Y.N.T. would like to acknowledge the Agency for Science, Technology and Research (A*STAR), Singapore for the financial support under the JCO CDA grant 13302FG063.

Achtung: Bitte beachten Sie die Sicherheitsdatenblätter (SDS) der beteiligten Chemikalien vor Gebrauch. Das Wirkstoff-Screening-Experiment beinhaltet die Synthese und Umgang mit Nanomaterialien, die im Vergleich zu ihren Groß Pendant zusätzlichen Gefahren verursachen. Stellen Sie sicher, alle erforderlichen Kontrollmaßnahmen, um während des Experiments durchgeführt werden, einschließlich der Verwendung von technischen Massnahmen verwendet (Abzug) und persönliche Schutzausrüstung (PSA, beispielsweise Sicherheitslänge Hosen, geschlossene Schuhe, chemikalienbeständige Handschuhe und Schutzbrille). 1. Herstellung von chemischen Reagenzien für die Drug Screening <ol><li> Vorprodukte für Au-NCs Synthese <ol><li> Auflösen 30 mg Gold (III) chlorid-Lösung (99,99% Spurenmetalle Basis, 30 Gew.% In verdünnter HCl) in 6,9 ml Reinstwasser zur Vorbereitung 15 mM von Gold (III) -chlorid-Lösung. Achtung: Goldchloridlösung ist ätzend und reizend. Tragen Sie angemessene PPE, um den direkten Kontakt mit Augen und der Haut vermeiden. </li><li> Dissolve 74 mg HSA oder BSA in 1 ml hochreinem Wasser bis 74 mg / ml Protein-Stammlösung vorzubereiten. </li></ol></li><li> Arzneimittellösungen <ol><li> Lösen die gewünschte Menge des Arzneimittels, zum Beispiel 1,9 mg für (a) Ibuprofen, 2,8 mg (b) Warfarin, 2,3 mg (c) Phenytoin und 1,5 mg für (d) Sulfanilamid werden in 20 ul DMSO zu 450 mm herzustellen der Arzneimittelstammlösungen. Anmerkung: DMSO als Lösungsmittel gewählt, da diese Arzneimittel sind hydrophob und haben eine schlechte Löslichkeit in Wasser. </li></ol></li><li> Andere Reagenzien <ol><li> Man löst 600 mg NaOH-Plätzchen in 10 ml Reinstwasser herzustellen 1,5 M NaOH-Lösung. Man löst 2,4 g Harnstoff in 2 ml hochreinem Wasser zur Vorbereitung 20 M Harnstofflösung. </li></ol></li></ol> 2. Synthese von Protein-gestützte Au NCs <ol><li> HSA-gestützten Au NCs (HSA-Au-NCs) <ol><li> Legen Sie zwei Glasfläschchen, die jeweils eine Mikro Magnetrührstab in ihm, auf zwei getrennten Temperatur steuerbaren Magnetrührstabsteller. </li><li> Stellen Sie die Temperatur von einem Magnetrührer bis 60 ° C, und beschriften Sie die Glasfläschchen auf es als &quot;60 ° C&quot;; während die andere Magnetrührer bei Raumtemperatur (RT), wobei die Glasfläschchen auf es wird als &quot;RT&quot; bezeichnet gehalten. </li><li> Mit 200 ul HSA-Lösung wurden 200 ul ultrareinem Wasser, und 200 ul der Gold (III) -chlorid-Lösung in jedes Röhrchen unter konstantem Rühren (als 360 UpM, wenn nicht anders angegeben), um die Einkapselung des Au-Ionen in der Proteintemplats ermöglichen. </li><li> 2 Minuten später werden 20 ul NaOH-Lösung in jedes Röhrchen, um so die Reduktionsfähigkeit von HSA bei der Bildung Au NCs aktivieren und um die Reaktionszeit 0 min aufzeichnen. </li><li> Alle 20 Minuten, ziehen 50 ul-Lösungen von jedem der Probe auf eine 384-Well-schwarze Platte und Messung der Emissionsspektrum mit einem Mikroplatten-Reader. Die typische Scan-Setup: λ ex = 370 nm, λ em = 410 bis 850 nm. </li><li> Stoppen Sie den Magnetrührer nach 100 min. </li><li> Cool down der Glasfläschchen unter fließendem Wasser in ein Waschbecken. Achtung: Die Glasflaschen sind heiß. Tragen Sie hitzebeständige Handschuhe, um Verbrennungen zu vermeiden. </li><li> Zeichnen Sie die Photoemissionsspektren zu jeder Zeit für jede Probe, um die Bildung Kinetik der Au NCs an verschiedenen Temperaturbedingungen zu erwerben. </li></ol></li><li> BSA-gestützten Au NCs (BSA-Au-NCs) <ol><li> Legen Sie ein Glasfläschchen, die eine Mikro Magnetrührstab auf der Oberseite der einen Magnetrührer. Lassen Sie die Temperatur als Raumtemperatur. </li><li> Mischungs 200 ul BSA-Lösung, 200 &amp; mgr; l Harnstoff und 200 &amp; mgr; l von Gold (III) -chlorid-Lösung in dem Glasfläschchen unter konstantem Rühren. </li><li> 2 Minuten später, fügen Sie 20 ul NaOH-Lösung, um die Verringerung der Fähigkeit der BSA zu aktivieren, um Au-NCs bilden, und starten Sie die Reaktionszeit als 0 min aufzuzeichnen. Messen die Photoemissionsspektrum der Reaktionsmischung pro Stunde. Hinweis: Die Photoemissionsspektrum der BSA-Au-Nanokristallen wird gemessenweniger häufig, weil