This work was supported by the Canadian Institutes of Health Research (CIHR grant #MOP142219).

1. Primary screening: cell culture and plate seeding
<ol>
	<li>To prepare poly-L-lysine (PLL)-coated plates, dispense 20 &#956;L/well of a 25 &#956;g/mL stock solution of PLL in white or black 384-well optical bottom plates using an electronic multichannel pipette or a reagent dispenser. Incubate the plates at room temperature for 0.5&#8211;2 h. 
	NOTE: If using the black 384-well plates, expect the background signal to be lower compared to the white plates. Black plates are recommended to reduce bleed-through of luminescence between adjacent wells.</li>
	<li>To preserve the coated plates and wash off the excess PLL, remove the PLL by flicking it over the sink, tap dry over a paper towel, and add 40 &#956;L/well of diluted 1x solution of antibiotic-antimycotic using an electronic multichannel pipette or a reagent dispenser. Store PLL-coated plates at 4 &#176;C until ready for plate seeding.</li>
	<li>Maintain HTLA cells (kindly provided by Dr. Richard Axel)&#8211;a human embryonic kidney cell line (HEK293T) stably expressing &#946;-arrestin2-TEV and tTA-driven luciferase&#8211;in complete Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 5% of Fetal Bovine Serum, 5% of Bovine Calf Serum, 2.5 &#956;g/mL of puromycin, 50 &#956;g/mL of hygromycin, 100 U/mL penicillin, and 100 &#956;g/mL streptomycin at 37 &#176;C in a humidified incubator containing 5% CO2.</li>
	<li>Culture HTLA cells in 150 mm dishes and pass cells twice a week at a dilution factor of 1:10, with optimal cell passage number of 5&#8211;25. Ensure that a sufficient number of 150 mm dishes are confluent the day of 384-well plate seeding, depending on the scale of the primary screen. 
	NOTE: Usage of HTLA cells greater than passage 25 may result in reduced viability, yielding suboptimal results.</li>
	<li>To seed HTLA cells for the primary screen, gently rinse the confluent 150 mm dish(es) with 1x phosphate-buffered saline (PBS), pH 7.4. Detach cells with approximately 6 mL of 0.05% Trypsin/0.53 mM EDTA, and transfer to a centrifuge tube containing at least equal amount of complete Dulbecco&#39;s modified Eagle medium (DMEM) to neutralize the trypsin.</li>
	<li>Spin down HTLA cells at 500 x g for 3 min and resuspend the cell pellet at a density of 0.22 x 106 cells/mL in complete DMEM, omitting the addition of 2.5 &#956;g/mL of puromycin and 50 &#956;g/mL of hygromycin as they can decrease transfection efficacy.</li>
	<li>Incubate the necessary 384-well PLL-coated plates at 37 &#176;C to warm them before seeding cells. Remove the storage solution of 1x antibiotic-antimycotic from the 384-well PLL-coated plate(s) by flicking the plate over the sink and taping it over a paper towel to dry.</li>
	<li>Seed cells into the 384-well PLL-coated plates at a final density of 10,000 cells/well by dispensing 45 &#956;L of the 0.22 x 106 cells/mL HTLA suspension using an electronic multichannel pipet. Incubate plates at 37 &#176;C overnight. If a same-day transfection is preferred, seed cells at a density of 16,000 cells/well and perform the transfection 4 h later. 
	NOTE: For high transfection efficiency, 50&#8211;70% cell confluency is optimal.</li>
</ol>
2. Primary screening: DNA plate preparation and transfections
<ol>
	<li>To prepare the 384-well DNA source plate for transfection as shown in Figure 2, distribute the plasmid cDNAs encoding the GPCR-Tango constructs of interest in a 96-well plate, with a different GPCR/well. The plasmid DNA should be suspended in 0.1x Tris-EDTA (TE) buffer at a concentration of 50 ng/&#956;L. 
	NOTE: The 96-well DNA plates can be sealed and stored at -20 &#176;C, and re-used for multiple screening experiments. All cDNA encoding GPCR-Tango constructs are available commercially (see the Table of Materials) and are cloned in the pcDNA3.1 neomycin plasmid. The PRESTO-Tango GPCR Kit consists of four 96-well plates, which include 80 GPCRs each, a couple of wells with an empty vector as negative controls, and positive control wells that hold the dopamine receptor D2 (DRD2), and wells that carry a plasmid encoding a fluorescent protein (YFP) to track transfection efficiency.</li>
	<li>Using a multichannel pipette, manually transfer DNA solution from the 96-well to the a 384-well DNA source plate, adding 10 &#956;L per 384-well. To ensure that each condition of the experiment is assayed in quadruplicate, half of the 96-well DNA plate (rows A-D or E-H) will cover a full 384-well plate by distributing each GPCR in two quadrants (first quadrant = - compound, second quadrant = + compound), such that the same GPCR will be transfected in 8 wells of the 384-well plate (see Figure 2 as a guide).</li>
	<li>Assemble the following transfection reagents needed for calcium phosphate precipitation method, as described by Jordan et al.20: 0.1x TE buffer (1 mM Tris-HCl and 0.1 mM EDTA); 2.5 M CaCl2 solution; 2x Hepes buffer, pH 7.05 (50 mM HEPES, 280 mM NaCl, 1.5 mM Na2HPO4). Sterilize all the solutions by filtration and store at 4 &#176;C. The day of transfection, allow the reagents to reach room temperature before use.</li>
