Name: a kinetic fluorescence-based ca2+ mobilization assay to identify g protein-coupled receptor agonists, antagonists, and allosteric modulators
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Description: Watch this Scientific Journal Video about A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators at JoVE.com

Forfatterne vil gerne takke Eric Fonteyn og Geert Schoofs for fremragende teknisk bistand. Dette arbejde er blevet støttet af KU Leuven (give nr. PF/10/018), Fonds voor Wetenschappelijk Onderzoek (CVE, give nr. G.485.08), og Fondation Dormeur Vaduz.

Bemærk: Alle trin beskrevet under afsnit 1 og 2 udføres under sterile forhold i en laminar flow kabinet.
1. vedligeholdelse af U87. CD4.hCXCR4 celler
<ol><li>Vokse celler i T75 kultur kolber på 37 ° C og 5% CO2 i en fugtig inkubator. Bemærk: Den i vitro cellelinje bruges i denne protokol er en U87 glioblastom cellelinie stabilt udtrykker klynge af differentiering 4 (CD4) og menneskelige CXCR4 og har været tidligere beskrevet17. Celle overf...

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Ca2 +-mobilisering assay beskrevet heri har tidligere vist sig at være et værdifuldt redskab til at identificere og karakterisere receptor antagonister målretning CXCR417. Det forventes dog, at denne metode kan være mere generelt anvendes til en stor gruppe af andre GPCRs, der udløser en cytosole Ca2 + release ved deres aktivering, som illustreret til relaterede chemokine receptor CCR5. For CCR5 præcis de samme forsøgsbetingelser kunne anvendes, skal flere trin i proto...