die Bildungsgeschwindigkeit von BSA-Au NCs ist langsamer als die des HSA-Au auf der gleichen Temperatur durch geringere Wirksamkeit von Harnstoff, um das Protein zu entfalten. </li><li> Stoppen Sie den Magnetrührer nach 7 Std. Zeichnen Sie die Photoemissionsspektren zu jeder Zeit, um die Bildung Kinetik der Au NCs durch Harnstoff Denaturierung zu sehen. </li></ol></li></ol> 3. Kleine Molecular Drug Screening <ol><li> Screen relative Bindungsaffinität von verschiedenen kleinen molekularen Drogen, um HSA <ol><li> Platzieren fünf Glasampullen, die jeweils einen magnetischen Rührstab in ihr, auf einer Temperatur steuerbares Mehr Magnetrührer. Beschriften Sie diese Ampullen als, b, c und jeweils für jedes Medikament, nämlich Ibuprofen, Warfarin, Phenytoin und Sulfanilamid d. Beschriften Sie die man mit reinem HSA als Kontrolle. </li><li> Fügen Sie 200 ul von HSA in jedes Fläschchen. 1 &amp; mgr; l Arzneimittellösung auf die vier entsprechenden Durchstechflaschen. 1 &amp; mgr; l DMSO zu der Kontrolle. Schalten Sie das Rührgerät stellen und das Schleuderdrehzahl auf 360min und Inkubation für 1 Stunde, damit das Medikament Bindung an HSA in Anspruch. </li><li> 1 h später mit 200 &amp; mgr; l Milli-Q-Wasser und 200 ul der Gold (III) -chlorid-Lösung in jedes Röhrchen unter konstantem Rühren. Die Temperatur auf 60 ° C unter konstantem Rühren für 10 min. Mit 20 &amp; mgr; l von 1,5 M NaOH zu jedem Gefäß und beginnt mit der Reaktionszeit 0 min aufzeichnen. </li><li> Ziehen schnell 50 ul Lösung aus jedem Fläschchen zu einer 384-Well-schwarze Platte und Messung der Emissionsspektrum (Ergebnisse in 0 min beschriftet). Aufzeichnung der Emissionsspektren von jeder Probe alle 10 min. Sammeln mindestens vier Spektren, die bei vier verschiedenen Zeit, das heißt 0, 10, 20 und 30 min. </li><li> Wiederholen Sie die Schritte 3.1.1 - 3.1.4 mehrmals, um Ergebnisse mit einer konsistenten Trend zu erhalten. Stoppen Sie den Magnetrührer. </li><li> Zeichnen Sie die zeitaufgelöste Photoemissionsspektren für jede Probe (A - D und Kontrolle). Identifizieren Sie die Spitzenintensität aller Proben und Verschwörung gegen die Zeit, um die Bildung Kinetik der Au-NCs in di vergleichenfferent wirkstoffbeladenen Protein-Vorlagen. </li></ol></li><li> Messung der Bindungskonstante eines bestimmten Arzneimittels an HSA <ol><li> Wiederholen Sie die Schritte 3.1.1 - 3.1.4. Ersetzen Sie die Medikamentenlösung mit Ibuprofen Lösungen bei vier verschiedenen Konzentrationen (einschließlich einer Kontrollprobe mit HSA nur ohne Wirkstoffbeladung). Beschriften Sie die Glasfläschchen nach der Medikamentenkonzentration entsprechend. </li><li> 10 Minuten später abkühlen die Glasfläschchen unter fließendem Wasser in ein Waschbecken. Achtung: Die Glasflaschen sind heiß. Tragen Sie hitzebeständige Handschuhe, um Verbrennungen zu vermeiden. </li><li> Zeichnen Sie 50 ul der oben genannten Lösungen aus jedem Fläschchen zu einer 384-Well-schwarze Platte und Messung der Emissionsspektrum. </li><li> Wiederholen Sie die Schritte 3.2.1 - 3.2.3 zweimal zwei weitere Sätze von Ergebnissen bei unterschiedlichen Wirkstoffkonzentrationen zu erhalten. </li><li> Stoppen Sie den Magnetrührer. Zeichnen Sie die Photoemissionsspektren und Analyse der Ergebnisse. <ol><li> Für die in jeder einzelnen Charge erhaltenen Rohdaten, zeichnen Sie die Photoemissionsspektren von jedemProbe in Software wie Origin-Software und finden Sie heraus die Spitzenintensität. </li><li> Berechnung und Darstellung der relativen Fluoreszenzintensität [(I 0 - I) / I 0] gegen die Arzneimittelkonzentration, wobei I und I 0 auf die Fluoreszenzintensität von Au NCs in wirkstoffbeladenen HSA und reine HSA gebildet, hinzuweisen. </li><li> Berechnung der Bindungskonstante durch Anpassen der Daten an einer einzigen Stelle Bindungsmodell unter Verwendung der Michaelis-Menten-Gleichung: Y = R max × C / (K D + C), wobei R max die maximale Antwortsignal angibt, C ist die Konzentration des Liganden und K D für die Bindungskonstante. Führen Sie diese in der Software wie Origin. Wählen Sie das Menü Analyse Montage Nichtlinearer Fit, dann wählen Sie Hill Funktion aus Kategorie Growth / Sigmoidal. Auf der Einstellungen: Auswahlseite Funktion, klicken Sie auf Fit zu zeigen, bis die Einbauergebnisse. </li></ol></li></ol></li></ol>