	<li>Dilute the 2.5 M CaCl2 stock solution in 0.1x TE (1:8 dilution) to a final concentration of 0.313 M CaCl2 and vortex. Transfer 40 &#956;L of 0.313 M CaCl2 to the 384-well DNA source plate and mix by pipetting up and down with a hand-held multichannel pipette or an automated benchtop 384-channel pipettor.</li>
	<li>Add 50 &#956;L of 2x Hepes buffer to the 384-well DNA source plate, mix again by pipetting up and down and let stand for 1 min; each 384-well will have an adequate amount of DNA/transfection mixture for the transfection of nine 384-well plates, depending on the number of compounds that need to be tested. Transfer 10 &#956;L of the DNA/transfection mixture from the 384-well DNA source plate to the seeded HTLA cells and incubate the plates overnight at 37 &#176;C.</li>
</ol>
3. Primary screening: Cell stimulation
<ol>
	<li>Twenty-four hours later, decant the transfected cell media by gently flicking the 384-well plate over the sink and taping it over a paper towel, or with an aspirator head. Slowly add 40 &#956;L of starving media (DMEM supplemented with 1% dialyzed fetal bovine serum (dFBS) and 1x antibiotic/antimycotic), being careful to avoid touching the cells directly.</li>
	<li>Pipet 20 &#956;L of the compound of interest at a 3x concentration (final concentration of the drug in the cell plate will be 1x) into the alternating rows with (+) stimulation, and 20 &#956;L of vehicle buffer for the alternating rows without (-) compound. Return the cell plate at 37 &#176;C in 5% CO2 and incubate for at least 16 h.</li>
</ol>
4. Primary screening: Luminescence reading
<ol>
	<li>Prepare the Glo reagent, modified from Baker and Boyce21: 108 mM Tris&#8211;HCl; 42 mM Tris-Base, 75 mM NaCl, 3 mM MgCl2, 5 mM Dithiothrei-tol (DTT), 0.2 mM Coenzyme A, 0.14 mg/ml D-Luciferin, 1.1 mM ATP, 0.25% v/v Triton X-100, 2 mM Sodium hydrosulfite. 
	NOTE: Stock solutions of the reagents can be made in advance, except for D-Luciferin, which is always freshly added to the Glo reagent in its powdered form. If black plates were used, the amount of D-Luciferin can be increased up to 0.25 mg/mL.</li>
	<li>At 16&#8211;24 h following stimulation, decant the transfected cell media by gently flicking the 384-well plate over the sink and taping it over a paper towel. Add 20 &#956;L/well of Glo reagent and incubate the plate at room temperature for 5&#8211;20 min. Read the plates using a microplate luminescence counter, with an integration time of 1 s/well.</li>
</ol>
5. Primary screening: Data analysis
<ol>
	<li>Export the saved files from the luminescence counter as a spreadsheet; results will be recorded in relative luminescence units (RLU). Based on the layout of the 384-well plate, calculate the activation (fold change) of each receptor using the following formula: 
	<img alt="Equation 1" src="/files/ftp_upload/60823/60823eq1.jpg" /> 
	NOTE: Here Sample RLU refers to value of each of the four replicate wells of the stimulated (+ compound) quadrant, Mean background RLU is the mean of the negative controls on the plate, and Mean basal RLU refers to the mean of the untreated quadrant of that same receptor (- compound). In addition, calculate the standard deviation of the 4 data points to verify the quality of the results. It is recommended to perform a log2 transformation on the mean of the fold changes to rectify any heteroskedasticity; the log2 base is a practical choice to help identify positive hits. Empirically set the positive hit thresholds; it has to be noted that some receptors can have as low as 2-fold increase and up to 40-fold increase for others with full agonist.</li>
	<li>Based on the results, select the GPCRs that are potential positive hits for secondary screening.</li>
</ol>
6. Secondary screening: Cell seeding and transfections
<ol>
	<li>Subculture HTLA cells in 100 mm dishes at a total cell density of 5 x 106 cells in 11 mL of complete media (4.55 x 105/mL) and incubate at 37 &#176;C for 24 h. If a same-day transfection is preferred, seed cells at a density of 7.5 x 106 cells and perform the transfection 4 h later.</li>
	<li>Pre-warm the reagents needed for calcium phosphate precipitation at room temperature. Combine 450 &#956;L of 0.1x TE buffer with 50 &#956;L of 2.5 M CaCl2 and quickly vortex; these amounts are specific for one 100 mm dish, based on the volume of growth medium it holds.</li>
	<li>In a tube, add 500 &#956;L of the TE/CaCl2 solution to 10 &#956;g of GPCR cDNA and vortex. Add 500 &#181;L of 2x Hepes buffer solution in the tube, shake vigorously (do not vortex), and incubate for 1 min. 
	NOTE: 1 &#181;g of any plasmid encoding a fluorescent protein (e.g. YFP, mCherry, etc.) can be co-transfect with 9 &#181;g of GPCR cDNA for a total of 10 &#956;g. The fluorescent protein is used to track transfection efficiency, and this minimal amount will not interfere with the assay.</li>
	<li>Immediately following the short incubation, dispense the 1 mL solution dropwise onto the cells. Gently rock the plate back and forth to evenly distribute the precipitate, taking care not to swirl the plate, and incubate at 37 &#176;C for 24 h.</li>
