Name: method for identifying small molecule inhibitors of the protein-protein interaction between hcn1 and trip8b
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This work was supported by National Institutes of Health Grant R21MH104471 and R01MH106511 (D.M.C.), Brain Research Foundation SG 2012-01 (D.M.C.), Northwestern University Clinical and Translational Sciences Institute 8UL1TR000150 (Y.H.), Chicago Biomedical Consortium HTS-004 (Y.H. and D.M.C.), and National Institutes of Health Grant 2T32MH067564 (K.L.). A part of this work was performed by the Northwestern University Medicinal and Synthetic Chemistry Core (ChemCore) at the Center for Molecular Innovation and Drug Discovery (CMIDD), which is funded by the Chicago Biomedical Consortium with support from the Searle Funds at the Chicago Community Trust and Cancer Center Support Grant P30 CA060553 from the National Cancer Institute awarded to the Robert H. Lurie Comprehensive Cancer Center. The high throughput screen work was performed in the High Throughput Analysis Laboratory which is also a core facility of the Robert H. Lurie Comprehensive Cancer Center.

1. Rensning af TRIP8b (241-602) Protein <ol><li> Transformer plasmid indeholdende TRIP8b (241-602) i den bakterielle protein ekspressionsvektoren pGS21 3 ind kompetent E. coli til proteinekspression i overensstemmelse med producentens anvisninger. Plade 300 pi kulturen på Luria Broth (LB) -agar med 5 ug / ml chloramphenicol og ampicillin. Inkubér pladen ved 37 ° C i 16 timer. </li><li> Den næste dag, vælge en enkelt koloni til at pode 50 ml LB med 50 ug / ml chloramphenicol og ampicillin. Inku...

