Name: high-throughput screening for small-molecule modulators of inward rectifier potassium channels
Uploaded: 2013-01-27T14:00:00
Description: Watch this Scientific Journal Video about High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels at JoVE.com

Dette arbejde blev støttet af midler fra National Institutes of Health tilskud 1R21NS073097-01 og 1R01DK082884 (JSD) og Institut for National Institutes tilskuddet PIER11VCTR.

1. Generering af stabile Polyklonale cellelinier <ol><li> Etableringen af ​​en høj kvalitet stabil cellelinie, der udtrykker Kir kanal af interesse er et vigtigt første skridt mod at udvikle en robust high-throughput screening assay. Konstitutiv K +-kanal overekspression kan føre til aktivering af celledød veje, stabil cellelinje degeneration og tab af analysens ydeevne. For at undgå disse potentielle problemer og tilvejebringe en bekvem intern kontrol til assay udvikling (se nedenfor), er en tetra...

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	<li>Ehrlich, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inward+rectifier+potassium+currents+as+a+target+for+atrial+fibrillation+therapy.">Inward rectifier potassium currents as a target for atrial fibrillation therapy.</a> J. Cardiovasc. Pharmacol. 52 (2), 129 (2008).</li><li>Bhave, G., Lonergan, D., Chauder, B. A., Denton, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+modulators+of+inward+rectifier+K++channels:+recent+advances+and+future+possibilities.">Small-molecule modulators of inward rectifier K+ channels: recent advances and future possibilities.</a> Future Med. Chem. 2 (5), 757 (2010).</li><li>Swartz, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tarantula+toxins+interacting+with+voltage+sensors+in+potassium+channels.">Tarantula toxins interacting with voltage sensors in potassium channels.</a> Toxicon. 49 (2), 213 (2007).</li><li>Jin, W., Lu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+high-affinity+inhibitor+for+inward-rectifier+K++channels.">A novel high-affinity inhibitor for inward-rectifier K+ channels.</a> Biochemistry. 37 (38), 13291 (1998).</li><li>Jin, W., Lu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+a+stable+form+of+tertiapin:+a+high-affinity+inhibitor+for+inward-rectifier+K++channels.">Synthesis of a stable form of tertiapin: a high-affinity inhibitor for inward-rectifier K+ channels.</a> Biochemistry. 38 (43), 14286 (1999).</li><li>Roy, A., McDonald, P. R., Sittampalam, S., Chaguturu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+access+high+throughput+drug+discovery+in+the+public+domain:+a+Mount+Everest+in+the+making.">Open access high throughput drug discovery in the public domain: a Mount Everest in the making.</a> Curr. Pharm. Biotechnol. 11 (7), 764 (2010).</li><li>Weaver, C. D., 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+thallium-sensitive,+fluorescence-based+assay+for+detecting+and+characterizing+potassium+channel+modulators+in+mammalian+cells.">A thallium-sensitive, fluorescence-based assay for detecting and characterizing potassium channel modulators in mammalian cells.</a> J. Biomol. Screen. 9 (8), 671 (2004).</li><li>Lewis, L. M., 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-throughput+screening+reveals+a+small-molecule+inhibitor+of+the+renal+outer+medullary+potassium+channel+and.">High-throughput screening reveals a small-molecule inhibitor of the renal outer medullary potassium channel and.</a> 76 (5), 1094 (2009).</li><li>Bhave, G., 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=Development+of+a+selective+small-molecule+inhibitor+of+Kir1.1,+the+renal+outer+medullary+potassium+channel.">Development of a selective small-molecule inhibitor of Kir1.1, the renal outer medullary potassium channel.</a> Mol. Pharmacol. 79 (1), 42 (2011).</li><li>Raphemot, R., 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=Discovery,+characterization,+and+structure-activity+relationships+of+an+inhibitor+of+inward+rectifier+potassium+(Kir)+channels+with+preference+for+Kir2.3,+Kir3.x,+and+Kir7.1.">Discovery, characterization, and structure-activity relationships of an inhibitor of inward rectifier potassium (Kir) channels with preference for Kir2.3, Kir3.x, and Kir7.1.</a> Front Pharmacol. 2, 75 (2012).</li><li>Niswender, C. M., 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+novel+assay+of+Gi/o-linked+G+protein-coupled+receptor+coupling+to+potassium+channels+provides+new+insights+into+the+pharmacology+of+the+group+III+metabotropic+glutamate+receptors.">A novel assay of Gi/o-linked G protein-coupled receptor coupling to potassium channels provides new insights into the pharmacology of the group III metabotropic glutamate receptors.</a> Mol. Pharmacol. 73 (4), 1213 (2008).</li></ol>

Databehandling: Når data indsamles, indbefatter et almindeligt trin i analysen normalisere hver brønd er fluorescensresponset, F, til den oprindelige værdi ved begyndelsen af eksperimentet, F 0. Dette omtales ofte som &quot;statisk forholdet&quot; og symboliseret &quot;F / F 0&quot;. Hvis F 0 domineres af indikatorfarvestoffet den statiske forhold operation vil det væsentlige korrigere for mange faktorer såsom disuniformities i belysningen, signal samling...

