This work was supported by the USDA-NIFA-AFRI Animal Health and Well-Being Award (Award number 2022-67015-36336, PVP [Project Director]) and from competitive funds from the Texas A&#38;M AgriLife Research Insect Vector Diseases Grant Program (FY&#39;22-23) to P.V.P. The A.W.E.S.O.M.E. faculty group of the College of Agriculture and Life Sciences, TAMU, is acknowledged for help editing the manuscript. Supplemental Table S2 contains data from an in-house, random, small-molecule library obtained from Dr. James Sacchettini&#39;s laboratory at Texas A&#38;M University and Texas A&#38;M AgriLife Research.

1. Cell maintenance
NOTE: A CHO-K1 cell line that stably expresses the kinin receptor from R. microplus, named BMLK3, was developed by Holmes et al.16. The details of the development of the cell line are presented elsewhere14. All the following steps are performed under sterile conditions in a class II biosafety cabinet.
<ol>
	<li>Grow the recombinant cell line in selective media (F-12K medium containing 10% fetal bovine serum [FBS] and 800 &#181;g/mL G418 sulfate) to ensure the expression of the target receptor. Store the cells stably expressing the receptor in 1 mL of freezing medium (90% FBS and 10% dimethyl sulfoxide [DMSO]) in a 2 mL cryovial in a &#8722;80 &#176;C freezer. 
	NOTE: For long-term storage of the frozen cells, store them in liquid nitrogen.</li>
	<li>Prewarm all the media before cell culture. In a biosafety cabinet, transfer 13 mL of prewarmed selective medium (F-12K medium containing 10% FBS and 800 &#181;g/mL G418 sulfate) into a T-75 flask, and keep it in the biosafety cabinet. Thaw one vial of the frozen BMLK3 cells (~1.5 &#215; 106 cells) in a 37 &#176;C water bath for 2-3 min. Transfer the thawed cells into the T-75 flask. Maintain the cells in a humidified incubator at 37 &#176;C and 5% CO2 unless specified otherwise.</li>
	<li>When the cells reach 90% confluency (1-2 days), prewarm all the media to 37 &#176;C except for Dulbecco&#39;s phosphate-buffered saline (DPBS), which is kept at room temperature. In a biosafety cabinet, remove the spent medium from the T-75 flask, wash the cells for 5 s with 10 mL of DPBS by swirling the flask gently, and then remove the DPBS with a serological pipette. 
	NOTE: An image of CHO-K1 cells at 90% confluency is shown in Lu et al.14.</li>
	<li>Detach the cells from the T-75 flask by adding 2 mL of 0.25% trypsin-EDTA, and incubate for 3-5 min at 37 &#176;C in the incubator. Then, add 8 mL of selective medium, and mix well by pipetting and releasing gently 2x-3x with the same serological pipette.</li>
	<li>Transfer 2 mL of the cell suspension (~1 &#215; 106 cells) from step 1.4 into a new T-75 flask containing 10 mL of warm selective medium. Grow the cells for 1-2 days in the incubator until they reach 90% confluency.</li>
	<li>Use the cells for the assay following the next steps, or repeat steps 1.4-1.5 once or twice before using the cells in the assay. 
	NOTE: Do not exceed three to four passages, as the assay signal with certain cell lines may become weaker with further passages.</li>
</ol>
2. Calcium fluorescence assay
<ol>
	<li>Coat the cell plate.
	<ol>
		<li>Use a liquid handling system placed inside a biosafety cabinet for all the pipetting steps in the 384-well plates. Create custom programs for pipetting into the 384-well plates31 (Supplemental Table S1). Coat the sterile 384-well plates in advance. In a biosafety cabinet, load 10 &#181;L/well of an aqueous solution of Poly-D-lysine (PDL) at 0.05 mg/mL into each plate, and incubate for 5 min at room temperature.</li>
		<li>Empty the plate by quickly inverting the plate and gently blotting it on sterile paper towels. Then, rinse each well with 10 &#181;L of water, empty the plate, and leave the plate to dry in the biosafety cabinet overnight without the lid. Close the plate with the lid, and store at 4 &#176;C in the refrigerator. 
		&#8203;NOTE: The coated plates can be stored at 4 &#176;C for up to 6 months.</li>
	</ol>
	</li>
	<li>Day 1
	<ol>
		<li>Take out the drug plate (100 &#181;M in 90% DPBS + 10% DMSO, final concentration in the well for HTS will be 2 &#181;M) stored in the &#8722;20 &#176;C freezer, and place it at room temperature. 
		NOTE: Layout of the drug plate: Each 384-well plate (24 columns x 16 rows) contains 320 wells with different library compounds and 64 wells with blank solvent (DPBS containing 10% DMSO), which are arranged in four columns, with two columns on the edge of each side. See Supplemental Table S2 for the plate layout.</li>
		<li>When cells reach ~70%-90% confluency in the T-75 flask, detach the cells from the T-75 flask as described below. Prewarm all the media to 37 &#176;C except for the DPBS (room temperature).
		<ol>
			<li>Remove the spent medium, wash the cells with 10 mL of DPBS, and then remove the DPBS. Detach the cells from the T-75 flask using 2 mL of 0.25% trypsin-EDTA for 3-5 min at 37 &#176;C in the incubator, add 8 mL of selective medium, and transfer the cell suspension into a 15 mL conical tube to centrifuge at 1,000 &#215; g for 3 min.</li>