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	<li>Biel, M., Wahl-Schott, C., Michalakis, S., Zong, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperpolarization-activated+cation+channels:+from+genes+to+function.">Hyperpolarization-activated cation channels: from genes to function.</a> Physiol Rev. 89 (3), 847-885 (2009).</li><li>Shah, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HCN1+channels:+a+new+therapeutic+target+for+depressive+disorders?.">HCN1 channels: a new therapeutic target for depressive disorders?.</a> Sci Signal. 5 (244), pe44 (2012).</li><li>Han, Y., 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=Identification+of+Small-Molecule+Inhibitors+of+Hyperpolarization-Activated+Cyclic+Nucleotide-Gated+Channels.">Identification of Small-Molecule Inhibitors of Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels.</a> J Biomol Screen. 20 (9), 1124-1131 (2015).</li><li>Postea, O., Biel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+HCN+channels+as+novel+drug+targets.">Exploring HCN channels as novel drug targets.</a> Nat Rev Drug Discov. 10 (12), (2011).</li><li>Stieber, J., Wieland, K., St&#246;ckl, G., Ludwig, A., Hofmann, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bradycardic+and+proarrhythmic+properties+of+sinus+node+inhibitors.">Bradycardic and proarrhythmic properties of sinus node inhibitors.</a> Mol Pharmacol. 69 (4), 1328-1337 (2006).</li><li>Santoro, B., Wainger, B. J., Siegelbaum, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+HCN+channel+surface+expression+by+a+novel+C-terminal+protein-protein+interaction.">Regulation of HCN channel surface expression by a novel C-terminal protein-protein interaction.</a> J Neurosci. 24 (47), 10750-10762 (2004).</li><li>Lewis, A. S., 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=Deletion+of+the+hyperpolarization-activated+cyclic+nucleotide-gated+channel+auxiliary+subunit+TRIP8b+impairs+hippocampal+Ih+localization+and+function+and+promotes+antidepressant+behavior+in+mice.">Deletion of the hyperpolarization-activated cyclic nucleotide-gated channel auxiliary subunit TRIP8b impairs hippocampal Ih localization and function and promotes antidepressant behavior in mice.</a> J Neurosci. 31 (20), 7424-7440 (2011).</li><li>Heuermann, R. 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=Reduction+of+thalamic+and+cortical+Ih+by+deletion+of+TRIP8b+produces+a+mouse+model+of+human+absence+epilepsy.">Reduction of thalamic and cortical Ih by deletion of TRIP8b produces a mouse model of human absence epilepsy.</a> Neurobiol Dis. 85, 81-92 (2016).</li><li>Steru, L., Chermat, R., Thierry, B., Simon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tail+suspension+test:+A+new+method+for+screening+antidepressants+in+mice.">The tail suspension test: A new method for screening antidepressants in mice.</a> Psychopharmacology. 85 (3), 367-370 (1985).</li><li>Petit-Demouliere, B., Chenu, F., Bourin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forced+swimming+test+in+mice:+a+review+of+antidepressant+activity.">Forced swimming test in mice: a review of antidepressant activity.</a> Psychopharmacology. 177 (3), 245-255 (2004).</li><li>Cryan, J. F., Mombereau, C., Vassout, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tail+suspension+test+as+a+model+for+assessing+antidepressant+activity:+Review+of+pharmacological+and+genetic+studies+in+mice.">The tail suspension test as a model for assessing antidepressant activity: Review of pharmacological and genetic studies in mice.</a> Neurosci Biobehav Rev. 29 (4-5), 571-625 (2005).</li><li>Han, Y., 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=Trafficking+and+gating+of+hyperpolarization-activated+cyclic+nucleotide-gated+channels+are+regulated+by+interaction+with+tetratricopeptide+repeat-containing+Rab8b-interacting+protein+(TRIP8b)+and+cyclic+AMP+at+distinct+sites.">Trafficking and gating of hyperpolarization-activated cyclic nucleotide-gated channels are regulated by interaction with tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) and cyclic AMP at distinct sites.</a> J Biol Chem. 286 (23), 20823-20834 (2011).</li><li>Hu, L., Santoro, B., Saponaro, A., Liu, H., Moroni, A., Siegelbaum, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+the+auxiliary+subunit+TRIP8b+to+HCN+channels+shifts+the+mode+of+action+of+cAMP.">Binding of the auxiliary subunit TRIP8b to HCN channels shifts the mode of action of cAMP.</a> J Gen Physiol. 142 (6), 599-612 (2013).</li><li>Saponaro, A., 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=Structural+basis+for+the+mutual+antagonism+of+cAMP+and+TRIP8b+in+regulating+HCN+channel+function.">Structural basis for the mutual antagonism of cAMP and TRIP8b in regulating HCN channel function.</a> Proc Natl Acad Sci U S A. 111 (40), 14577-14582 (2014).</li><li>Bankston, J. R., Camp, S. S., DiMaio, F., Lewis, A. S., Chetkovich, D. M., Zagotta, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+stoichiometry+of+an+accessory+subunit+TRIP8b+interaction+with+hyperpolarization-activated+cyclic+nucleotide-gated+channels.">Structure and stoichiometry of an accessory subunit TRIP8b interaction with hyperpolarization-activated cyclic nucleotide-gated channels.</a> Proc Natl Acad Sci U S A. 109 (20), 7899-7904 (2012).