GPCRs er en vigtig superfamilien af celle overflade proteiner, der aktiverer signaltransduktion kaskader af ekstracellulære ligand bindende. De kan aktiveres af en lang række stimuli herunder peptider, protein hormoner, biogene aminer og lipider, hvilket resulterer i indledningen af diverse intracellulær signalering veje og i sidste ende biologiske svar1,2 . Derudover GPCRs er involveret i mange, hvis ikke alle, udviklingsmæssige og fysiologiske processer og mange menneskelige sygdomme er forbundet med dysfunktionelle GPCR signalering eller receptor overekspression. GPCRs er derfor blandt de mest validerede farmakologiske mål i medicin1,3.
Typisk, en GPCR drug discovery arbejdsprocessen starter med cellulære screening-assays muliggør identificering af forbindelser som små molekyler, monoklonale antistoffer og peptider, der kan modulere aktiviteten af et bestemt GPCR. I GPCR drug discovery mange forskellige typer af assays findes for at søge efter sådanne forbindelser, er hvoraf de fleste kompatibel med midten af til high throughput screening kampagner. De mest anvendte assays omfatter receptor binding eksperimenter, fluorescens eller luminescence baseret assays afsløre udsving i niveauet af såkaldte sekundære budbringere (fx, Ca2 +, cyklisk adenosin fra (AMP)), fænotypisk screening-assays og β-arrestin ansættelse-undersøgelser vedrørende4. Valg for en bestemt type af analysen kan afhænge af flere faktorer, men også afhænger af forudgående viden om signaling egenskaberne af en given GPCR. Agonist binding til en GPCR inducerer en konformationel ændring katalysere udveksling af guanidin ud (BNP) for guanidin trifosfat (GTP) på α-subunit af heterotrimeric G proteiner. Efterfølgende Gα-GTP subunit dissocieres fra Gβγ subunit og begge underenheder vil indlede yderligere signaling veje. Hydrolyse af GTP-molekyle og efterfølgende re sammenslutning af Gα- BNP og Gβγ subunits vil gendanne G protein i sin inaktive kropsbygning5,6. Baseret på sekvensen ligheden af Gα -underenhed forskellige typer af G proteiner er defineret (Gs, Gjeg, Gq, G12/13)7. Signalering via Gα subunit giver anledning til flere typiske reaktioner såsom stigningen (via Gs) eller formindske (via G,jeg) af cykliske AMP produktion og intracellulære Ca2 + mobilisering (via Gq)5 ,7. Gβγ underenheder er også i stand til at fremkalde intracellulære effektor veje. For eksempel, ved aktivering af G,jeg-koblede GPCRs, Gβγ kan direkte stimulerer fosfolipase C (PLC-β) til at producere inositol trifosfat (IP3) der udløser frigivelsen af Ca2 + fra intracellulære butikker7 . Efter receptor aktivering, GPCRs er fosforyleret af GPCR kinaser (GRKs), som fremmer interaktion med β-arrestins. Denne proces afsluttes G protein signalering og fører til receptoren desensibilisering og til sidst internalisering. Β-arrestins er også i stand til at danne multi molekylære komplekser, der kan udløse andre signaling veje uafhængigt af G protein signalering8.
Inden for underfamilie af chemokine receptorer, Gjeg-koblede CXCR4 er en GPCR, der har rejst meget interesse som en lovende mål for drug discovery. Givet sin etablerede rolle som en større Co receptor for human immundefekt virus 1 (HIV-1) blev viral entry og infektion, forbindelser målretning CXCR4 oprindeligt udviklet som anti-HIV medicin kandidater9. For nylig, en voksende mængde af beviser har henvist til en vigtig rolle for CXCR4 i tumordannelse og kræft metastaser gør det et godt valideret terapeutiske mål inden for onkologi samt10. CXCR4 er stærkt udtrykt i mere end 20 typer af menneskelige kræft og kontrol tumor celle overlevelse, spredning, og migration og tumor-relaterede angiogenese10. CXCR4 antagonister af forskellige kemiske klasser tidligere har beskrevet11,12, men kun den lille molekyle AMD3100 er nu godkendt til brug i klinikken som en stamcelle mobilisering agent bruges under behandling af lymfom og myelomatose patienter13,14. Kliniske forsøg er løbende at vurdere sikkerheden og virkningen af flere andre CXCR4 antagonister i forskellige menneskelige sygdomme, men med et stærkt fokus på onkologi12. I betragtning af de mange potentielle anvendelser for CXCR4 antagonister, er søgen efter nye forbindelser med forbedret farmakokinetiske egenskaber, forbedret biotilgængelighed eller potentielt mindre bivirkninger berettiget.
Heri, en kinetisk fluorescens-baserede cellulære assay primært bruges til at screene for forbindelser i stand til at hæmme CXCR4 er beskrevet. Fluorescerende måling af denne metode er baseret på den forbigående stigning i den intracellulære Ca2 + koncentration fremkaldt ved CXCR4 aktivering af sin endogene agonist, chemokine ligand CXCL12 (tidligere kendt som stromale celle afledte faktor 1α ( SDF1-α)), og den potentielle hæmning af denne CXCL12-induceret Ca2 + svar af særlige forbindelser. I denne analyse anvendes U87 menneskelige glioblastom celler stabilt at udtrykke den menneskelige CXCR4 receptor. På samme tid mangler disse celler endogene udtryk for CXCR7, en relaterede chemokine receptor, som binder også CXCL1215,16,17. CXCR7 har tidligere også vist sig at være i stand til at danne heterodimers med CXCR4, hvorved modulerende signaling egenskaberne for denne sidstnævnte receptor18. Udsving i niveauet af intracellulære Ca2 + medieret af CXCR4 overvåges ved at indlæse CXCR4+ celler med fluo-2 acetoxymethyl (AM) ester, en celle-gennemtrængelige høj affinitet fluorescerende Ca2 +-bindende farvestof. Fluo-2 AM er et enkelt bølgelængde fluorescerende molekyle, der kan være glade på 490 nm mens dens emission Fluorescens er målt ved 520 nm. Denne emission fluorescens øger ved Ca2 + bindende, med et stort dynamikområde mellem Ca2 +-bundne og ubundne stat. Stigningen i fluorescerende signal er forbigående, forekommer med et interval på et par minutter, og vil henfalde bagefter. Tophøjde af fluorescerende emissioner yderligere korrelerer med niveau af receptor aktivering. Selve analysen udføres ved hjælp af et fluorescens mikrotiterplade læser udstyret med en intensiveret CCD (ICCD) kamera, der besidder en integreret pipettering system, der giver mulighed for standardisering af pipettering trin i analysen (jf. Tabel af materialer) . Derudover er samtidig måling af de fluorescerende signal i alle brønde på en mikrotiterplade en anden vigtig fordel af fluorescens-læseren, der bruges. Under først del af analysen forbindelser under undersøgelsen (fx, et panel af små molekyler på en fast koncentration eller i en fortyndingsrække) er tilføjet til fluo-2 AM indlæst CXCR4+ U87 celler efterfulgt af en ~ 10 min inkubationstiden hvor den potentielle agonistisk effekt af forbindelser måles kontinuerligt i realtid. Derefter, den endogene agonist (dvs.CXCL12) er føjet til cellerne til at fremmane en CXCR4-medieret forbigående stigning i niveauet af intracellulære Ca2 +. Under denne del af analysen kan den potentielle antagonistiske aktivitet af de testede forbindelser evalueres. En skematisk oversigt over analysens generelle arbejdsgang er præsenteret i figur 1.
Selv om denne Ca2 + mobilisering assay er primært blevet brugt til at identificere og bestemme den hæmmende styrken af konkurrencedygtige CXCR4 antagonister (dvs., forbindelser, der forhindrer den endogene agonist for at binde og stimulere receptor), det også kan identificere receptor agonister og derudover forbindelser at udøve deres funktion ved at binde på allosteriske websteder (dvs., websteder, der topografisk afviger fra det orthosteric bindingssted besat af den endogene agonist). Eksempler på den sidstnævnte kategori af forbindelser allosteriske agonister og PAMs og NAMs19,20. Mens receptor-specifikke antagonister samt NAMs ville hæmme CXCL12-induceret Ca2 + reaktionen, PAMs ville styrke dette svar (Se også diskussion sektion). Selvom analysen beskrevet heri specifikt mål CXCR4, det forventes, at denne metode kan anvendes på andre GPCRs med minimal optimering indsats, i det mindste hvis de signal via frigivelse af intracellulære Ca2 +.