An erratum was issued for: <a href="https://www.jove.com/video/4209/high-throughput-screening-for-small-molecule-modulators-inward">High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels</a>. The Protocol section has been updated.
The formula in step 7.1 of the Protocol has been updated from:
Z prime = 1- (3SDp + 3SDn)/|meanp + meann |
to:
Z prime = 1- (3SDp + 3SDn)/|meanp - meann |

An erratum was issued for: <a href="https://www.jove.com/video/4209/high-throughput-screening-for-small-molecule-modulators-inward">High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels</a>. The Protocol section has been updated.

Erratum: High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> pcDNA5/TO </td><td> Invitrogen </td><td> V1033-20 </td><td> Tetracyklin-inducerbare ekspressionsvektor </td></tr><tr><td> T-Rex-HEK293-celler </td><td> Invitrogen </td><td> R71007 </td><td> Tetracyklin-inducerbare cellelinie </td></tr><tr><td> Lipofectamin LTX / Plus Reagent </td><td> Invitrogen </td><td> 15338100 </td><td> Transfektionsreagens </td></tr><tr><td> FBS </td><td> ATLANTA Biologicals </td><td> S11550 </td><td> Celledyrkningsmedier </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11.965 </td><td> Celledyrkningsmedier </td></tr><tr><td> Hygromycin B </td><td> Invitrogen </td><td> 10687-010 </td><td> Celledyrkningsmedier </td></tr><tr><td> Blasticidin S </td><td> Invitrogen </td&gt; <td> R210-01 </td><td> Celledyrkningsmedier </td></tr><tr><td> Penicillin / streptomycin </td><td> Invitrogen </td><td> 15.140 </td><td> Celledyrkningsmedier </td></tr><tr><td> HBSS-divalent free </td><td> Mediatech </td><td> 21022CV </td><td> Cell vask </td></tr><tr><td> Trypsin-0,25% </td><td> Mediatech </td><td> 25053CI </td><td> Celledissociering </td></tr><tr><td> Tetracyclin-HCl </td><td> Sigma </td><td> T9823 </td><td> Induktion reagens </td></tr><tr><td> Dialyseret FBS </td><td> ATLANTA Biologicals </td><td> S12650 </td><td> Plademedier </td></tr><tr><td> FluoZin-2 </td><td> Invitrogen </td><td> F24189 </td><td> Fluorescerende farvestof </td></tr><tr><td> Pluronic F-127 </td><td> Invitrogen </td><td> P-3000MP </td><td> Dye loading </td></tr><tr><td> HBSS </td><td> Invitrogen </td><td> 14.175 </td><td> Assaypuffer </td></tr><tr><td> HEPES </td><td> Invitrogen </td><td> 15.630 </td><td> Assaypuffer </td></tr><tr><td> NaHCO3 </td><td> Sigma </td><td> S6297 </td><td> Tl + stimulus buffer </td></tr><tr><td> MgSO4 </td><td> Sigma </td><td> M2643 </td><td> Tl + stimulus buffer </td></tr><tr><td> CaSO 4 • 2H 2 O </td><td> Sigma </td><td> C3771 </td><td> Tl + stimulus buffer </td></tr><tr><td> D-glucose </td><td> Sigma </td><td> G7528 </td><td> Tl + stimulus buffer </td></tr><tr><td> Thallium sulfat </td><td> Aldrich </td><td> 204.625 </td><td> Tl + stimulus buffer </td></tr><tr><td> HEPES </td><td> Sigma </td><td> H4034 </td><td> Tl + stimulus buffer </td></tr><tr><td> DMSO </td><td> Sigma </td><td> D4540 </td><td> Opløsningsmiddel </td></tr><tr><td> Otte kanaler elektronisk pipette </td><td> Biohit </td><td> E300 </td><td> Cell udpladning på 384 -brøndsplader </td></tr><tr><td> BD PureCoat amin-coatede 384-brøndsplader </td><td> BD Biosciences </td><td> 356.719 </td><td> Assay mikroplader </td></tr><tr><td> Echo kvalificeret 384-Well polypropylen mikroplade (384PP) </td><td> Labcyte </td><td> P-05525 </td><td> Compound source mikroplader </td></tr><tr><td> 384-brønds polypropylen mikroplader </td><td> Greiner Bio-One </td><td> 781.280 </td><td></td></tr><tr><td> Multidrop Combi reagens dispenser </td><td> Thermo Scientific </td><td> 5840300 </td><td></td></tr><tr><td> ELx405 Micropladevasker </td><td> BioTek </td><td> ELx405HT </td><td> Automatiseret celle vask </td></tr><tr><td> Echo væskehåndteringsudstyr </td><td> Labcyte </td><td> Labcyte Echo 550 </td><td></td></tr><tr><td> Bravo automatiseret væskehåndtering platform </td><td> Agilent Technologies </td><td> Standardmodel </td><td></td></tr><tr><td> Hamamatsu FDSS 6000 </td><td> Hamamatsu </td><td></td><td> Kinetic imaging plate reader </td></tr></tbody></table> Tabel 1. Liste over materialer og reagenser.