</li><li>Gatto, G. J., Geisbrecht, B. V., Gould, S. J., Berg, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisomal+targeting+signal-1+recognition+by+the+TPR+domains+of+human+PEX5.">Peroxisomal targeting signal-1 recognition by the TPR domains of human PEX5.</a> Nat Struct Biol. 7 (12), 1091-1095 (2000).</li><li>Owicki, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+polarization+and+anisotropy+in+high+throughput+screening:+perspectives+and+primer.">Fluorescence polarization and anisotropy in high throughput screening: perspectives and primer.</a> J Biomol Screen. 5 (5), 297-306 (2000).</li><li>Smith, D. S., Eremin, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+polarization+immunoassays+and+related+methods+for+simple,+high-throughput+screening+of+small+molecules.">Fluorescence polarization immunoassays and related methods for simple, high-throughput screening of small molecules.</a> Anal Bioanal Chem. 391 (5), 1499-1507 (2008).</li><li>Roehrl, M. H. A., Wang, J. Y., Wagner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+framework+for+development+and+data+analysis+of+competitive+high-throughput+screens+for+small-molecule+inhibitors+of+protein-protein+interactions+by+fluorescence+polarization.">A general framework for development and data analysis of competitive high-throughput screens for small-molecule inhibitors of protein-protein interactions by fluorescence polarization.</a> Biochemistry. 43 (51), 16056-16066 (2004).</li><li>Arkin, M. R., Wells, J. A. <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+inhibitors+of+protein-protein+interactions:+progressing+towards+the+dream.">Small-molecule inhibitors of protein-protein interactions: progressing towards the dream.</a> Nat Rev Drug Discov. 3 (4), 301-317 (2004).</li><li>Zhang, J., Chung, T., Oldenburg, K. <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> J Biomol Screen. 4 (2), 67-73 (1999).</li><li>Eglen, R. 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=The+use+of+AlphaScreen+technology+in+HTS:+current+status.">The use of AlphaScreen technology in HTS: current status.</a> Curr Cheml Genomics. 1 (1), 2-10 (2008).</li><li>Lehninger, A. L., Nelson, D. L., Cox, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lehninger Principles of Biochemistry. , (2000).</li><li>Kim, C. S., Chang, P. Y., Johnston, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+dorsal+hippocampal+activity+by+knockdown+of+HCN1+channels+leads+to+anxiolytic-+and+antidepressant-like+behaviors.">Enhancement of dorsal hippocampal activity by knockdown of HCN1 channels leads to anxiolytic- and antidepressant-like behaviors.</a> Neuron. 75 (3), 503-516 (2012).</li><li>Wahl-Schott, C., Biel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HCN+channels:+structure,+cellular+regulation+and+physiological+function.">HCN channels: structure, cellular regulation and physiological function.</a> Cell Mol Life Sci. 66 (3), 470-494 (2009).</li><li>DeBerg, H. A., Bankston, J. R., Rosenbaum, J. C., Brzovic, P. S., Zagotta, W. N., Stoll, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Mechanism+for+the+Regulation+of+HCN+Ion+Channels+by+the+Accessory+Protein+TRIP8b.">Structural Mechanism for the Regulation of HCN Ion Channels by the Accessory Protein TRIP8b.</a> Structure. 23 (4), 734-744 (2015).</li><li>Weiss, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hill+equation+revisited:+uses+and+misuses.">The Hill equation revisited: uses and misuses.</a> FASEB J. 11 (11), 835-841 (1997).</li><li>Prinz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hill+coefficients,+dose-response+curves+and+allosteric+mechanisms.">Hill coefficients, dose-response curves and allosteric mechanisms.</a> J Chem Biol. 3 (1), 37-44 (2009).</li><li>Pollard, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Guide+to+Simple+and+Informative+Binding+Assays.">A Guide to Simple and Informative Binding Assays.</a> Mol Biol Cell. 21 (23), 4061-4067 (2010).</li><li>Rossi, A. M., Taylor, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+protein-ligand+interactions+by+fluorescence+polarization.">Analysis of protein-ligand interactions by fluorescence polarization.</a> Nat Protoc. 6 (3), (2011).</li><li>Ryan, A. J., Gray, N. M., Lowe, P. N., Chung, C. -. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Detergent+on+"Promiscuous"+Inhibitors.">Effect of Detergent on "Promiscuous" Inhibitors.</a> J Med Chem. 46 (16), 3448-3451 (2003).</li><li>McGovern, S. L., Caselli, E., Grigorieff, N., Shoichet, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+common+mechanism+underlying+promiscuous+inhibitors+from+virtual+and+high-throughput+screening.">A common mechanism underlying promiscuous inhibitors from virtual and high-throughput screening.</a> J Med Chem. 45 (8), 1712-1722 (2002).</li><li>Hertzberg, R. P., Pope, A. J. <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:+new+technology+for+the+21st+century.">High-throughput screening: new technology for the 21st century.</a> Curr Opin Chem Biol. 4 (4), 445-451 (2000).</li><li>Sundberg, S. A. <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+and+ultra-high-throughput+screening:+solution-and+cell-based+approaches.">High-throughput and ultra-high-throughput screening: solution-and cell-based approaches.</a> Curr Opin Biotechnol. 11 (1), (2000).</li></ol>