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Am. Chem. Soc. 135, 4978-4981 (2013).</li><li>Negishi, 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=Controlled+Loading+of+Small+Aun+Clusters+(n+=+10&#8211;39)+onto+BaLa4Ti4O15+Photocatalysts:+Toward+an+Understanding+of+Size+Effect+of+Co-Catalyst+on+Water+Splitting+Photocatalytic+Activity.">Controlled Loading of Small Aun Clusters (n = 10&#8211;39) onto BaLa4Ti4O15 Photocatalysts: Toward an Understanding of Size Effect of Co-Catalyst on Water Splitting Photocatalytic Activity.</a> J. Phys. Chem. C. , (2015).</li><li>Xie, J., Zheng, Y., Ying, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-Directed+Synthesis+of+Highly+Fluorescent+Gold+Nanoclusters.">Protein-Directed Synthesis of Highly Fluorescent Gold Nanoclusters.</a> J. Am. Chem. Soc. 131, 888-889 (2009).</li><li>Celej, M. S., Montich, G. G., Fidelio, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Stability+Induced+by+Ligand+Binding+Correlates+with+Changes+in+Protein+Flexibility.">Protein Stability Induced by Ligand Binding Correlates with Changes in Protein Flexibility.</a> Protein Sci. 12, 1496-1506 (2003).</li><li>Layton, C. J., Hellinga, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamic+Analysis+of+Ligand-Induced+Changes+in+Protein+Thermal+Unfolding+Applied+to+High-Throughput+Determination+of+Ligand+Affinities+with+Extrinsic+Fluorescent+Dyes.">Thermodynamic Analysis of Ligand-Induced Changes in Protein Thermal Unfolding Applied to High-Throughput Determination of Ligand Affinities with Extrinsic Fluorescent Dyes.</a> Biochemistr. 49, 10831-10841 (2010).</li><li>Yu, Y., New, S. Y., Xie, J., Su, X., Tan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-Based+Fluorescent+Metal+Nanoclusters+for+Small+Molecular+Drug+Screening.">Protein-Based Fluorescent Metal Nanoclusters for Small Molecular Drug Screening.</a> Chem. Commun. 50, 13805-13808 (2014).</li><li>Shortridge, M. 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> Nuclear Magnetic Resonance Affinity Screening Methods for Functional Annotation of Proteins and Drug Discover. , (2010).</li></ol>

The authors have nothing to disclose.

