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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</td>
		</tr>
		<tr>
			<td>10 mL serological pipette</td>
			<td>Corning</td>
			<td>29443-047</td>
			<td>Corning Costar Stripette individually wrapped&#160;</td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Falcon</td>
			<td>352196</td>
			<td>sterile</td>
		</tr>
		<tr>
			<td>20 &#181;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&#160;</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 &#181;L low retention tips</td>
			<td>Integra Bioscience</td>
			<td>6405</td>
			<td>long, sterile filter</td>
		</tr>
		<tr>
			<td>384/ 12.5 &#181;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, &#181;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 &#181;L chamber</td>
		</tr>
		<tr>
			<td>CO2 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, &#62;99.9%</td>
		</tr>
		<tr>
			<td>Drug plate</td>
			<td>Corning</td>
			<td>3680</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;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&#160;</td>
			<td>Corning</td>
			<td>45000-354</td>
			<td>(Kaighn&#39;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&#39;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&#160;</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 &#181;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...

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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 ...

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Research

JoVE Journal

Biochemistry

2.0K Views.  Texas A&M University. 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.

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

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a major pharmacological target for multiple indications, including analgesia, blood pressure regulation, and the treatment of psychiatric disease. Upon ligand binding, GPCRs catalyze the activation of intracellular G-proteins by stimulating the incorporation of guanosine triphosphate (GTP). Activated G-proteins then stimulate signaling pathways that elicit cellular responses. GPCR signaling can be monitored by measuring the incorporation of a radiolabeled and non-hydrolyzable form of GTP, [35S]guanosine-5&#39;-O-(3-thio)triphosphate ([35S]GTP&#947;S), into G-proteins. Unlike other methods that assess more downstream signaling processes, [35S]GTP&#947;S binding measures a proximal event in GPCR signaling and, importantly, can distinguish agonists, antagonists, and inverse agonists. The present protocol outlines a sensitive and specific method for studying GPCR signaling using crude membrane preparations of an archetypal GPCR, the &#181;-opioid receptor (MOR1). Although alternative approaches to fractionate cells and tissues exist, many are cost-prohibitive, tedious, and/or require non-standard laboratory equipment. The present method provides a simple procedure that enriches functional crude membranes. After isolating MOR1, various pharmacological properties of its agonist, [D-Ala, N-MePhe, Gly-ol]-enkephalin (DAMGO), and antagonist, naloxone, were determined.

Guanosine triphosphate (GTP) binding is one of the earliest events in G-Protein-Coupled Receptor (GPCR) activation. This protocol describes how to pharmacologically characterize specific GPCR-ligand interactions by monitoring the binding of the radio-labeled GTP analog, [35S]guanosine-5&#39;-O-(3-thio)triphosphate ([35S]GTP&#947;S), in response to a ligand of interest.

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, consequently, cardiovascular disease. Although PCSK9 proteolysis is required for its full hypercholesterolemic effect, the evaluation of its proteolytic function is challenging: PCSK9 is only known to cleave itself, undergoes only a single turnover, and after proteolysis, retains its substrate in its active site as an auto-inhibitor. The methods presented here describe an assay which overcomes these challenges. The assay focuses on intermolecular proteolysis in a cell-based context and links successful cleavage to the secreted luciferase activity, which can be easily read out in the conditioned medium. Via sequential steps of mutagenesis, transient transfection, and a luciferase readout, the assay can probe PCSK9 proteolysis under conditions of either genetic or molecular perturbation in a high-throughput manner. This system is well suited for both the biochemical evaluation of clinically discovered missense single-nucleotide polymorphisms (SNPs), as well as for the screening of small-molecule inhibitors of PCSK9 proteolysis.