På grund af dens potentiale som et terapeutisk mål i MDD 24, har der været betydelig interesse for farmakologiske tilgange, som antagoniserer HCN kanal funktion i centralnervesystemet 4. Imidlertid har disse bestræbelser blevet bremset af den vigtige rolle, HCN-kanaler i hjertets pacemaking og risikoen for arytmi 25. Vi ræsonnerede, at forstyrre vekselvirkningen mellem HCN og dens hjerne specifik medhjælper underenhed, TRIP8b 8, kan være tilstrækkelig til at producere ...

Hyperpolarisationsaktiveret cykliske nukleotid-gatede (HCN) kanaler udtrykkes i hjertet og centralnervesystemet, hvor de spiller en vigtig rolle i reguleringen af membranen ophidselse 1. HCN kanaler har været impliceret i patogenesen af depression (MDD) 2, hvilket har ført flere grupper til at foreslå, at en begrænsning HCN-kanal funktion farmakologisk kan være effektiv som en ny behandling af MDD 3. Men direkte rettet mod HCN-kanaler ikke er levedygtig på grund af deres vigtige rolle i hjertets handling potentiale 4. Ivabradin, den eneste FDA-godkendte HCN-kanal antagonist, anvendes til behandling af hjertesvigt for at frembringe en bradycardiske virkning 5. Som sådan er der et behov for farmakologiske midler, der begrænser HCN kanalfunktion udelukkende i centralnervesystemet. Tetratricopeptide gentage-holdige Rab8b interagerende protein (TRIP8b) er en hjerne speciFIC medhjælper underenhed af HCN-kanaler, der styrer overfladeekspression og lokalisering af HCN-kanaler 6,7. Genetisk knockout af TRIP8b forårsager en reduktion af hjernens HCN kanaler 7 uden at påvirke HCN udtryk i hjertet 8. Interessant, TRIP8b knockout-mus bruger mindre tid immobil på tvungen svømme opgaven og hale suspension opgave 7, to almindeligt anvendte screeningstest for antidepressiv effekt 9-11. Disse resultater antyder, at i stedet for direkte målretning HCN-kanaler med et lille molekyle antagonist af HCN-kanal funktion, forstyrrer interaktionen mellem TRIP8b og HCN kan være tilstrækkelig til at frembringe antidepressiv-lignende opførsel. TRIP8b binder sig til HCN ved to forskellige bindingssteder. Den cykliske nukleotid bindingsdomæne (CNBD) af HCN interagerer med et konserveret domæne af TRIP8b placeret N-terminal til TPR domæner af TRIP8b 12,13. Selv om rester af CNBD, der er involveret i denneinteraktion er blevet kortlagt 14, har regionen TRIP8b som er involveret ikke blevet indsnævret over en-80-aminosyre fragment 13. En anden vekselvirkning opstår mellem tetratricopeptide gentagelse (TPR) domæner af TRIP8b og C-terminal tripeptid af HCN ( &#39;SNL&#39; i HCN1, HCN2, og HCN4, men &#39;ANM&#39; i HCN3) 3,12. Den nyligt løst krystalstruktur 15 i denne C hale interaktion afslørede betydelig strukturel lighed med interaktionen mellem peroxisomal import-receptoren, peroxin 5 (PEX5), og dens interagerende partnere, som indeholder type 1 peroxisomale målrettet sekvenser (PTS1) 16. Selvom begge interaktionssteder er nødvendige for HCN-kanal funktion, samspillet mellem de TPR-domænerne af TRIP8b og den C-terminale tripeptid HCN1 tjener som den dominerende bindingssted og regulerer HCN overfladeekspression. Derfor blev denne interaktion valgt som målrettende sted i denne undersøgelse.For resten af ​​manuskriptet, når der henvises til samspillet mellem TRIP8b og HCN, det er denne interaktion, der bliver henvist til. Denne interaktion rekapituleres af en yderst opløseligt fragment af TRIP8b svarende til sin konserverede C-terminale indeholdende TPR domæner er nødvendige for binding af C-terminale hale af HCN (resterne 241-602 af 1A-4 isoformer af TRIP8b) 3. For at udvikle en høj produktion skærmen for at identificere små molekyler, der kan forstyrre denne interaktion, en fluorescenspolarisering (FP) -baseret assay blev anvendt 17. Fluorescenspolarisering er baseret på excitation af en fluorofor-mærket ligand med polariseret lys og måle graden af polarisering af den udsendte fluorescens 18. I nærvær af en bindingspartner, er den roterende bevægelse af det fluorescerende ligand begrænset og polariseret lys udsendes 19. I mangel af en bindingspartner, than roterende bevægelse af liganden fører til emission af depolariserede lys. I vedlagte protokol, er en metode til rensning af N-terminal-mærket (6xHis) TRIP8b (241-602) ved hjælp af nikkel-Nitrilotrieddikesyre (Ni-NTA) perler præsenteres. En lignende protokol blev anvendt til at rense Glutathion-S-transferase (GST) tagged C terminal 40 aminosyrer af HCN1 (HCN1 C40), der anvendes i trin 7 i protokollen. For pladshensyn, blev en detaljeret beskrivelse af denne procedure udeladt. I trin 2 til 7 i protokollen, er en high throughput screening workflow præsenteret (se figur 1). Protein-protein interaktioner er en notorisk vanskeligt mål for high throughput screening og læsere rådes til at opsøge yderligere ressourcer om emnet 20. Trin 2 og 3 i proceduren karakterisere in vitro affiniteten af det oprensede TRIP8b (241-602) konstruere fora fluoresceinisothiocyanat (FITC) -mærket elleve aminosyrer peptid svarende til den C-terminale hale af HCN1 (HCN1 FITC). Baseret på krystalstrukturen af TRIP8b-HCN-kompleks 15, dette elleve aminosyrer segment er tilstrækkelig til at frembringe binding med TRIP8b (241-602). I trin 2 er Kd af interaktionen måles ved titrering TRIP8b (241-602) til en fast koncentration af HCN1 FITC. I trin 3 er en umærket version af HCN peptid anvendt i trin 2 titreret til en fast koncentration af både TRIP8b (241-602) og HCN1 FITC at undersøge, om den FITC-tag forstyrrer binding. Disse eksperimenter er afgørende for at vælge passende koncentrationer af TRIP8b (241-602) og HCN1 FITC anvendes i high throughput skærmen. Udgangspunktet for high throughput skærm er, at et lille molekyle stand til at forstyrre interaktionen mellem TRIP8b (241-602) og HCN1 FITC vil producere en decemberrease i polariseret lys. I trin 4 er Z faktor assayet beregnet 21 for en given koncentration af TRIP8b (241-602) og HCN1 FITC at sikre, at analysen er egnet til high throughput screening (trin 5). Trin 6 og 7 er validering analyser for at bekræfte, at de hits identificeret i den primære high throughput skærm handler ved at forstyrre interaktionen mellem TRIP8b (241-602) og HCN1 FITC snarere end gennem en uspecifik mekanisme. I trin 6, carboxytetramethylrhodamin (TAMRA) -mærket HCN1 peptid (HCN1 TAMRA) er anvendt i en ellers identisk fluorescens polarisering assay at filtrere fluorescerende forbindelser, der kompromitterer FP-analysen ved hjælp af FITC-tag. Trin 7 anvender et større HCN1 C-terminale fragment (HCN1 C40) og beskæftiger en perle-baseret nærhed assay, som er baseret på &quot;tunnelering&quot; af en singlet oxygen fra en donor bead til en acceptor bead bragt tæt på hinanden ved interagerende proteiner 22.