			<li>Discard the supernatant, and resuspend the cell pellet in 10 mL of F-12K medium containing 1% FBS and 400 &#181;g/mL G418 sulfate. Keep the suspension in the biosafety cabinet while determining the cell count.</li>
		</ol>
		</li>
		<li>Determine the cell density of the suspension for further dilution: Mix 20 &#181;L of cell suspension into 20 &#181;L of 0.4% trypan blue, and then load 20 &#181;L of the mixture into a cell counting chamber to be read by a cell counter for cell density.</li>
		<li>Dilute the cell suspension in the same medium (F-12K medium containing 1% FBS and 400 &#181;g/mL G418 sulfate) to a final volume of at least 15 mL at a density of 4 &#215; 105 cells/mL.</li>
		<li>Seed the cells in the PDL-coated 384-well plate. Transfer 15 mL of the above cell suspension (4&#160;&#215; 105 cells/mL) into a 150 mL auto-friendly reagent reservoir. Dispense 25 &#181;L of the cell suspension (~10,000 cells/well) into each well of the 384 wells of the plate using a liquid handling system in two steps. Load 384/12.5 &#181;L low-retention tips onto the liquid handler head, aspirate 12.5 &#181;L from the reservoir (speed 5.2 &#181;L/s), and dispense into each well (speed: 3.1 &#181;L/s).</li>
		<li>Repeat the pipetting as in previous step 2.2.5 to reach 25 &#181;L per well. Then, incubate the plate overnight (14-16 h) at 37 &#176;C and 5% CO2 in the humidified incubator. 
		NOTE: As the maximum pipetting volume is 12.5 &#181;L for this specific head, pipetting for 25 &#181;L is performed in two steps.</li>
	</ol>
	</li>
	<li>Day 2
	<ol>
		<li>The next morning, check the covered cell plate under a microscope; if not confluent, wait until the cells reach 90% confluency.</li>
		<li>Prepare a stock solution of fluorescent dye: Resuspend lyophilized fluorescent dye in 100 &#181;L of DMSO, and avoid direct light on the stock solution. Wrap the tube with aluminum foil to prevent photobleaching. 
		NOTE: The stock can be aliquoted into 15 &#181;L aliquots for each plate assay to avoid repeated freezing and thawing; the aliquots can be stored at &#8722;80 &#176;C for up to 1 month.</li>
		<li>In the biosafety cabinet with its lights off, prepare loading dye (1x) in a 15 mL conical tube wrapped with aluminum foil, combining 15 &#181;L of the fluorescent dye stock solution (from step 2.3.2) and the rest of the prewarmed kit components (37 &#176;C): 13.5 mL of 1x HHBS (Hank&#39;s buffer with 20 mM HEPES) and 1.5 mL of reagent B (dye efflux inhibitor). 
		NOTE: The room light is on during this step.</li>
		<li>Close the tube and mix well by gently inverting the tube several (typically 3-5x) times. 
		NOTE: Keep at room temperature, and use the loading dye mixture within 30 min.</li>
		<li>When the cells reach 90% confluency, remove the spent medium from the 384-well assay plate by quickly inverting the plate and gently blotting on sterile paper towels; repeat this movement 2x-3x to remove all the liquid from the plate. Discard the wet towels.</li>
		<li>Turn off most artificial direct lights in the room (leaving a soft, dim desk lamp on or similar to allow visible work conditions) and in the biosafety cabinet for all the steps until the end of the assay (approximately for 1.5 h).</li>
		<li>Place 15 mL of the loading dye (1x) from step 2.3.3 into a 150 mL auto-friendly reservoir.
		<ol>
			<li>Transfer 25 &#181;L of the loading dye (1x) from the reservoir into each well using a liquid handling system with 384/12.5 &#181;L low-retention tips by dispensing 12.5 &#181;L into each well of the plate (aspiration and dispensing speed: 3.8 &#181;L/s).</li>
		</ol>
		</li>
		<li>Repeat the pipetting step as in 2.3.7.1 to reach a final volume of 25 &#181;L in each well of the plate. Cover and wrap the plate with aluminum foil to protect it from ambient light.</li>
		<li>Incubate the covered cell plate at 37 &#176;C in the CO2 humidified incubator for 30 min, remove it from the incubator, and equilibrate it at room temperature inside the plate reader or on the bench, keeping the plate covered and wrapped with foil for another 30 min. The cells are then ready for high-throughput screening (HTS).</li>
		<li>Chemical preparation: Spin the drug plate from step 2.2.1 at 1,200 &#215; g in a plate centrifuge for 1 min at room temperature.
		<ol>
			<li>Prepare a 10x agonist peptide solution of Rhimi-K-1 (QFSPWGamide) (10 &#181;M) by resuspending 100 nmoles of lyophilized peptide in 10 mL of 1x HHBS containing 0.1% DMSO, and transfer the solution into a 150 mL auto-friendly reservoir.</li>
		</ol>
		</li>
		<li>Measure the background signal: For the entire HTS assay in the plate reader, under Protocols, choose end-point fluorescence mode. 
		NOTE: The fluorescent signal is read from the bottom of the plate at excitation/emission wavelengths of 495 nm/525 nm.
		<ol>