Es gibt mehrere wichtige Schritte, die in diesem Verfahren markiert werden müssen. In das Protokoll der Screening der relativen Bindungsaffinität von verschiedenen kleinen molekularen Drogen, Schritte 3.1.2, 3.1.3 und 3.1.4 sind entscheidend für gute Ergebnisse zeigt einheitlichen Trend für die relative Bindungsstärke zu erhalten. In diesen Schritten, sollten die Maßnahmen der Zugabe von Chemikalien und Darstellung von Reaktionslösungen für die Messung, so schnell wie möglich sein, um die Zeitverzögerung Wirku...

Serum Albumine, wie Humanserumalbumin (HSA) und Rinderserumalbumin (BSA) sind das häufigste Protein im Plasma und spielen eine entscheidende Rolle bei der Aufrechterhaltung der osmotische Druck der Blutkammer. Sie werden auch als Trägerproteine ​​für kleine Moleküle mit geringer Wasserlöslichkeit, wie Steroiden, Fettsäuren, Schilddrüsenhormone, und eine Vielzahl von Medikamenten erkannt. Die Bindungseigenschaft (beispielsweise Bindungsstellen, Bindungsaffinität oder die Stärke) dieser Moleküle an Albumine im Serum bildet ein wichtiges Thema in der Pharmakokinetik. 1-4 mehrere Analyseverfahren entwickelt worden, um die Bindungseigenschaften von verschiedenen Medikamenten zu untersuchen Albumine im Serum, beispielsweise Röntgenkristallographie, 5,6 kernmagnetische Resonanz (NMR), 7-11 und Oberflächenplasmonresonanz (SPR), 12,13 usw. Jedoch sind diese Verfahren entweder durch eine mühsame und zeitraubende Analyseprozess ist (Zwangs zB Wachstum von Einkristall-Röntgen kristalloGrafik-Studie), Erfordernis spezialisierte und teure Ausrüstung (SPR), oder in der Notwendigkeit von teuren Isotopenmarkierung (NMR) für die Erkennung. Es ist daher höchst wünschenswert, alternative Möglichkeiten zur kleinen molekularen Wirkstoff-Screening auf eine schnelle, unkomplizierte und kostengünstige Art und Weise zu entwickeln. Gold-Nanocluster (Au ​​NCs) sind eine spezielle Art von Nanomaterialien, die mehrere Zehnmetallatome mit einer Größe von weniger als 2 nm. 14-17 Sie haben umfangreiche Forschungsinteressen zogen enthalten aufgrund ihrer diskreten und größenabhängige elektronische Struktur, 18, 19 und molekularartigen Absorptionen und Emissionen. 20-23 Solche einzigartigen Materialeigenschaften, insbesondere die starke Fluoreszenz, haben diverse Anwendungen wie Sensorik und Bildgebung in biologischen Systemen. 24-32 Ultrasmall Fluoreszenz Au NCs können mit Hilfe der funktionellen Proteine ​​synthetisiert werden gefunden, wie Serumalbumine, als Vorlage. 33 In einem typischen Protein-gestützten Synthese<html> Au NCs, eine bestimmte Menge an Au-Salze werden zunächst innerhalb der Protein eingekapselt und anschließend durch das Protein selbst reduziert. Die reduzierende Fähigkeit des Proteins zugeschrieben wird, um funktionelle Aminosäurereste (zB Tyrosin), die durch eine Erhöhung des pH-Lösung alkalisch aktivierbaren Bestandteil enthält. Entfaltung der Proteinstruktur wird als ein kritischer Schritt für die Bildung von Au NCs betrachtet. Dies liegt daran, dass in einem ungefalteten Protein kann mehrere Reduktions funktionellen Gruppen zu den eingekapselten Au-Salzen ausgesetzt werden. Proteinentfaltung kann durch Wärmebehandlung oder durch Einwirkung von Denaturierungsmitteln erfolgen. Einführung von kleinen Molekül Medikamente können auch auf den Entfaltungsvorgang, dh Änderung der Mittelpunkt Denaturierungstemperatur und die Enthalpie der Entfaltung. 34,35 die Wirkung aller dieser Faktoren kann wiederum durch die Bildungskinetiken fluoreszierender Au NCs reflektiert und manifestieren die Fluoreszenzintensität der resultierenden Au NCs. 36 e_content &quot;&gt; Das Video zeigt das Verfahren zum Wirkstoff-Screening durch Synthetisieren Au NCs mit Wirkstoff beladenen Albuminproteine ​​bei einer höheren Temperatur (60 ° C) oder in Gegenwart denaturierender Agenzien (zB Harnstoff). Die Fluoreszenzintensität der resultierenden Au NCs ist die Signalauslese. Zuerst Au NCs werden HSA und BSA-Vorlagen, auf 60 ° C oder in Gegenwart von Harnstoff behandelt wird synthetisiert, um zu zeigen, wie Proteinentfaltung (durch Wärmebehandlung oder Denaturierungsmitteln induziert) wirkt sich auf die Bildung Kinetik Au NCs. Zweitens Au NCs in Protein-Vorlagen mit unterschiedlichen Medikamenten vorbelastet synthetisiert und die Wirkstoffbeladung auf die relative Fluoreszenzintensitäten von resultierenden Au NCs sucht wird, das die Messung der relativen Bindungsstärke bereitzustellen. Schließlich wird der Au-NC-Wirkstoff-Screening-Protokoll für die modifizierte quantitative Messung der Wirkstoff-Protein-Bindungskonstante (K D) durch Variieren der Arzneistoffgehalt in dem Protein von einer festen Konzentration vorgespannt.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Gold (III) chloride solution, 30%</td>
			<td>Sigma-Aldrich</td>
			<td>484385</td>
			<td>Corrosive, irritant</td>
		</tr>
		<tr>
			<td>Human serum albumin, 96%</td>
			<td>Sigma-Aldrich</td>
			<td>A1887</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum albumin, 96%</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Ibuprofen, 98%</td>
			<td>Sigma-Aldrich</td>
			<td>I4883&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>warfarin, 98%</td>
			<td>Sigma-Aldrich</td>
			<td>A2250</td>
			<td></td>
		</tr>
		<tr>
			<td>phenytoin</td>
			<td>Sigma-Aldrich</td>
			<td>PHR1139</td>
			<td></td>
		</tr>
		<tr>
			<td>sulphanilamide, 99%</td>
			<td>Sigma-Aldrich</td>
			<td>S9251</td>
			<td></td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>urea</td>
			<td>Sigma-Aldrich</td>
			<td>U5128</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>221465</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stirrer</td>
			<td>IKA</td>
			<td>RT5</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Tecan</td>
			<td>Infinite M200</td>
			<td></td>
		</tr>
		<tr>
			<td>384-well plate</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL air displacement pipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1000 mL air displacement pipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL air displacement pipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5000 mL Eppendorf tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1000 mL Eppendorf tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL Eppendorf tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL micro tube</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL glass vial with screw cap</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 mL glass vial with screw cap</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a rapid and quantitative fluorimetric method for protein-targeting small molecule drug screening