	<li>The following day, observe the transfection efficiency by looking at the expression of the fluorescent protein under a fluorescent cell imager; transfections greater than 50% coverage are ideal.</li>
	<li>Incubate the necessary 384-well PLL-coated plate(s) in the incubator at 37 &#176;C to warm it before seeding cells. Remove the storage solution of 1x antibiotic-antimycotic from the 384-well PLL-coated plate(s) by flicking the plate over the sink and taping it over a paper towel to dry.</li>
	<li>Gently rinse the transfected cells with Versene solution (1X PBS, pH 7.4; 0.53 mM EDTA), and detach by adding 3 mL of 0.05% trypsin/0.53 mM EDTA to the dish. Transfer the contents to a centrifuge tube containing at least an equal amount of complete DMEM to neutralize the trypsin.</li>
	<li>Spin down the cells at 500 x g for 3 min and resuspend the cells at a density of 0.4 x 106 cells/mL in starving media. Seed cells into the 384-well PLL-coated plate(s) at a final density of 25,000 cells/well by dispensing 45 &#956;L of the cell suspension using an electronic multichannel pipet. Return the plates to the 37 &#176;C for a minimum of 4 h, allowing the cells to properly attach to the wells before proceeding to the stimulation.</li>
</ol>
7. Secondary screening: Drug plate preparation for 16-point (half log) dose-curve
<ol>
	<li>In a 96-well plate, add 270 &#956;L of 1X HBSS drug buffer (1x Hank&#39;s Balanced Salt Solution [HBSS], 20 mM HEPES pH 7.4, 1x antibiotic-antimycotic), excluding the last row (row H) of the plate, as shown in Figure 4. 
	NOTE: For peptides, colloidal molecules, and poorly water-soluble compounds, the addition of 0.1&#8211;1% BSA is suggested. To prevent drug oxidation, up to 0.01% ascorbic acid can also be added.</li>
	<li>From the drug stock, prepare a drug solution (referred to as the &#34;High&#34; concentration) by calculating a final 3x concentration (final concentration of the drug in the cell plate will be 1x). As an example, for a dose&#8211;response curve with 10 &#956;M as its highest concentration, prepare the &#34;High&#34; concentration at 30 &#956;M. Pipet 300 &#956;L of &#34;High&#34; concentration into wells in row H.</li>
	<li>In another tube, prepare the &#34;Low&#34; concentration, which represents the &#34;High&#34; concentration divided by 3.16 (half-log). Based on the previous example, the &#34;Low&#34; concentration would be 9.49 &#956;M. Pipet 300 &#956;L of &#34;Low&#34; concentration into wells in row H, adjacent to the &#34;High&#34; wells. 
	NOTE: The total number of 96-wells needed in row H will depend on the number of cells and stimulation conditions. Four wells (two &#34;High&#34; and two &#34;Low&#34;) will have ample drug solution to stimulate an entire 384 well plate.</li>
	<li>Perform a serial dilution by pipetting 30 &#956;L of drug solution from the &#34;High&#34; and &#34;Low&#34; wells of row H to the previous row (row G) and mix by manually pipetting up and down, or as recommended, using an electronic multichannel pipette with the &#34;Pipette and Mix&#34; function. Repeat this step up until the first and most diluted row (row A), while discarding tips between dilutions. 
	NOTE: If desired, the serial dilutions can be stopped before row A, representing an internal control with no drug, in other words, a &#34;true zero&#34;.</li>
	<li>Using Figure 4 as a reference, stimulate transfected cells by pipetting 20 &#956;L of the &#34;Low&#34; column dilutions from the 96-well plate to rows A&#8211;O of the previously seeded 384-well plate, as well as 20 &#956;L of the &#34;High&#34; column dilutions to wells B&#8211;P. Incubate the plate at 37 &#176;C for a minimum of 16 h.</li>
</ol>
8. Secondary screening: Luminescence reading and data analysis
<ol>
	<li>At 16&#8211;24 h following stimulation, decant the transfected cell media by gently flicking the 384-well plate over the sink and taping it over a paper towel. Add 20 &#956;L/well of Glo reagent and incubate the plate at room temperature for 5&#8211;20 min. Read the plates using a microplate luminescence counter, with an integration time of 1 s/well.</li>
	<li>Export the saved files from the luminescence counter as a spreadsheet; results will be recorded in relative luminescence units (RLU). Transfer the data of the 384-well plate to a statistics software to analyze the results using its built-in XY analysis for non-linear regression curve fit. Select the built-in 3-parameter dose-response stimulation function &#34;Log(agonist) vs. response (three parameters)&#34;, 
	<img alt="Equation 2" src="/files/ftp_upload/60823/60823eq2.jpg" /> 
	NOTE: Here Top and Bottom are plateaus in the units of the Y axis, respectively the maximal response and basal level, EC50 is the concentration of the agonist that that generates 50% response between Top and Bottom, and X refers to the log concentration of the agonist. This model assumes that the dose-response curve has a standard Hill slope of 1.</li>
</ol>