<table>
	<tbody>
		<tr>
			<td>Fluo-2 AM</td>
			<td>Abcam</td>
			<td>ab142775</td>
			<td>fluorescent Ca2+ sensitive dye</td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Sigma</td>
			<td>P2443-250G</td>
			<td>pluronic acid</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma</td>
			<td>G9391</td>
			<td></td>
		</tr>
		<tr>
			<td>AMD3100</td>
			<td>Sigma</td>
			<td>A5602-5 mg</td>
			<td>specific CXCR4 antagonist</td>
		</tr>
		<tr>
			<td>Maraviroc</td>
			<td>kind gift of AnorMed</td>
			<td></td>
			<td>antiretroviral drug, CCR5 antagonist</td>
		</tr>
		<tr>
			<td>Chemokine ligand CXCL12</td>
			<td>PeproTech</td>
			<td>300-28A</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemokine ligand CCL5</td>
			<td>PeproTech</td>
			<td>300-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco (Life Technologies)</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>A1933-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Gibco (Life Technologies)</td>
			<td>41965-039</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (10 x), calcium, magnesium, no phenol red</td>
			<td>Gibco (Life Technologies)</td>
			<td>14065-049</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Gibco (Life Technologies)</td>
			<td>15630-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0,25 %), phenol red</td>
			<td>Gibco (Life Technologies)</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (DPBS)</td>
			<td>Gibco (Life Technologies)</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes, 50 mL</td>
			<td>Greiner Bio-One</td>
			<td>227 261</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture flask (T75)</td>
			<td>Corning</td>
			<td>353024</td>
			<td></td>
		</tr>
		<tr>
			<td>Black plate, 96-well, clear bottom, with lid</td>
			<td>Costar/Fisher Scientific</td>
			<td>10530753</td>
			<td>assay plate (96-well), for cell seeding</td>
		</tr>
		<tr>
			<td>Polypropylene (PP) plates</td>
			<td>Thermo Scientific (VWR)</td>
			<td>732-2661</td>
			<td>plates used to prepare the compound plates and chemokine plates, round bottom</td>
		</tr>
		<tr>
			<td>FLIPR Tetra high throughput cellular screening system</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>Fluorescent plate reader with integrated pipettor head and ICCD camera</td>
		</tr>
		<tr>
			<td>FLIPR Tetra LED Module 470 - 495 nm</td>
			<td>Molecular Devices</td>
			<td>0200-6128</td>
			<td>Light emitting diodes for excitation of the fluorescent Ca2+ sensitive dye</td>
		</tr>
		<tr>
			<td>FLIPR Tetra Emission Filter 515 - 575 nm</td>
			<td>Molecular Devices</td>
			<td>0200-6203</td>
			<td>emission filter compatible with the fluorescent dye</td>
		</tr>
		<tr>
			<td>FLIPR Tetra 96 Head</td>
			<td>Molecular Devices</td>
			<td>0310-4536</td>
			<td>96-well pipettor head, integrated within the fluorescent plate reader</td>
		</tr>
		<tr>
			<td>ScreenWorks</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>software package used for data analysis and visualization on the FLIPR Tetra</td>
		</tr>
		<tr>
			<td>Vi-CELL</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>cell viability analyzer</td>
		</tr>
		<tr>
			<td>Corning CellBIND 96 Well Flat Clear Bottom Black Polystyrene Microplates, with Lid, Sterile</td>
			<td>Corning</td>
			<td>3340</td>
			<td>Pre-coated 96-well assay plates that may represent an alternative for manual coating of the assay plate.</td>
		</tr>
	</tbody>
</table>