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	<tbody>
		<tr>
			<td>Ni-NTA agarose&#160;</td>
			<td>Qiagen</td>
			<td>30210</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis cassette&#160;</td>
			<td>ThermoFisher</td>
			<td>66456</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl b-D-1-thiogalactopyranoside&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>I5502-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>384 Well Black plate&#160;</td>
			<td>Corning</td>
			<td>3820</td>
			<td></td>
		</tr>
		<tr>
			<td>Proxiplate&#160;</td>
			<td>Perkin-Elmer&#160;</td>
			<td>6008289</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GST Acceptor beads&#160;</td>
			<td>Perkin-Elmer&#160;</td>
			<td>6760603C</td>
			<td></td>
		</tr>
		<tr>
			<td>NiChelate</td>
			<td>Perkin-Elmer&#160;</td>
			<td>AS101D</td>
			<td></td>
		</tr>
		<tr>
			<td>pGS21-a</td>
			<td>Genscript</td>
			<td>SD0121</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma-Aldrich</td>
			<td>10837091001</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Kit</td>
			<td>ThermoFisher</td>
			<td>23200</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein concentrator</td>
			<td>ThermoFisher</td>
			<td>88527</td>
			<td></td>
		</tr>
		<tr>
			<td>Perkin Elmer Enspire Multimode Plate reader</td>
			<td>Perkin-Elmer&#160;</td>
			<td>#2300-001M</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21 (DE3) Competent Cells</td>
			<td>Agilent</td>
			<td>200131</td>
			<td></td>
		</tr>
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</table>

method for identifying small molecule inhibitors of the protein-protein interaction between hcn1 and trip8b

