model.

We describe here protocols for the measurement of antibody-antigen binding affinity and kinetics using four commonly used biosensor platforms.

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the ...

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Bioengineering

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane1. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein&#39;s behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

Optical biosensor techniques can detect changes in mass near the plasma membrane in living cells and allow one to follow cellular responses in both individual cells and populations of cells. This protocol will describe detection of the modulation of potassium channels by G-protein coupled receptors in intact cells using this approach.

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma ...

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

Chemistry

Monitoring GPCR-&#946;-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery

Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. Currently, the characterization of novel drugs towards their interactions with &#946;-arrestins and other cytosolic proteins is extremely valuable in the field of GPCR drug discovery particularly during the study of GPCR biased agonism. Here, we show the application of a novel structural complementation assay to accurately monitor receptor-&#946;-arrestin interactions in real time living systems. This method is simple, accurate and can be easily extended to any GPCR of interest and also it has the advantage that it overcomes unspecific interactions due to the presence of a low expression promoter present in each vector system. This structural complementation assay provides key features that allow an accurate and precise monitoring of receptor-&#946;-arrestin interactions, making it suitable in the study of biased agonism of any GPCR system as well as GPCR c-terminus &#8216;phosphorylation codes&#8217; written by different GPCR-kinases (GRKs) and post-translational modifications of arrestins that stabilize or destabilize the receptor-&#946;-arrestin complex.

GPCR-&#946;-arrestin interactions are an emerging field in GPCR drug discovery. Accurate, precise and easy to set up methods are necessary to monitor such interactions in living systems. We show a structural complementation assay to monitor GPCR-&#946;-arrestin interactions in real time living cells, and it can be extended to any GPCR.

Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. ...

Parallel Interrogation of &#946;-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent one of the most promising targets for the pharmaceutical industry. Ergo, the design, implementation, and optimization of GPCR ligand screening assays is crucial, as they represent remote-control tools for drug discovery and for manipulating GPCR pharmacology and outcomes. In the past, G-protein dependent assays typified this area of research, detecting ligand-induced events and quantifying the generation of secondary messengers. However, since the advent of functional selectivity, as well as an increased awareness of several other G protein-independent pathways and the limitations associated with G-protein dependent assays, there is a greater push towards the creation of alternative GPCR ligand screening assays. Towards this endeavor, we describe the application of one such resource, the PRESTO-Tango platform, a luciferase reporter-based system that enables the parallel and simultaneous interrogation of the human GPCR-ome, a feat which was previously considered technically and economically unfeasible. Based on a G-protein independent &#946;-arrestin2 recruitment assay, the universality of &#946;-arrestin2-mediated trafficking and signaling at GPCRs makes PRESTO-TANGO an apt tool for studying approximately 300 non-olfactory human GPCRs, including approximately 100 orphan receptors. PRESTO-Tango&#39;s sensitivity and robustness make it suitable for primary high-throughput screens using compound libraries, employed to uncover new GPCR targets for known drugs or to discover new ligands for orphan receptors.

Given that GPCRs are attractive druggable targets, GPCR ligand screening is thus indispensable for the identification of lead compounds and for deorphanization studies. Towards these efforts, we describe PRESTO-Tango, an open-source resource platform used for simultaneous profiling of transient &#946;-arrestin2 recruitment at approximately 300 GPCRs using a TEV-based reporter assay.

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent ...

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.
The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.
The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a &#34;dual-addition&#34; approach can detect functional agonist and antagonist &#34;hit&#34; molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z&#39; factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 &#181;M final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include well-validated targets for therapeutic intervention. Discovery of lead compounds, including small synthetic molecules, that specifically inhibit the receptor&#39;s function, is an important initial step in drug development and relies on sensitive, specific, and robust cell-based assays. Here, we describe a kinetic cellular assay with a fluorescent readout primarily designed to identify receptor-specific antagonists that inhibit the intracellular Ca2+ release evoked upon the activation of the CXC chemokine receptor 4 (CXCR4) by its endogenous ligand, the CXC chemokine ligand 12 (CXCL12). A key advantage of this method is that it also enables screening of compounds endowed with intrinsic agonistic properties (i.e., compounds eliciting an increase in intracellular Ca2+ concentration in the absence of CXCL12) or compounds modulating the receptor&#39;s function via interaction with allosteric binding sites (i.e., positive and negative allosteric modulators (PAMs and NAMs, respectively)). On the down side, autofluorescent compounds might interfere with the assay&#39;s readout, thereby hampering reliable data interpretation. Most likely this assay can be implemented, with minimal adaptations, as a generic drug discovery assay for many other GPCRs of which the activation leads to a release of intracellular Ca2+.

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

The described cellular assay is designed for the identification of CXC chemokine receptor 4 (CXCR4)-interacting agents that inhibit or stimulate, either competitively or allosterically, the intracellular Ca2+ release initiated by CXCR4 activation.

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include ...

Medicine

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays

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