a kinetic fluorescence-based ca2+ mobilization assay to identify g protein-coupled receptor agonists, antagonists, and allosteric modulators

G protein koblede receptorer (GPCRs) er af stor betydning for den farmaceutiske industri, som de er involveret i mange menneskelige sygdomme og omfatter godt validerede mål for terapeutisk intervention. Opdagelsen af blyforbindelser, herunder små syntetiske molekyler, der specifikt hæmmer den receptor funktion, er et vigtigt første skridt i udvikling af lægemidler og er afhængig af følsomme, specifikke og robust cellebaserede assays. Her, vi beskriver en kinetisk cellulære assay med en fluorescerende udlæsning primært designet til at identificere receptor-specifikke antagonister, der hæmmer den intracellulære Ca2 + release fremkaldt ved aktivering af CXC chemokine receptor 4 (CXCR4) af sin endogene ligand, CXC chemokine ligand 12 (CXCL12). En vigtig fordel ved denne metode er, at det også gør screening af forbindelser udrustet med iboende agonistiske egenskaber (dvs., forbindelser fremkalde en stigning i intracellulære Ca2 + koncentration i mangel af CXCL12) eller forbindelser modulerende receptor's funktion via interaktion med allosteriske bindingssteder (dvs., positive og negative allosteriske modulatorer (PAMs og NAMs, henholdsvis)). På den negative side, kan autofluorescent forbindelser interferere med assayets udlæsning, dermed hæmmer pålidelige data fortolkning. Mest sandsynligt kan dette assay gennemføres med minimale tilpasninger, som et generisk lægemiddel discovery assay for mange andre GPCRs, hvoraf aktivering fører til en frigivelse af intracellulære Ca2 +.

Effekten af CXCL12 stimulation på den intracellulære Ca2 + mobilisering i U87. CD4.CXCR4+ og U87. CD4-celler blev evalueret med Ca2 + mobilisering assay. I stedet for 20 µL af prøven stof, der normalt ville blive tilføjet under den første pipettering trin i protokollen (figur 1), analysebuffer blev føjet til fluo-2 AM indlæst U87. CD4.CXCR4+ celler i måling plade. Under det andet udlevering skridt, forskell...

Watch this Scientific Journal Video about A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators at JoVE.com

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

Den beskrevne cellulære assay er designet til identifikation af CXC chemokine receptor 4 (CXCR4)-interagerende agenter, der hæmmer eller stimulere enten konkurrencedygtige eller allosterically, den intracellulære Ca2 + release initieret af CXCR4 aktivering.

En kinetisk fluorescens-baserede Ca2 + mobilisering Assay at identificere G Protein-koblet Receptor agonister og antagonister allosteriske modulatorer