Zeigen wir eine neue Wirkstoff-Screening-Verfahren zur Bestimmung der Bindungsaffinität von kleinen Arzneimittelmolekülen an ein Zielprotein durch Bilden Fluoreszenzgoldcluster (Au NCs) innerhalb der mit Wirkstoff beladenen Proteins, basierend auf den Differenzfluoreszenzsignal von der Au NCs emittiert. Albuminproteine ​​wie humanes Serumalbumin (HSA) und Rinderserumalbumin (BSA) als die Modellproteinen ausgewählt ist. Vier kleinen Molekular Medikamente (zum Beispiel Ibuprofen, Warfarin, Phenytoin und Sulfanilamid) unterschiedlicher Bindungsaffinitäten zur Albumin-Proteine ​​getestet. Es wurde gefunden, daß die Bildungsrate von fluoreszierenden Au NCs Inneren Wirkstoff beladen Albuminprotein unter denaturierenden Bedingungen (dh 60 ° C oder in Gegenwart von Harnstoff) langsamer ist als die in dem ursprünglichen Protein (ohne Arzneimittel) gebildet. Darüber hinaus ist die Fluoreszenzintensität der so gebildeten Nanokristalle festgestellt wird, umgekehrt an die Bindungsaffinitäten dieser Medikamente auf die Albuminproteine ​​korreliert werden. Insbesondereje höher die Drogen-Protein-Bindungsaffinität, desto langsamer die Rate der Au NCs-Bildung und damit eine niedrigere Fluoreszenzintensität der erhaltenen Au NCs beobachtet. Die Fluoreszenzintensität der erhaltenen Au NCs stellt daher ein einfaches Maß der relativen Bindungsstärke von verschiedenen Medikamenten getestet. Dieses Verfahren ist auch erweiterbar auf die spezifischen Drogen-Protein-Bindungskonstante (K D), die durch einfache Variation der Wirkstoffgehalt im Protein an einer festen Proteinkonzentration vorbelastet messen. Die gemessenen Ergebnisse entsprechen auch die Verwendung anderer Prestige, sondern komplizierter Verfahren erhaltenen Werte.