high-throughput screening for small-molecule modulators of inward rectifier potassium channels

Specifikke medlemmer af aktiv ensretter kalium (Kir) kanal familien postuleres lægemiddelmål for en række lidelser, herunder hypertension, atrieflimren, og smerte 1,2. For det meste er der dog fremskridt i retning af at forstå deres terapeutiske potentiale eller endda basale fysiologiske funktioner er blevet bremset på grund af manglen på gode farmakologiske værktøjer. Faktisk har den molekylære farmakologi indad ensretter familien haltet langt bagefter S4 superfamilie af spændingsstyrede kalium (KV) kanaler, hvor en række nanomolær affinitet og højselektive peptid toxin modulatorer er blevet opdaget 3. The bigift toksin tertiapin og dets derivater er kraftige inhibitorer af Kir1.1 og Kir3 kanaler 4,5, men peptider er af begrænset nytte terapeutisk såvel som eksperimentelt på grund af deres antigene egenskaber og ringe biotilgængelighed, metaboliske stabilitet og væv penetrans. Udviklingen af ​​potenteog selektive små molekyler prober med forbedrede farmakologiske egenskaber vil være en nøgle til fuldt ud at forstå fysiologi og terapeutiske potentiale af Kir-kanaler. The Molecular Biblioteker Probes Production Center Network (MLPCN) støttet af National Institutes of Health (NIH) fælles fond har skabt muligheder for akademiske forskere at indlede probe opdagelse kampagner for molekylære mål og signalveje, der har behov for bedre farmakologi 6. Den MLPCN giver forskere adgang til industri-skala screening centre og medicinsk kemi og informatik støtte til at udvikle små molekyler sonder at belyse funktionen af ​​gener og gen netværk. Det kritiske trin i at få adgang til MLPCN er udviklingen af ​​en robust mål-eller pathwaygen-specifikke assay, der er modtagelig for high-throughput screening (HTS). Her beskriver vi hvordan man udvikler en fluorescens-baseret thallium (Tl +) flux ASSAy af Kir kanalfunktion til high-throughput screening forbindelse 7,8,9,10. Assayet er baseret på permeabiliteten af K +-kanal pore til K + congener Tl +. Et kommercielt tilgængeligt fluorescerende Tl + rapportør-farvestof anvendes til påvisning transmembrane flux af Tl + gennem poren. Der er mindst tre kommercielt tilgængelige farvestoffer, som er egnede til Tl + flux assays: BTC, FluoZin-2, og FluxOR 7,8. Denne protokol beskriver assay udvikling ved hjælp FluoZin-2. Selvom oprindeligt udviklet og markedsført som en zink-indikator, FluoZin-2 udviser en robust og dosisafhængig stigning i fluorescensemission ved Tl + binding. Vi begyndte at arbejde med FluoZin-2 før FluxOR var tilgængelig 7,8 og har fortsat med at gøre det 9,10. Men trinene i assay udvikling er det væsentlige identiske for alle tre farvestoffer, og brugerne bør afgøre, hvilke farve der er mest hensigtsmæssig for deres specifikke needs. Vi diskuterer også assayet s benchmarks, som skal opfyldes for at komme i betragtning til optagelse i MLPCN. Da Tl + let gennemtrænger fleste K +-kanaler, bør assayet kunne tilpasses til de fleste K +-kanal mål.

Anvendelsen af en tetracyklin-inducerbare ekspressionssystem tilvejebringer en passende intern kontrol for at skelne Tl + flux gennem endogene veje og Kir kanal af interesse. Figur 1 viser nogle eksempler på Celleudpladning kort, der anvendes i forskellige typer af eksperimenter. Positionerne af brønde indeholdende uinducerede eller tetracyclin-inducerede celler angives med forskellige farver. Figur 2A viser kilden pladediagram anvendes til at bestemme den optimale Tl ...

Watch this Scientific Journal Video about High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels at JoVE.com

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Metoder til udvikling og validering af en kvantitativ fluorescens assay til måling af aktiviteten af ​​indadgående ensretter kalium (Kir) kanaler til high-throughput sammensatte screening er præsenteret.

High-throughput screening for små-molekyle modulatorer af indadgående Ensretter kaliumkanaler

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

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