			<li>Insert the cell plate into the plate reader. In the instrument panel, select adjust gain, select a random well on the plate, assign it as 5%-10% of the maximum measurable fluorescence value, and select adjust (reading) height.</li>
			<li>Click start measurement to read the whole plate for the background fluorescence signal in relative fluorescence units (RFU).</li>
		</ol>
		</li>
		<li>&#34;Dual-addition&#34; assay
		<ol>
			<li>Using the liquid handling system with 384/12.5 &#181;L tips, to mix the drug solution in each well of the drug plate, pipette 3x up and down 10 &#181;L of the drug solution (in DPBS containing 10% DMSO) (pipetting speed: 5.2 &#181;L/s), and &#34;aspirate&#34; 1.5 &#181;L from each well of the drug plate (aspiration speed: 1.0 &#181;L/s).</li>
			<li>&#34;Dispense&#34;&#160;0.5 &#181;L of the compounds into the cell assay plate (pipetting speed: 1 &#181;L/s) to reach a final concentration of 2 &#181;M in 0.2% DMSO.</li>
			<li>Place the assay plate immediately into the plate reader after adding the screening compounds. Read the same plate in both the forward and reverse reading directions. To obtain these readouts, define a program to read from &#34;well 1-384&#34; and immediately from &#34;384-1&#34;. 
			NOTE: The readout in both directions takes 2 min in total. This readout design is to compensate for the decrease in signal strength that occurs during plate reading in each direction. See step 3.1 for the readout analyses.</li>
			<li>Dispose of the remaining 1 &#181;L of drug solution in the tips by immersing the tips into a 150 mL waste reservoir containing ~50 mL of DPBS (dispensing speed: 1.0 &#181;L/s).</li>
			<li>Incubate the screening compounds with the cells for a total of 5 min (includes the 2 min of plate reading) at room temperature in the biosafety cabinet with the lights off. Add 3 &#181;L of the agonist peptide, Rhimi-K-1 (QFSPWGamide), from the reservoir (aspiration speed: 3.1 &#181;L/s) into the assay plate using the liquid handling system (dispensing speed: 3.1 &#181;L/s) with 384/12.5 &#181;L tips.</li>
			<li>Place the plate into the plate reader immediately after the addition of the agonist peptide. Read the plate in both the forward and reverse directions using the same program as in step 2.3.12.3</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Data analysis
<ol>
	<li>Using the analysis software associated with the plate reader installed on a computer, calculate the cellular fluorescence responses (in RFU) for both readings after the first compound addition (First read; RFUago&#160;[Supplemental Table S2]) and after the agonist addition steps (Second read = RFUant [Supplemental Table S2]). Each read is obtained by averaging the two values obtained (not shown) by the forward and reverse plate readings from step 2.3.12.3 and step 2.3.12.6, respectively. 
	NOTE: RFUago, RFUant&#160;refer to the relative fluorescence units of the potential agonist hits (read in step 2.3.12.3) and the potential antagonist hits (read in step 2.3.12.6), respectively.</li>
	<li>Export all three sets of data, RFUbg, RFUago, and RFUant, from the analysis software into three separate spreadsheets. Each spreadsheet will have only two columns: well position and raw RFU (files not shown here). 
	NOTE: RFUbg refers to the RFU of the background read in step 2.3.11.2.</li>
	<li>Format the data from the three spreadsheets, and organize the above data in one csv file (see the example in Supplemental Table S2). Subtract the background signal read from the RFUago and RFUant, respectively (G and H columns in Supplemental Table S2).</li>
	<li>Import the csv file into a commercially available &#34;online HTS data platform&#34; (see the Table of Materials) for downstream data analysis (Table 1) and storage.</li>
	<li>Manually calculate the Z&#39;-factor for the quality control of each plate assay using equation (1): 
	equation (1): 
	<img alt="Equation 1" src="/files/ftp_upload/64505/64505eq01.jpg" style="margin: auto;" />&#160; &#160; (1) 
	NOTE: &#181;c- and&#160;&#181;c+ represent the average RFUs of readings of the same wells, which serve as negative controls in the first addition after adding the solvent (blank solvent, n = 64; numbers in blue in Supplemental Table S2) and serve as positive controls after the addition of the agonist (blank solvent + agonist, n = 64; numbers in magenta), respectively. Additionally,&#160;&#963;c- and &#963;c+ represent their corresponding standard deviations (SDs).</li>
	<li>Manually select the hit molecules from the heat maps on the &#34;online HTS data platform,&#34; and calculate the normalized percent activation (NPA) and inhibitory activity (Io) for the agonist hits and antagonist hits using equation (2) and equation (3): 
	<img alt="Equation 2" src="/files/ftp_upload/64505/64505eq02.jpg" style="margin: auto;" />&#160; &#160; (2) 
	<img alt="Equation 3" src="/files/ftp_upload/64505/64505eq03.jpg" style="margin: auto;" />&#160; &#160; (3)</li>
</ol>