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

The overall goal of this cell-based assay is to identify and characterize molecules capable of stimulating or inhibiting G protein-coupled receptor-mediated intracellular calcium mobilization. This method can be a valuable assay for G protein-coupled receptor drug discovery because it can be used to screen for molecules that modify receptor-mediated intracellular calcium bodies. The main advantage of this technique is that it allows molecules with agonistic or antagonistic properties to be identified within the same assay.This assay may help in the discovery of novel drug candidates that act on G protein-coupled receptors promising therapeutic targets that play a role in many human diseases. On the day of cell passaging, coat each well of a black walled polystyrene 96-well plate with clear bottoms with 100 microliters of 0.1%gelatin solution and incubate the plates for two hours at room temperature. While the gelatin is solidifying, use a trypsin EDTA solution to lift the cells of interest from the bottom of their culture flask, resuspending the cells in fresh growth medium in a 50 milliliter conical tube once they have detached.After counting, collect the cells by centrifugation and resuspend the pellet in fresh growth medium to a one times 10 to the fifth viable cells per milliliter concentration. When the plates are ready, discard any excess gelatin solution and dry the plates on a lab tissue. Then wash the wells with 200 microliters of PBS and replace the wash with 200 microliters of cells per well for an overnight incubation at 37 degrees Celsius and 5%carbon dioxide.The next morning, tap the plate to discard the growth medium from the wells and dry the plate upside on a paper towel. Then label the cells in each well with 100 microliters of loading dye solution per well and incubate the plate for 45 minutes in the dark at room temperature. While the cells are incubating, add 75 microliters of CXCL12 per well to the chemokine plate and 50 microliters of the compound of interest to each well of the compound plate.Next, turn on the cooler unit of the fluorescence plate reader followed by the plate reader itself. After allowing the system a few minutes to initiate, open the plate reader software. To set up the assay protocol, under the settings tab select read mode and the appropriate excitation and emission wavelengths for the loading dye.Select the mix with transfer fluid tab for automatic mixing of the compounds in the compound plate. In the transfer fluid tab, set 20 microliters of each well from the compound plate to be transferred into the measurement plate and position the pipette tips at 20 microliters below the liquid surface. Set the speed of aspiration to 50 microliters per second and the speed of dispensing to 25 microliters per second and click read with transfer fluid.During the first interval, select 60 reads with a read interval time of one second with 10 reads to be recorded before the compounds are dispensed into the measurement plate and 50 reads to be completed after. Set the second interval with a read interval time of 30 seconds and 18 reads. Select three tip washes for the protocol, setting each wash step to include the appropriate wash fluid, one wash cycle, a fast pump speed, and five strokes per wash.Use the pause pipettor function to include the pipettor pause of 300 seconds and use the mix with transfer fluid setting to allow automatic mixing of the CXCL12 solution within the chemokine plate. In the subsequent transfer fluid tab, set 25 microliters from each well of the chemokine plate to be transferred to the measurement plate and position the tips 20 microliters below the liquid surface. Set the aspiration speed to 50 microliters per second and the dispensing speed to 25 microliters per second.Use the read with transfer fluid button to set the first interval to include 145 reads with a read interval time of one second. Set the second interval to a read interval time of six seconds and 20 reads. Select three more wash tip cycles.Then click set stage temperature and set the device temperature to 37 degrees Celsius. At the end of the loading dye incubation, discard the buffer from the measurement plate and dry the plate on a lab tissue. Wash the cells with 150 microliters per well of assay buffer replacing the buffer with 80 microliters of assay buffer after two minutes.Place the measurement plate in the read position, the compound plate at the source two position, the chemokine plate at the source three position, and a box of black tips at the source one position. Close the device and click protocol signal test to determine the background relative light units and fluorescence variance over the plate. Select test signal in the popup window.If background values of 8, 000 to 10, 000 relative light units are obtained, select update and click save to save the main protocol. Then push run to start the assay. In this graph, a dose-dependent increase in fluorescence correlating with the dose-dependent increase in the release of calcium can be observed.Increasing levels of CXCL12-induced fluorescence allow the generation of a dose response curve based on the difference between the minimum and maximum response over baseline between the first measurement after addition addition of the chemokine and the final measurement in the protocol. No fluorescent calcium-related response was evoked by CXCL12 even at a high concentration when cells lacking the corresponding functional chemokine receptor CXCR4 were used demonstrating the receptor specificity of the CXCL12-evoked measurement. A key application for this cell-based assay is to identify small molecules that, following pre-incubation, dose dependently inhibit CXCL12-induced calcium mobilization in the cells as compared to control non-active compounds that do not inhibit the response.To determine the inhibitory potency of small molecules, a dose response curve can be generated for the inhibitory molecules using nonlinear regression. This assay can be used to study other G protein-coupled receptors with no or only minimal further optimization as well as in this experiment in which a dose-dependent increase in fluorescence was observed when another chemokine receptor, CCR5, was stimulated with its natural chemokine agonist CCL5. Once mastered, this technique can be used to study many other G-protein coupled receptors that signal via the mobilization of intracellular calcium ions upon receptor activation.After watching this video, you should have a good understanding of how this cellular assay can be used to identify agonists and antagonists of the CXCL4 chemokine receptor.

Research

JoVE Journal

Medicine

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

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