Proteinentfaltung ist ein wichtiges Verfahren für die Bildung von Protein-gestützten Au NCs, da mehr reaktive funktionelle Gruppen (zB Tyrosin) eines Proteins, um die eingekapselte Au Ionen zu reduzieren und damit Beschleunigung der Bildungsgeschwindigkeit von Au NCs ausgesetzt werden. Heiz- und externen Denaturierungsmitteln gibt zwei übliche Mittel, um das Protein Entfaltungsvorgangs zu fördern. Figur 1 zeigt die Wirkung von Erhitzen und Hinzufügen externer Denaturierungsmitteln auf die ...

Watch this Scientific Journal Video about A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening at JoVE.com

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

Eine schnelle und quantitative fluorimetrische Verfahren zur Protein-Targeting kleines Molekül als Wirkstoff-Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

a-rapid-quantitative-fluorimetric-method-for-protein-targeting-small

Research

JoVE Journal

Bioengineering

9.9K Aufrufe.  Agency for Science, Technology and Research (A*STAR). A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

Serie: A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

Verbesserte Visualisierung und quantitative Analyse von Arzneimittelwirkungen mit mikrostrukturierten Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

Forschung

Biotechnik

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

Candida albicans Biofilm Chip (Ca BChip) für das Hochdurchsatz-Screening-Antimykotikum

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

Drug-induzierte Sensibilisierung der Adenylatzyklase: Assay Straffung und Miniaturisierung für Small Molecule-und siRNA-Screening-Anwendungen

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 &#176;C for 2 h in order to investigate the products&#39; high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to &#946;-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry.

Rapid-Mix Herstellung von Bioinspirierte Nanoskalige Hydroxyapatit für biomedizinische Anwendungen

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

Automatische Kontraktions Analyse menschlicher Engineered Herzgewebe für Herz Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

3D Microtissues für injizierbare Regenerative Therapie und Hochdurchsatz-Drogen-Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Gentranskription durch Umleiten zellulären Maschinerie mit chemischen epigenetische Modifikatoren zu unterdrücken

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Reinigung und Analyse eines monoklonalen Antikörpers aus chinesischen Hamster-Ovarialzellen mit einem automatisierten Mikrobioreaktorsystem

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Applications for Open Source Microplate-Compatible Illumination Panels

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well as large-scale high-throughput screening (HTS) campaigns. Though laboratory automation has greatly increased the utility of microplates there remain instances where automation-based instrumentation is not feasible, cost-effective or compatible with microplate formatting needs. In these cases, microplates must be manually prepared. Problematic to manual microplate manipulations is that a number of difficulties can arise pertaining to the accurate tracking of sample operations, data record keeping and quality control (QC) inspection for well artifacts or formatting errors. As microplate well densities increase (i.e., 96-well, 384-well, 1536-well) the potential for introducing errors also drastically increases.&#160; Moreover, for small bench-top laboratory operations there exists a need to improve the ease and accuracy of sample handling in a cost-effective fashion. Herein, we describe a system that acts as a semi-automated pipetting guide referred to as the Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.). &#160;M.A.P.L.E. has multiple uses for supporting compound hit-picking and microplate preparation for assay development in high-throughput screening or laboratory benchtop operations, as well as QC/quality assurance (QA) diagnostic evaluation of microplate quality or visualizing well formatting errors.

Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.) is a computer-driven device that systematically illuminates microtiter wells to provide guidance for the manual preparation of microplates.&#160; M.A.P.L.E. improves the accuracy of microplate preparation while automating data recordkeeping. &#160;In addition, it can assist with examining microplate quality or aid in the detection of errors.&#160; &#160;

Anwendungen für Open Source Microplate-Compatible Illumination Panels

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...