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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=Central+peptidergic+ensembles+associated+with+organization+of+an+innate+behavior.">Central peptidergic ensembles associated with organization of an innate behavior.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (38), 14211-14216 (2006).</li><li>Al-Anzi, 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=The+leucokinin+pathway+and+its+neurons+regulate+meal+size+in+Drosophila.">The leucokinin pathway and its neurons regulate meal size in Drosophila.</a> Current Biology. 20 (11), 969-978 (2010).</li><li>Yurgel, M. E., 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+single+pair+of+leucokinin+neurons+are+modulated+by+feeding+state+and+regulate+sleep-metabolism+interactions.">A single pair of leucokinin neurons are modulated by feeding state and regulate sleep-metabolism interactions.</a> PLoS Biology. 17 (2), 2006409 (2019).</li><li>N&#228;ssel, D. R., Zandawala, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+neuropeptide+signaling+in+Drosophila,+from+genes+to+physiology+and+behavior.">Recent advances in neuropeptide signaling in Drosophila, from genes to physiology and behavior.</a> Progress in Neurobiology. 179, 101607 (2019).</li><li>Okusawa, S., Kohsaka, H., Nose, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+and+downstream+leucokinin+neurons+modulate+larval+turning+behavior+in+Drosophila.">Serotonin and downstream leucokinin neurons modulate larval turning behavior in Drosophila.</a> Journal of Neuroscience. 34 (7), 2544-2558 (2014).</li><li>Kersch, C. N., Pietrantonio, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosquito+Aedes+aegypti+(L.)+leucokinin+receptor+is+critical+for+in+vivo+fluid+excretion+post+blood+feeding.">Mosquito Aedes aegypti (L.) leucokinin receptor is critical for in vivo fluid excretion post blood feeding.</a> FEBS letters. 585 (22), 3507-3512 (2011).</li><li>Kwon, 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=Leucokinin+mimetic+elicits+aversive+behavior+in+mosquito+Aedes+aegypti+(L.)+and+inhibits+the+sugar+taste+neuron.">Leucokinin mimetic elicits aversive behavior in mosquito Aedes aegypti (L.) and inhibits the sugar taste neuron.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (25), 6880-6885 (2016).</li><li>Xiong, C., Baker, D., Pietrantonio, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+random+small+molecule+library+screen+identifies+novel+antagonists+of+the+kinin+receptor+from+the+cattle+fever+tick,+Rhipicephalus+microplus+(Acari:+Ixodidae).">A random small molecule library screen identifies novel antagonists of the kinin receptor from the cattle fever tick, Rhipicephalus microplus (Acari: Ixodidae).</a> Pest Management Science. 77 (5), 2238-2251 (2021).</li><li>Torfs, 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=The+kinin+peptide+family+in+invertebrates.">The kinin peptide family in invertebrates.</a> Annals of the New York Academy of Sciences. 897 (1), 361-373 (1999).</li><li>Ma, Q., Ye, L., Liu, H., Shi, Y., Zhou, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+Ca2++mobilization+assays+in+GPCR+drug+discovery.">An overview of Ca2+ mobilization assays in GPCR drug discovery.</a> Expert Opinion on Drug Discovery. 12 (5), 511-523 (2017).</li><li>Zhang, J. -. H., Chung, T. D., Oldenburg, K. 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+simple+statistical+parameter+for+use+in+evaluation+and+validation+of+high+throughput+screening+assays.">A simple statistical parameter for use in evaluation and validation of high throughput screening assays.</a> Journal of Biomolecular Screening. 4 (2), 67-73 (1999).</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 (3), 372-384 (2012).</li><li>Offermanns, S., Simon, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G&#945;15+and+G&#945;16+couple+a+wide+variety+of+receptors+to+phospholipase+C.">G&#945;15 and G&#945;16 couple a wide variety of receptors to phospholipase C.</a> Journal of Biological Chemistry. 270 (25), 15175-15180 (1995).</li><li>Murgia, M. V., 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=High-content+phenotypic+screening+identifies+novel+chemistries+that+disrupt+mosquito+activity+and+development.">High-content phenotypic screening identifies novel chemistries that disrupt mosquito activity and development.</a> Pesticide Biochemistry and Physiology. 182, 105037 (2022).</li><li>Lismont, E., 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+BRET-based+biosensors+be+used+to+characterize+G-protein+mediated+signaling+pathways+of+an+insect+GPCR,+the+Schistocerca+gregaria+CRF-related+diuretic+hormone+receptor.">Can BRET-based biosensors be used to characterize G-protein mediated signaling pathways of an insect GPCR, the Schistocerca gregaria CRF-related diuretic hormone receptor.</a> Insect Biochemistry and Molecular Biology. 122, 103392 (2020).</li></ol>