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The authours declare no competing interests.

The conformationally dynamic GPCRs are powerhouses of signal transduction. The physiochemical properties of the binding pockets of these heptahelical receptors, as well as their physiological relevance underscore the need for GPCR ligand screening tools. As presented above, the PRESTO-Tango assay is rapid, sensitive and user-friendly, lending itself to drug development. Not only does this assay measure agonist-induced activation, but it can also be used to quantify the activity of antagonists and allosteric modulators<su...

G-protein-coupled receptors (GPCRs) constitute the largest and most diverse family of transmembrane proteins, operating as communication interfaces between a cell and its environment1. The versatility of GPCRs is highlighted by their ability to detect a diverse array of ligands&#8211;from neurotransmitters to nucleotides, peptides to photons, and many more&#8211;as well as their ability to regulate numerous downstream signaling cascades involved in cellular growth, migration, differentiation, apoptosis, cell firing, etc.2,3. Considering their ubiquity and involvement in a multitude of physiological processes, this receptor family is of utmost therapeutic importance, showcased by the fact that more than a third of currently available prescribed medications target GPCRs4. However, these existing therapeutics only target a small subset of the superfamily (an estimated 10%), and the pharmacology of many GPCRs remains unelucidated. Moreover, more than 100 GPCRs exist as orphan receptors, as they have not been matched with an endogenous ligand5. Thus, GPCR ligand screening is critical in deorphanization and drug development, as it paves the path towards lead discovery and optimization, and possibly to the clinical trial phase.
Methods for GPCR ligand screening have traditionally fallen in one of two categories, G-protein dependent or G-protein independent functional assays6. GPCR signaling is regulated by heterotrimeric G-proteins (G&#945;&#946;&#947;), which are activated by the exchange of GTP for GDP bound on the G&#945; subunit7. Signals from the activated receptor are transduced by G-proteins via secondary messengers, such as cAMP, Calcium, DAG, and IP3, to mediate downstream signaling at downstream effectors8. The nature of the functional consequences of G-protein signaling has been exploited to create cell-based assays that reflect receptor activation. These methods, which measure proximal (direct) or distal (indirect) events in G-protein signaling, are most frequently used for GPCR ligand screening and have been principally employed in deorphanization studies6. Examples of assays that directly measure GPCR-mediated G-protein activation include the [35S]GTP&#947;S binding assay, which measures binding of a radiolabeled and non-hydrolyzable GTP analog to the G&#945; subunit, and F&#246;rster/bioluminescence resonance energy transfer (FRET/BRET, respectively) probes to monitor GPCR-G&#945; and G&#945;/G&#947; interactions, which have been gaining more traction over the years9,10. Assays that monitor distal events are the most commonly used tools for GPCR profiling; for example, cAMP and IP1/3 assays measure intracellular accumulation of G-protein dependent secondary messengers, whereas [Ca2+] flux and reporter assays involving specific response elements implicated in G-protein activation (CRE, NFAT-RE, SRE, SRF-RE) examine events further downstream the signaling cascade11. While most of the aforementioned assays can be performed at a high-throughput level, are fairly sensitive, and boast certain assay-specific advantages (e.g., discrimination between full/partial agonists, neutral antagonists and inverse agonists in the case of GTP&#947;S binding, or assay functionality on live cells such as [Ca2+] and IP1/3)6, there are unfortunately no existing G-protein dependent methods befitting the interrogation of the entire druggable GPCR-ome. This is largely due to the native coupling of multiple G-protein subfamilies to GPCRs, resulting in signaling at several cascades and the unknown G-protein coupling at orphan GPCRs. To mitigate this issue, assays have been developed to force promiscuous G-protein coupling through a single common signaling read-out, such as cAMP, and Ca2+, albeit most of them are low-throughput12.