This protocol presents a method to evaluate the proteolytic activity of an intrinsically low-activity, single turnover protease in a cellular context. Specifically, this method is applied to evaluate the proteolytic activity of PCSK9, a key driver of lipid metabolism whose proteolytic activity is required for its ultimate hypercholesterolemic function.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced contrast due to optical sectioning. The conventional approach is to use high numerical aperture microscope TIRF objectives for both excitation and collection, severely limiting the field of view and throughput. We present a novel approach to generating TIRF excitation for imaging with optical waveguides, called chip-based nanoscopy. The aim of this protocol is to demonstrate how chip-based imaging is performed in an already built setup. The main advantage of chip-based nanoscopy is that the excitation and collection pathways are decoupled. Imaging can then be done with a low magnification lens, resulting in large field of view TIRF images, at the price of a small reduction in resolution. Liver sinusoidal endothelial cells (LSECs) were imaged using direct stochastic optical reconstruction microscopy (dSTORM), showing a resolution comparable to traditional super-resolution microscopes. In addition, we demonstrate the high-throughput capabilities by imaging a 500 &#181;m x 500 &#181;m region with a low magnification lens, providing a resolution of 76 nm. Through its compact character, chip-based imaging can be retrofitted into most common microscopes and can be combined with other on-chip optical techniques, such as on-chip sensing, spectroscopy, optical trapping, etc. The technique is thus ideally suited for high throughput 2D super-resolution imaging, but also offers great opportunities for multi-modal analysis.

Chip-based super-resolution optical microscopy is a novel approach to fluorescence microscopy and offers advantages in cost effectiveness and throughput. Here, the protocols for chip preparation and imaging are shown for TIRF microscopy and localization-based super-resolution microscopy.

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced ...

Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral membrane enzymes, resulting in triacylated proteins. The first step in the lipoprotein modification pathway involves the transfer of a diacylglyceryl group from phosphatidylglycerol onto the prolipoprotein, resulting in diacylglyceryl prolipoprotein. In the second step, the signal peptide of prolipoprotein is cleaved, forming an apolipoprotein, which in turn is modified by a third fatty acid derived from a phospholipid. This last step is catalyzed by apolipoprotein N-acyltransferase (Lnt). The lipoprotein modification pathway is essential in most &#947;-proteobacteria, making it a potential target for the development of novel antibacterial agents. Described here is a sensitive assay for Lnt that is compatible with high-throughput screening of small inhibitory molecules. The enzyme and substrates are membrane-embedded molecules; therefore, the development of an in vitro test is not straightforward. This includes the purification of the active enzyme in the presence of detergent, the availability of alkyne-phospholipids and diacylglyceryl peptide substrates, and the reaction conditions in mixed micelles. Furthermore, in order to use the activity test in a high-throughput screening (HTS) setup, direct readout of the reaction product is preferred over coupled enzymatic reactions. In this fluorometric enzyme assay, the alkyne-triacylated peptide product is rendered fluorescent through a click-chemistry reaction and detected in a multiwell plate format. This method is applicable to other acyltransferases that use fatty acid-containing substrates, including phospholipids and acyl-CoA.

Presented here is a sensitive fluorescence assay to monitor apolipoprotein N-acyltransferase activity using diacylglyceryl peptide and alkyne-phospholipids as substrates with click-chemistry.

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase with critical roles in human health and disease. Here we present a versatile enzyme-coupled SAMHD1 activity assay, deployed in a 384-well microplate format, that allows for the evaluation of small molecules and nucleotide analogues as SAMHD1 substrates, activators, and inhibitors.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...

High-Throughput Screening for Protein Crystallization

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into an ordered lattice amenable to diffraction remains challenging. The crystallization of biomolecules is largely experimentally defined, and this process can be labor-intensive and prohibitive to researchers at resource-limited institutions. At the National High-Throughput Crystallization (HTX) Center, highly reproducible methods have been implemented to facilitate crystal growth, including an automated high-throughput 1,536-well microbatch-under-oil plate setup designed to sample a wide breadth of crystallization parameters. Plates are monitored using state-of-the-art imaging modalities over the course of 6 weeks to provide insight into crystal growth, as well as to accurately distinguish valuable crystal hits. Furthermore, the implementation of a trained artificial intelligence scoring algorithm for identifying crystal hits, coupled with an open-source, user-friendly interface for viewing experimental images, streamlines the process of analyzing crystal growth images. Here, the key procedures and instrumentation are described for the preparation of the cocktails and crystallization plates, imaging the plates, and identifying hits in a way that ensures reproducibility and increases the likelihood of successful crystallization.

Author Spotlight: High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography  

This protocol details high-throughput crystallization screening, ranging from the 1,536 microassay plate preparation to the end of a 6 week experimental time window. Details are included about the sample setup, the imaging obtained, and how users can perform analyses using an artificial intelligence-enabled graphical user interface to quickly and efficiently identify macromolecular crystallization conditions.

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into ...