The authors have no conflict of interest to disclose.

The goal of HTS is to identify hit molecules through screening massive numbers of small molecules. Therefore, the results from this example only represent a small part of a conventional HTS experiment. Furthermore, the hit molecules identified need to be validated in downstream assays such as a dose-dependent assay on the same recombinant cell line and on a CHO-K1 cell line carrying only the empty vector, which can be performed simultaneously to save small molecules. Cytotoxicity assays will help demonstrate that a lack ...

G protein-coupled receptors (GPCRs), which are present from yeast to humans, represent the largest superfamily of receptors in many organisms1. They play critical roles in regulating nearly all biological processes in animals. There are 50-200 GPCRs in the genome of arthropods, meaning they represent the largest membrane receptor superfamily2. They are classified into six major classes, A-F, based on their sequence similarity and functions3. GPCRs transduce various extracellular signals, such as those of hormones, neuropeptides, biogenic amines, glutamate, proton, lipoglycoproteins, and photons4. GPCRs couple to heterotrimer G proteins (G&#945;, G&#946;, and G&#947;) to transmit downstream signals. GPCRs coupled to G&#945;s or G&#945;i/o proteins increase or decrease, respectively, the intracellular 3&#39;, 5&#39;-cyclic adenosine monophosphate (cAMP) levels by activating or inhibiting adenylyl cyclase. GPCRs coupled to G&#945;q/11 induce calcium release from the endoplasmic reticulum calcium stores by activating the phospholipase C (PLC)-inositol-1,4,5-triphosphate (IP3) pathway. GPCRs coupled to G&#945;12/13 activate RhoGTPase nucleotide exchange factors5,6. GPCRs are the target of more than 50% of human drugs and an acaricide, amitraz4. As GPCRs transduce such diverse signals, they are promising targets for developing novel pesticides that disrupt invertebrate-specific physiological functions.
The goal of HTS is to identify hit molecules that can modulate receptor functions. HTS involves assay development, miniaturization, and automation7. Arthropod neuropeptide GPCRs are involved in most physiological functions, such as development, molting and ecdysis, excretion, energy mobilization, and reproduction4. Most of the neuropeptide GPCRs of arthropods and metazoans signal through the calcium signaling cascade2,6,8,9,10, such as in the myoinhibitory peptide and SIFamide receptors of the blacklegged tick Ixodes scapularis; their ligands are antagonistic in hindgut motility assays, with SIF eliciting contraction and MIP inhibiting it11,12. An NPY-like receptor of the yellow fever mosquito, Aedes aegypti, regulates female host seeking13. Compared to other alternative calcium mobilization assays such as the aequorin calcium bioluminescence assay14, the calcium fluorescence assay is easy to perform, does not require the transfection of other recombinant calcium detecting proteins, and is cost-effective. The calcium fluorescence assay produces a prolonged signal compared to the fast kinetic signal obtained in the aequorin calcium bioluminescence assay14,15.
In the example here, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, was recombinantly expressed in the CHO-K1 cell line and used for the calcium fluorescence assay. There is only one kinin receptor gene found in R. microplus; the receptor signals through a Gq protein-dependent signaling pathway and triggers the efflux of Ca2+ from calcium stores into the intracellular space16. This process can be detected and quantified by a fluorophore, which elicits a fluorescence signal when binding calcium ions (Figure 1).
The kinin receptor is an invertebrate-specific GPCR, which belongs to the Class A Rhodopsin-like receptors. Kinin is an ancient signaling neuropeptide that is present in Mollusca, Crustacea, Insecta, and Acari4,17,18. Coleopterans (beetles) lack the kinin signaling system; in the mosquito Aedes aegypti, there is only one kinin receptor that binds three aedeskinins, while Drosophila melanogaster has one kinin receptor with drosokinin as a unique ligand19,20,21. There are no homologous kinins or kinin receptors in vertebrates. Although the exact function of kinin is unknown in ticks, the kinin receptor RNAi-silenced females of R. microplus show significantly reduced reproductive fitness22. Kinins are pleotropic peptides in insects. In Drosophila melanogaster, they are involved in both the central and peripheral nervous regulatory systems23, pre-ecdysis24, feeding25, metabolism26, and sleep activity patterns26,27, as well as larval locomotion28. Kinins regulate hindgut contraction, diuresis, and feeding in the mosquito A. aegypti29,30,31. The kinin peptides have a conserved C-terminal pentapeptide Phe-X1-X2-Trp-Gly-NH2, which is the minimum required sequence for biological activity32. The arthropod specificity, the small size of the endogenous ligand, which makes them amenable to small-molecule interference, and the pleiotropic functions in insects make the kinin receptor a promising target for pest control4.
The &#34;dual-addition&#34; assay (Figure 2) allows the identification of agonists or antagonists in the same HTS assay15. It is adapted from a &#34;dual-addition&#34; assay that is commonly used in the pharmaceutical industry for drug discovery33. In brief, the first addition of drugs into the cell plate allows the identification of potential agonists in the chemical library when a higher fluorescence signal is detected compared to the application of the solvent control. After 5 min of incubation with these small molecules, a known agonist (kinin peptide) is applied to all the wells. Those wells that randomly received an antagonist from the drug plate display a lower fluorescence signal upon agonist addition compared to the control wells that received the solvent in the first addition. This assay then allows the identification of potential agonists and antagonists with the same cells. In a standard HTS project, these hit molecules would be further validated through dose-response assays and by additional biological activity assays, which are not shown here.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64505/64505fig01.jpg" /> 