Hyperpolarisationsaktiveret cykliske nukleotid-gatede (HCN) kanaler udtrykkes ubikvitært i hele hjernen, hvor de fungerer til at regulere ophidselse af neuroner. Den subcellulære fordeling af disse kanaler i pyramideformede neuroner i hippocampale område CA1 reguleres af tetratricopeptide gentagelser og Rab8b interagerende protein (TRIP8b), en ekstra underenhed. Genetisk knockout af HCN poredannende underenheder eller TRIP8b, både føre til en stigning i antidepressiv-lignende opførsel, hvilket antyder, at begrænse funktionen af ​​HCN-kanaler kan være nyttigt som en behandling for depression (MDD). Trods betydelig terapeutisk interesse, er HCN-kanaler også til udtryk i hjertet, hvor de regulerer rytme. For at omgå off-target spørgsmål i forbindelse med blokering kardiale HCN-kanaler, har vores laboratorium for nylig foreslået målretning af protein-protein-interaktion mellem HCN og TRIP8b for specifikt afbryde HCN-kanal funktion i hjernen.TRIP8b binder sig til HCN pore danner underenheder på to forskellige interaktion sites, selvom her er fokus på samspillet mellem tetratricopeptide gentagelse (TPR) domæner af TRIP8b og C-terminal hale på HCN1. I denne protokol, en udvidet beskrivelse af en fremgangsmåde til oprensning TRIP8b og udføre en høj produktion skærmen for at identificere småmolekylære inhibitorer af interaktionen mellem HCN og TRIP8b, beskrives. Fremgangsmåden til screening med højt gennemløb anvender en fluorescenspolarisering (FP) -baseret assay til overvågning af binding af et stort TRIP8b fragment til en fluorofor-mærket elleve aminosyre peptid svarende til HCN1 C-terminale hale. Denne metode giver &quot;hit&quot; forbindelser, der skal identificeres på grundlag af ændringen i polariseringen af ​​udsendt lys. Validation analyser udføres derefter for at sikre, at &quot;hit&quot; forbindelser er ikke kunstig.

For at undgå ophavsretlige spørgsmål med vores tidligere publikation blev en TAMRA-mærket probe HCN1 TAMRA bruges til at generere figur 2 og 3. Bemærk, at denne substitution ikke gøre en mærkbar forskel i resultaterne, og protokollerne er identiske med dem beskrevet ovenfor med HCN1 FITC. At evaluere samspillet med HCN1 TAMRA, TRIP8b (241-602) blev titreret til en fast koncentration af HCN1 TAMRA anvende...

Watch this Scientific Journal Video about Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b at JoVE.com

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

Samspillet mellem HCN kanaler og deres ekstra subunit er blevet identificeret som en terapeutisk mål i depression. Her, en fluorescenspolarisering-baseret fremgangsmåde til identifikation af småmolekylære inhibitorer af dette protein-protein-interaktion, er præsenteret.

Fremgangsmåde til at identificere småmolekylære inhibitorer af protein-protein-vekselvirkning mellem HCN1 og TRIP8b

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

Research

JoVE Journal

Biochemistry

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Brug Stillads Liposomer at Rekonstituer Lipid-proksimal protein-protein interaktioner<em&gt; In vitro</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Visualisering af protein-protein interaktion i nukleare og Cytoplasmiske Brøker af Co-immunopræcipitation og<em&gt; In Situ</em&gt; Proximity ligeringsassayet

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay

Indfange interaktion kinetik af en Ion-kanal Protein med små molekyler af Bio-lag interferometri Assay

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe ...

Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction

Titrering ELISA som en metode til at bestemme dissociationskonstant Receptor Ligand interaktion

The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

Kovalente immobilisering af proteiner for enkelt molekyle kraft spektroskopi

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Proteom-wide kvantificering af mærkning homogenitet på enkelt-molekyle niveau

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

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

En semi-High-gennemløb tilpasning af NADH-koblede ATPase assay til screening små molekyle hæmmere

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

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Native Cell Membrane Nanopartikler System for Membran Protein-Protein Interaktion Analyse

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay (DRaCALA)

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay DRaCALA

Identifikation af bindingsproteiner fra små ligands med Ligand Assays differentierede radiale kapillærhandling (DRaCALA)

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins ...

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 ...