An important aspect of the GPCR lifecycle is the termination of G-protein-dependent signaling, which occurs in large part through the recruitment of &#946;-arrestins which induces dissociation of the G-protein, and ultimately desensitizing the receptor, which is targeted for clathrin-coated internalization13. The most ubiquitously expressed isoforms of &#946;-arrestin are the non-visual &#946;-arrestin1 and &#946;-arrestin2, also denoted as arrestin-2 and arrestin-3, respectively14. Enter G-protein independent cell-based assays, which add a new dimension to GPCR ligand screening; receptor trafficking, label-free whole cell, and &#946;-arrestin recruitment assays are all notable examples. GPCR trafficking assays employ fluorophore-labeled ligands or co-internalized antibodies targeting the receptor15, whereas label-free whole cell assays use biosensors which translate cellular changes induced by ligand binding into quantifiable outputs, such as electrical or optical signals16. Notably, quintessential GPCR- &#946;-arrestin interactions fashion the &#946;-arrestin recruitment assay as an attractive tool in the repertoire of functional assays17. The Tango system, first developed by Barnea et al. only a decade ago, involves the introduction of three exogenous genetic elements: a protein fusion consisting of &#946;-arrestin2 with a tobacco etch virus protease (TEVp), a tetracycline transactivator (tTA) that is tethered to a GPCR via a tobacco etch virus protease cleavage site (TEVcs) and is preceded by a sequence from the C-terminus of the V2 vasopressin receptor (V2 tail) to promote arrestin recruitment, and a reporter luciferase gene whose transcription is triggered by the tTA transcription factor translocation to the nucleus, which is freed from the membrane anchoring following &#946;-arrestin2 recruitment (Figure 1)18. Quantitative readings of GPCR activation and &#946;-arrestin2 recruitment can be subsequently determined by reading for luminescence. A notable distinction is that while receptor trafficking and label-free whole cell methods are relatively low-throughput, the Tango has several advantages, including selective read-out that is specific to the target receptor and sensitivity due to signal integration, which make it a suitable candidate for ligand screening on a larger scale18.
In view of these strategic features, Kroeze et al. developed PRESTO-Tango (Parallel Receptor-ome Expression and Screening via Transcriptional Output-Tango), a high-throughput open-source platform that uses the Tango approach to profile the druggable GPCR-ome in a parallel and simultaneous manner19. Exploiting the &#34;promiscuous&#34; recruitment of &#946;-arrestin2 to nearly all GPCRs, PRESTO-Tango is the first-of-its-kind in terms of cell-based functional assays, enabling rapid &#34;first-round&#34; screening of small molecule compounds at almost all non-olfactory GPCRs, including orphans, independent of the G-protein subfamily coupling.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>384 Well Optical Bottom Plates, Polystyrene Polymer Base, Cell Culture Treated, BLACK, with lid, Sterile</td>
			<td>NUNC</td>
			<td>12-566</td>
			<td></td>
		</tr>
		<tr>
			<td>384 Well Optical Bottom Plates, Polystyrene Polymer Base, Cell Culture Treated, White, with lid, Sterile</td>
			<td>NUNC</td>
			<td>12-566-1</td>
			<td></td>
		</tr>
		<tr>
			<td>384 Well Round Bottom, Polypropylene, Non-Treated, Blue, non-sterile, without lid</td>
			<td>ThermoFisher</td>
			<td>12-565-390</td>
			<td></td>
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			<td>Antibiotic-Antimycotic</td>
			<td>Wisent</td>
			<td>450-115-EL</td>
			<td></td>
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			<td>D-Luciferin, sodium salt</td>
			<td>GoldBio</td>
			<td>LUCNA</td>
			<td></td>
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			<td>DMEM with L-Glutamine, 4.5g/L Glucose and Sodium Pyruvate</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
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			<td>Eppendorf Xplorer, 12-channel, variable, 15&#8211;300 &#181;L</td>
			<td>Eppendorf</td>
			<td>4861000155</td>
			<td></td>
		</tr>
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			<td>Eppendorf Xplorer, 12-channel, variable, 5&#8211;100 &#181;L</td>
			<td>Eppendorf</td>
			<td>4861000139</td>
			<td></td>
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			<td>Matrix Platemate 2x3</td>
			<td>ThermoFisher</td>
			<td>801-10001</td>
			<td></td>
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			<td>MicroBeta 1450 Wallac</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td></td>
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			<td>Penicilin-Streptomycin</td>
			<td>Wisent</td>
			<td>450-201-EL</td>
			<td></td>
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			<td>Poly-L-Lysine hydrobromide</td>
			<td>Millipore-Sigma</td>
			<td>P2636-500MG</td>
			<td></td>
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			<td>Roth Lab PRESTO-Tango GPCR Kit</td>
			<td>Addgene</td>
			<td>Kit #1000000068</td>
			<td></td>
		</tr>
	</tbody>
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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.