Figure 1: Illustration of the calcium fluorescence assay mechanism. The Gq protein triggers the intracellular calcium signaling pathway. The kinin receptor (G protein-coupled receptor) was recombinantly expressed in CHO-K1 cells. When the agonist ligand binds to the receptor, the Gq protein associated with the kinin receptor activates PLC, which catalyzes the conversion of a PIP2 molecule into IP3 and DAG. IP3 then binds to the IP3R on the surface of the endoplasmic reticulum, leading to the release of Ca2+ into the cytoplasm, where Ca2+ ions bind to the fluorophores and elicit a fluorescence signal. The fluorescence signal can be obtained by excitation at 490 nm and detected at 514 nm. Abbreviations: GPCR = G protein-coupled receptor; PLC = phospholipase C; PIP2 = phosphatidylinositol 4,5-bisphosphate; IP3 = inositol trisphosphate; DAG = diacylglycerol; IP3R = IP3 receptor. Created with BioRender.com.&#160;<a href="https://www.jove.com/files/ftp_upload/64505/64505fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64505/64505fig02.jpg" /> 
Figure 2: The workflow for the high-throughput screening of small molecules on a G protein-coupled receptor expressed in CHO-K1 cells. (A)&#160;Recombinant CHO-K1 cells stably expressing the kinin receptor were added to the 384-well plate (10,000 cells/well) using a liquid handling system (25 &#181;L/well) and incubated in a humidified CO2 incubator for 12-16 h. (B)&#160;The assay buffer containing the fluorescent dye (25 &#181;L/well) was added into the cell plate using a liquid handling system. The plate was incubated for 30 min at 37 &#176;C for 30 min and equilibrated at RT for another 30 min. (C) The background fluorescence signal of the cells in each well was measured with a plate reader. (D)&#160;Drug solutions from a 384-well library plate and blank solvent (all at 0.5 &#181;L/well) were added into the cellular assay plate using a liquid handling system. (E) Cellular calcium fluorescence responses were measured with the plate reader immediately after the addition of the drug solutions; compound(s) eliciting higher than average fluorescence signals were picked out as agonist hit(s). Antagonist hits that block the GPCR (icon below) were revealed after the addition of the peptide agonist during step G. (F)&#160;In the same assay plate, after 5 min of incubation of the cells with screening compounds, an endogenous agonist peptide Rhimi-K-1 (QFSPWGamide) of the tick kinin receptor was added to each well (1 &#181;M). (G) Cellular fluorescence responses after the agonist peptide addition were measured by the plate reader immediately. Compound(s) inhibiting the fluorescence signal were selected as antagonist hit(s). Abbreviations: GPCR = G protein-coupled receptor; RT = room temperature; RFU = relative fluorescence units. Created with BioRender.com.&#160;<a href="https://www.jove.com/files/ftp_upload/64505/64505fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>0.25% trypsin-EDTA</td><td>Gibco Invitrogen</td><td>15050-065</td><td>with phenol red</td></tr><tr><td>0.4% trypan blue</td><td>MilliporeSigma</td><td>T8154</td><td>liquid, sterile</td></tr><tr><td>1.5 mL microcentrifuge tubes</td><td>Thermo Fisher</td><td>AM12400</td><td>RNase-free Microfuge Tubes</td></tr><tr><td>5 mL serological pipette</td><td>Corning</td><td>29443-045</td><td>Corning Costar Stripette individually wrapped&amp;nbsp;</td></tr><tr><td>10 mL serological pipette</td><td>Corning</td><td>29443-047</td><td>Corning Costar Stripette individually wrapped&amp;nbsp;</td></tr><tr><td>15 mL conical tubes</td><td>Falcon</td><td>352196</td><td>sterile</td></tr><tr><td>20 &amp;micro;L filter tips</td><td>USA Scientifc Inc.</td><td>P1121</td><td>sterile, barrier</td></tr><tr><td>25 mL serological pipette</td><td>Corning</td><td>29443-049</td><td>Corning Costar Stripette individually wrapped&amp;nbsp;</td></tr><tr><td>50 mL conical tubes</td><td>Corning</td><td>430828</td><td>graduated, sterile</td></tr><tr><td>150 mL auto-friendly reservior</td><td>Integra Bioscience</td><td>6317</td><td>sterile, individually wrapped for cell seeding in day 1</td></tr><tr><td>150 mL auto-friendly reservior</td><td>Integra Bioscience</td><td>6318</td><td>sterile, stacked, for loading dye in day 2</td></tr><tr><td>384/ 12.5 &amp;micro;L low retention tips</td><td>Integra Bioscience</td><td>6405</td><td>long, sterile filter</td></tr><tr><td>384/ 12.5 &amp;micro;L tips</td><td>Integra Bioscience</td><td>6404</td><td>long, sterile filter</td></tr><tr><td>384-well plate</td><td>Greiner</td><td>781091</td><td>CELLSTAR, clear polystyrene, &amp;micro;Clear, Black/Flat</td></tr><tr><td>Aluminum plate seals</td><td>Axygen Scientific</td><td>PCR-AS-200</td><td>polyester-based</td></tr><tr><td>Aluminum foil wrap</td><td>Walmart</td><td></td><td></td></tr><tr><td>Biosafty cabinet II</td><td>NuAire</td><td>NU-540-300</td><td></td></tr><tr><td>Cell counter</td><td>Nexcelom</td><td>AutoT4</td><td></td></tr><tr><td>cell counting slides</td><td>Nexcelom</td><td>SD-100</td><td>20 &amp;micro;L chamber</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; humidified incubator</td><td>Thermo Fisher</td><td>Forma Series II</td><td></td></tr><tr><td>Desk Lamp</td><td>SunvaleeyTEK</td><td>RS1000B</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>MilliporeSigma</td><td>276855</td><td>anhydous, &gt;99.9%</td></tr><tr><td>Drug plate</td><td>Corning</td><td>3680</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline</td><td>Corning</td><td>21-031-CV</td><td>DPBS, 1x without calcium amd magnesium</td></tr><tr><td>Ethanol</td><td>Koptec</td><td>2000</td><td></td></tr><tr><td>F-12K Nutrient Mixture&amp;nbsp;</td><td>Corning</td><td>45000-354</td><td>(Kaighn&#039;s Mod.) with L-glutamine</td></tr><tr><td>Fetal bovine serum</td><td>Equitech-Bio</td><td>SFBU30</td><td></td></tr><tr><td>Fluorescent calcium assay kit</td><td>ENZO Lifescience</td><td>ENZ-51017</td><td>10x96 tests</td></tr><tr><td>G418 sulfate</td><td>Gibco Invitrogen</td><td>10131-027</td><td>Geneticin selective antibiotic 50 mg/mL</td></tr><tr><td>Hank&#039;s buffer</td><td>MilliporeSigma</td><td>55037C</td><td>HBSS modified, with calcium, with magnesium, without phenol read</td></tr><tr><td>HEPES buffer</td><td>Gibco Invitrogen</td><td>15630-080</td><td>1 Molar</td></tr><tr><td>HTS data storage plateform</td><td>CDD vault&amp;nbsp;</td><td></td><td>https://www.collaborativedrug.com/</td></tr><tr><td>Liquid handling system</td><td>Integra Bioscience</td><td>Viaflo</td><td>384/12.5 &amp;micro;L</td></tr><tr><td>Plate centrifuge</td><td>Thermo Fisher</td><td>Sorvall ST8</td><td></td></tr><tr><td>Plate reader</td><td>BMG technology</td><td>Clariostar</td><td></td></tr><tr><td>Poly-D-lysine</td><td>MilliporeSigma</td><td>P6407</td><td></td></tr><tr><td>Rhimi-K-1 agonist peptide</td><td>Genscript</td><td>custom order</td><td>QFSPWGamide</td></tr><tr><td>T-75 flask</td><td>Falcon</td><td>353136</td><td></td></tr></tbody></table>

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.

An in-house drug plate (SAC2-34-6170) composed of 320 random small molecules was used for demonstrating this HTS assay as an example. The HTS had excellent assay quality with a Z&#39; factor of 0.7 (Table 1). This&#160;Z&#39; factor reflects the assay quality independent of the tested compounds34. A Z&#8242;-factor of 0.5 or greater indicates a good assay signal dynamic range between the RFUs of the positive controls and the negative controls. Assays with a Z...

Watch this Scientific Journal Video about A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors at JoVE.com

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

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-dual-addition-calcium-fluorescence-assay-for-high-throughput

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

2.1K Views.  Texas A&M University. De High-Throughput screening calciumfluorescentie dubbele additietest maakt identificatie mogelijk van nieuwe liganden met kleine moleculen van een G-eiwitgekoppelde receptor die door de intracellulaire calciumcascade signaleert. Het belangrijkste voordeel van de dual addition assay is de detectie van agonist en antagonist HTS in een enkele assay met dezelfde cellen mits het fluorescentiesignaal twee minuten duurt. Deze methode kan worden toegepast op elke GPCR die signaleert via de calciumca...

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

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates. It is ideal for medium- to high-throughput screens in academic settings. It has an integrated fluid transfer module equipped with a multi-channel pipetter and the machine reads one column at a time to monitor fluorescence changes of a variety of fluorescent reagents. For example, FlexStation 3 has been used to study the function of Ca2+-permeable ion channels and G-protein coupled receptors by measuring the changes of intracellular free Ca2+ levels. Transient receptor potential (TRP) channels are a large family of nonselective cation channels that play important roles in many physiological and pathophysiological functions. Most of the TRP channels are calcium permeable and induce calcium influx upon activation. In this video, we demonstrate the application of FlexStation 3 to study the pharmacological profile of the TRPA1 channel, a molecular sensor for numerous noxious stimuli. HEK293 cells transiently or stably expressing human TRPA1 channels, grown in 96-well plates, are loaded with a Ca2+-sensitive fluorescent dye, Fluo-4, and real-time fluorescence changes in these cells are measured before and during the application of a TRPA1 agonist using the FLEX mode of the FlexStation 3. The effect of a putative TRPA1 antagonist was also examined. Data are transferred from the SoftMax Pro software to construct concentration-response relationships of TRPA1 activators and inhibitors.