Using the PRESTO-Tango protocol presented herein, a chromaffin granule (CG) extract was screened against 168 non-olfactory GPCR targets, with the majority being orphan receptors. Profiling of said extract was performed by examining &#946;-arrestin2 mobilization at the chosen receptors, based on the principle designed by Barnea et al.18 (Figure 1). Plasmid cDNA of the GPCRs of interest was taken from the PRESTO-Tango GPCR Kit and assembled in two 96 well-plates in the ...

Watch this Scientific Journal Video about Parallel Interrogation of ÃŽÂ²-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay at JoVE.com

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

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 ...

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Research

JoVE Journal

Biochemistry

11.8K Views.  University of Ottawa. 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 β-arrestin2 recruitment at approximately 300 GPCRs using a TEV-based reporter assay.

Video: Parallel Interrogation of β-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay

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 ...

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 ...

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts of recombinant RNAs are limited. Here, we describe a protocol to produce large amounts of recombinant RNA in Escherichia coli based on co-expression of a chimeric molecule that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. Viroids are relatively small, non-coding, highly base-paired circular RNAs that are infectious to higher plants. The host plant tRNA ligase is an enzyme recruited by viroids that belong to the family Avsunviroidae, such as Eggplant latent viroid (ELVd), to mediate RNA circularization during viroid replication. Although ELVd does not replicate in E. coli, an ELVd precursor is efficiently transcribed by the E. coli RNA polymerase and processed by the embedded hammerhead ribozymes in bacterial cells, and the resulting monomers are circularized by the co-expressed tRNA ligase reaching a remarkable concentration. The insertion of an RNA of interest into the ELVd scaffold enables the production of tens of milligrams of the recombinant RNA per liter of bacterial culture in regular laboratory conditions. A main fraction of the RNA product is circular, a feature that facilitates the purification of the recombinant RNA to virtual homogeneity. In this protocol, a complementary DNA (cDNA) corresponding to the RNA of interest is inserted in a particular position of the ELVd cDNA in an expression plasmid that is used, along the plasmid to co-express eggplant tRNA ligase, to transform E. coli. Co-expression of both molecules under the control of strong constitutive promoters leads to production of large amounts of the recombinant RNA. The recombinant RNA can be extracted from the bacterial cells and separated from the bulk of bacterial RNAs taking advantage of its circularity.