This video provides a detailed protocol for studying the pharmacological profile of human TRPA1 channels using FlexStation 3. The protocol covers details of cell preparation, dye loading and operation of the microplate reader, FlexStation 3.

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates.  It is ...

Bioengineering

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

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 ...

Studying Protease-Activated Receptor Ligands in Endothelial Cells

Characterizing Modulators of Protease-Activated Receptors with a Calcium Mobilization Assay Using a Plate Reader

Changes in calcium concentration in cells are rapidly monitored in a high-throughput fashion with the use of intracellular, fluorescent, calcium-binding dyes and imaging instruments that can measure fluorescent emissions from up to 1,536 wells simultaneously. However, these instruments are much more expensive and can be challenging to maintain relative to widely available plate readers that scan wells individually. Described here is an optimized plate reader assay for use with an endothelial cell line (EA.hy926) to measure the protease-activated receptor (PAR)-driven activation of G&#945;q signaling and subsequent calcium mobilization using the calcium-binding dye Fluo-4. This assay has been used to characterize a range of PAR ligands, including the allosteric PAR1-targeting anti-inflammatory "parmodulin" ligands identified in the Dockendorff lab. This protocol obviates the need for an automated liquid handler and permits the medium-throughput screening of PAR ligands in 96-well plates and should be applicable to the study of other receptors that initiate calcium mobilization.

Author Spotlight: Developing Parmodulins to Target Protease-Activated Receptors for Inflammation Control

An improved protocol for a calcium mobilization assay with endothelial cells, used to identify ligands of protease-activated receptors (PARs), has been developed. The new protocol reduces total assay time by 90-120 min and yields reproducible concentration-response curves.

Changes in calcium concentration in cells are rapidly monitored in a high-throughput fashion with the use of intracellular, fluorescent, calcium-binding ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

Biology

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR malfunction, expressed as either over- or under-activity caused by mutations, altered expression, trafficking, or localization, can contribute to numerous diseases, especially in the central nervous system. Therefore, understanding the receptor&#39;s biology as well as facilitating the discovery of compounds and small molecules is crucial in ongoing efforts to combat neurological diseases. Current approaches to studying the receptor have limitations including low throughput, high cost, and the inability to study its functional abilities due to the necessary presence of channel blockers to prevent NMDAR-mediated excitotoxicity. Additionally, the existing assay systems are sensitive to stimulation by glutamate only and lack sensitivity to stimulation by glycine, the other co-ligand of the NMDAR. Here, we present the first plate-based assay with high-throughput power to study an NMDA receptor with sensitivity to both co-ligands, glutamate and D-serine/glycine. This approach allows the study of different NMDAR subunit compositions and allows functional studies of the receptor in glycine- and/or glutamate-sensitive modes. Additionally, the method does not require the presence of inhibitors during measurements. The effects of positive and negative allosteric modulators can be detected with this assay and the known pharmacology of NMDAR has been replicated in our system. This technique overcomes the limitations of existing methods and is cost-effective. We believe that this novel technique will accelerate the discovery of therapies for NMDAR-mediated pathologies.

The goal of this protocol is to facilitate the study of NMDA-receptors (NMDAR) at a larger scale and allow the examination of modulatory effects of small molecules and their therapeutic applications.

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR ...

Neuroscience

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

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Screening Peptides that Activate MRGPRX2 using Engineered HEK Cells

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new therapeutics. Mas related G-protein receptor-X2 (MRGPRX2) is an important receptor that regulates mast cell activation and, thus, directs the general immune response. Numerous ligands for MRGPRX2 have been identified and include endogenous peptides like PAMPs, defensins, LL-37 and other protein fragments (i.e., degraded albumin). Further identification of MRGPRX2 specific ligands requires the screening of a large number of peptides (i.e., peptide library); however, mast cells are difficult and expensive to maintain in vitro and, therefore, not economical to use for screening large numbers of molecules. The present paper demonstrates a method to design, develop, and screen a library of small peptide molecules using MRGPRX2 expressing HEK cells. This cell line is relatively easy and inexpensive to maintain and can be used for in vitro high-throughput analysis. A calcium sensitive Fura-2 fluorescent dye to mark intracellular calcium flux upon activation was used to monitor the activation. The ratio of fluorescence intensity of Fura-2 at 510 nm against excitation wavelengths of 340 and 380 nm was used to calculate calcium concentration. The peptide library used to verify this system was based on the endogenous proadrenomedullin N-terminal 12 (PAMP-12) secretagogue, which is known to bind MRGPRX2 with high specificity and affinity. Subsequent peptides were generated through amino acid truncation and alanine scanning techniques applied to PAMP-12. The method described here is simple and inexpensive yet robust for screening a large library of compounds to identify binding domains and other important parameters that play an important role in receptor activation.

Techniques for generating a library of short peptides that can activate mast cells via the MRGPRX2 receptor are described. Associated techniques are easy, inexpensive, and can be extended to other cell receptors.

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new ...

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

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