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

Here, we present a protocol to produce large amounts of recombinant RNA in Escherichia coli by co-expressing a chimeric RNA that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. The main product is a circular molecule that facilitates purification to homogeneity.

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable.
Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes&#39; design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.

Altered nuclease activity has been associated with different human conditions, underlying its potential as a biomarker. The modular and easy to implement screening methodology presented in this paper allows the selection of specific nucleic acid probes for harnessing nuclease activity as a biomarker of disease.

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

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 ...

Screening for Phytoestrogens using a Cell-based Estrogen Receptor  &#946; Reporter Assay

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in the vertebrates that consume them, such as endocrine function. Phytoestrogens, the most well studied endocrine-active phytochemicals, directly interact with the hypothalamo-pituitary gonadal axis of the vertebrate endocrine system. Here we present the novel use of a cell-based assay to screen plant extracts for the presence of compounds that have estrogenic biological activity. This assay uses mammalian cells engineered to highly express estrogen receptor beta (ER&#946;) and that have been transfected with a luciferase gene. Exposure to compounds with estrogenic activity results in the cells producing light. This assay is a reliable and simple way to test for biological estrogenic activity. It has several improvements over transient transfection assays, most notably, ease of use, the stability of the cells, and the sensitivity of the assay.

We have optimized a commercially available estrogen receptor &#946; reporter assay for screening human and nonhuman primate foods for estrogenic activity. We validated this assay by showing that the known estrogenic human food soy registers high, while other foods show no activity.

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in ...

On-Chip Crystallization and Large-Scale Serial Diffraction at Room Temperature

Biochemical reactions and biological processes can be best understood by demonstrating how proteins transition among their functional states. Since cryogenic temperatures are non-physiological and may prevent, deter, or even alter protein structural dynamics, a robust method for routine X-ray diffraction experiments at room temperature is highly desirable. The crystal-on-crystal device and its accompanying hardware and software used in this protocol are designed to enable in situ X-ray diffraction at room temperature for protein crystals of different sizes without any sample manipulation. Here we present the protocols for the key steps from device assembly, on-chip crystallization, optical scanning, crystal recognition to X-ray shot planning and automated data collection. Since this platform requires no crystal harvesting nor any other sample manipulation, hundreds to thousands of protein crystals grown on chip can be introduced into an X-ray beam in a programmable and high-throughput manner.

This contribution describes how to set up protein crystallization on crystal-on-crystal devices and how to perform automated serial data collection at room temperature using the on-chip crystallization platform.

Biochemical reactions and biological processes can be best understood by demonstrating how proteins transition among their functional states. Since ...

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

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

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

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

Development of a Click-Based Assay for Screening HAT1 Enzyme Activity

An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome assembly. It is required for tumor growth in various systems, making it a potential target for cancer treatment. To facilitate the identification of compounds that can inhibit HAT1 enzymatic activity, we have devised an acetyl-click assay for rapid screening. In this simple assay, we employ recombinant HAT1/Rbap46, which is purified from activated human cells. The method utilizes the acetyl-CoA analog 4-pentynoyl-CoA (4P) in a click-chemistry approach. This involves the enzymatic transfer of an alkyne handle through a HAT1-dependent acylation reaction to a biotinylated H4 N-terminal peptide. The captured peptide is then immobilized on neutravidin plates, followed by click-chemistry functionalization with biotin-azide. Subsequently, streptavidin-peroxidase recruitment is employed to oxidize amplex red, resulting in a quantitative fluorescent output. By introducing chemical inhibitors during the acylation reaction, we can quantify enzymatic inhibition based on a reduction of the fluorescence signal. Importantly, this reaction is scalable, allowing for high throughput screening of potential inhibitors for HAT1 enzymatic activity.

Author Spotlight: Developing Acetyl-Click Assay for HAT1 Inhibitor Screening

Quick and accurate chemical assays to screen for specific inhibitors are an important tool in the drug development arsenal. Here, we present a scalable acetyl-click chemistry assay to measure the inhibition of HAT1 acetylation activity.

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome ...