generation-high-throughput-three-dimensional-tumor-spheroids-for-drug

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

Novartis Institutes for BioMedical Research

Compound sample preparation and delivery are the most critical steps in high-throughput screening (HTS) campaigns. Historically, several methods of compound delivery to assays have been used for HTS, including intermediate plates with prediluted compounds, assay-ready plates (ARPs) using either preplated dried compound films or nanoliter DMSO spots of compounds, as well as pin tool-delivered compounds. We and others have observed differences in apparent compound potency depending on the compound delivery method. To quantitatively measure compound potency differences due to the chosen delivery methods, we conducted a controlled study using a validated biochemical luciferase assay and compared potencies when compounds were delivered in either ARPs (using acoustic dispensed nanoliter spots) or by pin tool. Here we compare hit rates, confirmation rates, false-positive rates, and false-negative rates between the two delivery methods using the luciferase assay. We compared polystyrene (PS) and cyclic olefin copolymer (COC) plates using both delivery methods and examined whether ARPs stored at 4 °C were superior to those stored frozen at -20 °C. The data show that the choice of compound delivery method to the assay has an effect on the apparent IC(50)'s and that pin tool delivery results in more confirmed hits than preplated compounds, resulting in a lower false-negative rate. However, this effect is minimized through the use of COC plates and by obtaining plates in a "just-in-time" mode. Overall, this report provides guidance on using assay-ready compound plates and has affected the way HTS campaigns are using acoustically dispensed plates in our department.

Comparison of compound administration methods in biochemical assays: effects on apparent compound potency using either assay-ready compound plates or pin tool-delivered compounds.

&#x78F7;&#x8102;&#x9170;&#x808C;&#x9187;&#x6FC0;&#x9176;&#x8C03;&#x8282;&#x591A;&#x79CD;&#x7EC6;&#x80DE;&#x529F;&#x80FD;&#x4E0E;&#x7CD6;&#x5C3F;&#x75C5;&#x548C;&#x764C;&#x75C7;&#x7B49;&#x75BE;&#x75C5;&#x7684;&#x6CBB;&#x7597;&#x53D1;&#x5C55;&#x7684;&#x91CD;&#x8981;&#x76EE;&#x6807;&#x3002;&#x8102;&#x8D28;&#x57FA;&#x7247;&#x5236;&#x5907;&#x7684;&#x9C81;&#x68D2;&#x548C; miniaturizable &#x8102;&#x8D28;&#x6FC0;&#x9176;&#x6D4B;&#x5B9A;&#x7684;&#x53D1;&#x5C55;&#x81F3;&#x5173;&#x91CD;&#x8981;&#x3002;&#x78F7;&#x8102;&#x9170;&#x808C;&#x9187;&#x6FC0;&#x9176;&#x9176;&#x68C0;&#x6D4B;&#x65B9;&#x6CD5;&#x7ECF;&#x5E38;&#x4F7F;&#x7528;&#x8102;&#x8D28;&#x57FA;&#x677F;&#x4ECE;&#x51BB;&#x7684;&#x8102;&#x7B79;&#x5907;&#x7F16;&#x5199;&#x7684;&#x8D85;&#x58F0;&#x6CE2;&#xFF0C;&#x8102;&#x8D28;&#x4F53;&#x5927;&#x5C0F;&#x7684;&#x53D8;&#x5316;&#x5BFC;&#x81F4;&#x5236;&#x5907;&#x7684;&#x5236;&#x5907;&#x3002;&#x672C;&#x6587;&#x6240;&#x8FF0;&#xFF0C;&#x6211;&#x4EEC;&#x62A5;&#x544A;&#x5747;&#x8D28; 1536&#x5E74;&#x4E95;&#x8367;&#x5149;&#x7D20;&#x9176;&#x8026;&#x5408;&#x751F;&#x7269;&#x53D1;&#x5149;&#x6D4B;&#x5B9A;&#x4E3A; PI5P4K&alpha;&#x3002;&#x57FA;&#x677F;&#x5236;&#x5907;&#x662F;&#x5C0F;&#x8BF4;&#xFF0C;&#x5E76;&#x5141;&#x8BB8;&#x5FEB;&#x901F;&#x751F;&#x4EA7;&#x7684;&#x4E8C;&#x7532;&#x57FA;&#x4E9A;&#x781C;&#x542B;&#x57FA;&#x677F;&#x89E3;&#x51B3;&#x65B9;&#x6848;&#x65E0;&#x9700;&#x5197;&#x957F;&#x7684;&#x8102;&#x8D28;&#x4F53;&#x5236;&#x5907;&#x7684;&#x534F;&#x8BAE;&#xFF0C;&#x4ECE;&#x800C;&#x4F7F;&#x8FD9;&#x79CD;&#x4F20;&#x7EDF;&#x4E0A;&#x5F88;&#x96BE;&#x6D4B;&#x5B9A;&#x7684;&#x7C7B;&#x578B;&#x7684;&#x653E;&#x5927;&#x3002;Z&#39;-&#x56E0;&#x5B50;&#x7684;&#x503C;&#x662F;&#x5927;&#x4E8E; 0.7 PI5P4K&alpha; &#x6D4B;&#x5B9A;&#xFF0C;&#x8868;&#x660E;&#x5176;&#x662F;&#x5426;&#x9002;&#x5408;&#x9AD8;&#x901A;&#x91CF;&#x7B5B;&#x9009;&#x7684;&#x5E94;&#x7528;&#x7A0B;&#x5E8F;&#x3002;&#x901A;&#x8FC7;&#x8BC4;&#x4F30;&#x7684; PI5P &#xFF1B; &gamma;-(32) P-ATP &#x79FB;&#x4EA4;&#x78F7;&#x9178;&#x5316;&#x7A0B;&#x5EA6;&#x4F5C;&#x4E3A;&#x6291;&#x5236;&#x5242; PI5P4K&alpha; &#x786E;&#x5B9A;&#x4E86; Tyrphostin AG 82&#x5176;&#x5BF9; PI5P4K&alpha; &#x7684;&#x6291;&#x5236;&#x6D3B;&#x6027;&#x8BC1;&#x5B9E;&#x672C;&#x5C0F;&#x578B;&#x5316;&#x6D4B;&#x5B9A;&#x3002;&#x4ECE;&#x8BD5;&#x70B9;&#x7684;&#x751F;&#x7269;&#x6D3B;&#x6027;&#x5316;&#x5408;&#x7269;&#xFF0C;&#x53E6;&#x4E00;&#x4E2A; tyrphostin&#xFF0C;-&#x9752;&#x6885; tyrphostin AG 538 &#x5E93; &#xFF08;&#x6211;-&#x9752;&#x6885;-&#x94F6;-538&#xFF09;&#xFF0C;&#x5C4F;&#x5E55;&#x88AB;&#x786E;&#x5B9A;&#x4E3A; ATP &#x7ADE;&#x4E89;&#x6027;&#x6291;&#x5236;&#x5242; PI5P4K&alpha; &#x4E0E; IC(50) &#x7684; 1 &micro; M&#xFF0C;&#x7533;&#x660E;&#x6291;&#x5236;&#x5242;&#x53D1;&#x73B0;&#x8FD0;&#x52A8;&#x6D4B;&#x5B9A;&#x65B9;&#x6CD5;&#x7684;&#x9002;&#x7528;&#x6027;&#x3002;&#x8FD9;&#x79CD;&#x540C;&#x8D28;&#x6D4B;&#x5B9A;&#x65B9;&#x6CD5;&#x53EF;&#x80FD;&#x4E0D;&#x9002;&#x7528;&#x4E8E;&#x5176;&#x4ED6;&#x8102;&#x8D28;&#x6FC0;&#x9176;&#x548C;&#x5E94;&#x901A;&#x8FC7;&#x542F;&#x7528;&#x9AD8;&#x901A;&#x91CF;&#x7B5B;&#x9009;&#x52AA;&#x529B;&#x5E2E;&#x52A9;&#x6B64;&#x7C7B;&#x9176;&#x7684;&#x6F5C;&#x5728;&#x987E;&#x5BA2;&#x7684;&#x6807;&#x8BC6;&#x3002;

&#x78F7;&#x8102;&#x9170;&#x808C;&#x9187; 5-&#x78F7;&#x9178; 4 &#x6FC0;&#x9176;&#x5177;&#x6709;&#x65B0;&#x9896;&#x3001; &#x5FEB;&#x901F;&#x57FA;&#x677F;&#x5236;&#x5907;&#x5747;&#x8D28;&#x3001; &#x9AD8;&#x901A;&#x91CF;&#x68C0;&#x6D4B;&#x3002;

Phosphoinositide kinases regulate diverse cellular functions and are important targets for therapeutic development for diseases, such as diabetes and cancer. Preparation of the lipid substrate is crucial for the development of a robust and miniaturizable lipid kinase assay. Enzymatic assays for phosphoinositide kinases often use lipid substrates prepared from lyophilized lipid preparations by sonication, which result in variability in the liposome size from preparation to preparation. Herein, we report a homogeneous 1536-well luciferase-coupled bioluminescence assay for PI5P4KÎ±. The substrate preparation is novel and allows the rapid production of a DMSO-containing substrate solution without the need for lengthy liposome preparation protocols, thus enabling the scale-up of this traditionally difficult type of assay. The Z'-factor value was greater than 0.7 for the PI5P4KÎ± assay, indicating its suitability for high-throughput screening applications. Tyrphostin AG-82 had been identified as an inhibitor of PI5P4KÎ± by assessing the degree of phospho transfer of Î³-(32)P-ATP to PI5P; its inhibitory activity against PI5P4KÎ± was confirmed in the present miniaturized assay. From a pilot screen of a library of bioactive compounds, another tyrphostin, I-OMe tyrphostin AG-538 (I-OMe-AG-538), was identified as an ATP-competitive inhibitor of PI5P4KÎ± with an IC(50) of 1 ÂµM, affirming the suitability of the assay for inhibitor discovery campaigns. This homogeneous assay may apply to other lipid kinases and should help in the identification of leads for this class of enzymes by enabling high-throughput screening efforts.

A homogeneous, high-throughput assay for phosphatidylinositol 5-phosphate 4-kinase with a novel, rapid substrate preparation.

Phosphoinositide kinases r&eacute;gulent diverses fonctions cellulaires et sont des objectifs importants pour le d&eacute;veloppement de th&eacute;rapeutique pour les maladies comme le diab&egrave;te et le cancer. Pr&eacute;paration du substrat lipidique est cruciale pour le d&eacute;veloppement d&#39;une analyse de kinase de lipides robuste et miniaturizable. Des essais enzymatiques des phosphoinositides kinases utilisent souvent les substrats lipidiques, pr&eacute;par&eacute;s &agrave; partir de pr&eacute;parations lyophilis&eacute;es lipidique par sonication, entra&icirc;nant la variabilit&eacute; de la taille de liposomes de la pr&eacute;paration &agrave; la pr&eacute;paration. Ici, nous rapportons un dosage homog&egrave;ne 1536 puits coupl&eacute; lucif&eacute;rase bioluminescence pour PI5P4K&alpha;. La pr&eacute;paration du substrat est roman et permet la production rapide d&#39;une solution de substrat contenant du DMSO sans n&eacute;cessiter de longues liposome protocoles de pr&eacute;paration, permettant ainsi &agrave; l&#39;intensification de ce type traditionnellement difficile de dosage. Le Z&#39;-valeur du facteur &eacute;tait sup&eacute;rieure &agrave; 0,7 pour le dosage de PI5P4K&alpha;, en indiquant ses qualit&eacute;s pour les applications de criblage &agrave; haut d&eacute;bit. Tyrphostine AG-82 avait &eacute;t&eacute; identifi&eacute; comme un inhibiteur de la PI5P4K&alpha; en &eacute;valuant le degr&eacute; de transfert de phospho de &gamma; - P-ATP (32) &agrave; PI5P ; son activit&eacute; inhibitrice contre PI5P4K&alpha; a &eacute;t&eacute; confirm&eacute;e dans le pr&eacute;sent test miniaturis&eacute;. Partir d&#39;un pilote &eacute;cran d&#39;une biblioth&egrave;que de compos&eacute;s bioactifs, un autre tyrphostine, je-OMe tyrphostine AG-538 (I-OMe-AG-538), a &eacute;t&eacute; identifi&eacute; comme un inhibiteur comp&eacute;titif-ATP de PI5P4K&alpha; avec un IC (50) de 1 &micro;M, qui a confirm&eacute; la pertinence du test pour les campagnes de d&eacute;couverte d&#39;inhibiteur. Ce dosage homog&egrave;ne peut s&#39;appliquer &agrave; d&#39;autres kinases de lipides et devrait aider &agrave; l&#39;identification de prospects pour cette classe d&#39;enzymes en permettant aux efforts de criblage &agrave; haut d&eacute;bit.

Une analyse homog&egrave;ne et &agrave; haut d&eacute;bit pour phosphatidylinositol phosphate-5 4-kinase avec une pr&eacute;paration de substrat innovatrice et rapide.

Phosphoinositid-Kinasen diverse Zellfunktionen regulieren und sind wichtige Ziele f&uuml;r die therapeutische Entwicklung f&uuml;r Krankheiten wie Diabetes oder Krebs. Vorbereitung des Untergrunds Lipid ist entscheidend f&uuml;r die Entwicklung eines robusten und miniaturizable Lipid Kinase Assay. Enzymatische Assays f&uuml;r Phosphoinositid-Kinasen verwenden h&auml;ufig Lipid Substrate aus lyophilisierten Lipid-Vorbereitungen durch Sonorisierung, vorbereitet, die Variabilit&auml;t in der Gr&ouml;&szlig;e Liposomen aus Vorbereitung zur Vorbereitung f&uuml;hren. Hier berichten wir eine homogene 1536-Well-Luciferase-gekoppelter Biolumineszenz-Assay f&uuml;r PI5P4K&alpha;. Die Vorbereitung des Untergrunds ist Roman und erm&ouml;glicht die schnelle Herstellung einer DMSO-haltige Substrat-L&ouml;sung ohne langwierige Liposomen Vorbereitungs-Protokolle, wodurch das Scale-up-traditionell schwierigen solcher Assay. Die Z&#39;-Faktor Wert war gr&ouml;&szlig;er als 0,7 f&uuml;r den PI5P4K&alpha;-Assay, der angibt, seine Eignung f&uuml;r High-Throughput-Screening-Anwendungen. Tyrphostin AG-82 war als Inhibitor der PI5P4K&alpha; identifiziert worden, durch die Beurteilung der Phospho-&Uuml;bergabe der &gamma;-(32) P-ATP an PI5P; seiner hemmenden Wirkung gegen PI5P4K&alpha; wurde in der vorliegenden miniaturisierte Probe best&auml;tigt. Von einem pilot-Bildschirm einer Bibliothek von bioaktiven Verbindungen, ein weiteres Tyrphostin-OMe-Tyrphostin AG-538 (I-OMe-AG-538) wurde als ATP-kompetitiver Inhibitor der PI5P4K&alpha; mit einem IC (50) von 1 &micro;M, best&auml;tigen die Eignung des Assays f&uuml;r Hemmer Entdeckung Kampagnen identifiziert. Dieses homogene Assays kann gelten f&uuml;r andere Lipid-Kinasen und sollte helfen bei der Identifizierung von Leads f&uuml;r diese Klasse von Enzymen durch Aktivieren von High-Throughput-Screening Bem&uuml;hungen.

Eine homogene, Hochdurchsatz-Assay f&uuml;r Phosphatidylinositol-5-Phosphat 4-Kinase mit einer neuartigen, schnelle Verlegeuntergr&uuml;nde.

Chinasi di phosphoinositide regolano diverse funzioni cellulari e sono importanti obiettivi di sviluppo terapeutico per malattie come diabete e cancro. Preparazione del substrato lipidico &egrave; cruciale per lo sviluppo di un&#39;analisi della chinasi lipidica robusto e miniaturizable. Dosaggi enzimatici per chinasi di phosphoinositide spesso utilizzano substrati lipidici preparati da preparati liofilizzati lipidica tramite sonicazione, che risultano in variabilit&agrave; delle dimensioni di liposomi da preparazione a preparazione. Qui, segnaliamo un dosaggio omogeneo 1536 pozzetti accoppiato luciferase bioluminescenza per PI5P4K&alpha;. La preparazione del substrato &egrave; romanzo e permette la rapida produzione di una soluzione di substrato contenente DMSO senza la necessit&agrave; di lunga liposoma protocolli di preparazione, consentendo in tal modo la scala di questo tipo tradizionalmente difficile di dosaggio. La Z&#39;-fattore valore era maggiore di 0,7 per il dosaggio di PI5P4K&alpha;, che indica l&#39;idoneit&agrave; per applicazioni high-throughput screening. Tyrphostin AG-82 era stato identificato come un inibitore della PI5P4K&alpha; di valutazione del grado di fosfo trasferimento di &gamma;-(32) P-ATP a PI5P; sua attivit&agrave; inibitoria contro PI5P4K&alpha; &egrave; stato confermato nel presente saggio miniaturizzato. Da un pilota schermo di una libreria di composti bioattivi, un&#39;altra tyrphostin, I-OMe tyrphostin AG-538 (I-OMe-AG-538), &egrave; stato identificato come un inibitore competitivo-ATP di PI5P4K&alpha; con un IC(50) di 1 &micro;M, affermando l&#39;adeguatezza del dosaggio per campagne di inibitore della scoperta. Questo dosaggio omogeneo pu&ograve; applicare ad altre chinasi lipidica e dovrebbe aiutare nell&#39;identificazione di cavi per questa classe di enzimi abilitando gli sforzi di high-throughput screening.

Un&#39;analisi omogenea, ad alta velocit&agrave; per la 5-fosfato di fosfatidilinositolo 4-chinasi con una preparazione del substrato romanzo, rapido.

&#x30DB;&#x30B9;&#x30DB;&#x30A4;&#x30CE;&#x30B7;&#x30C1;&#x30C9; &#x30AD;&#x30CA;&#x30FC;&#x30BC;&#x306B;&#x7CD6;&#x5C3F;&#x75C5;&#x3084;&#x764C;&#x306A;&#x3069;&#x306E;&#x75BE;&#x60A3;&#x306E;&#x6CBB;&#x7642;&#x958B;&#x767A;&#x306E;&#x305F;&#x3081;&#x306E;&#x91CD;&#x8981;&#x306A;&#x76EE;&#x6A19;&#x304C;&#x3042;&#x308A;&#x3001;&#x591A;&#x69D8;&#x306A;&#x7D30;&#x80DE;&#x6A5F;&#x80FD;&#x3092;&#x8ABF;&#x7BC0;&#x3057;&#x307E;&#x3059;&#x3002;&#x8102;&#x8CEA;&#x57FA;&#x677F;&#x306E;&#x4F5C;&#x88FD;&#x306F;&#x3001;&#x5805;&#x7262;&#x306A;&#x3068; miniaturizable &#x306E;&#x8102;&#x8CEA;&#x306E;&#x30AD;&#x30CA;&#x30FC;&#x30BC;&#x8A66;&#x91D1;&#x306E;&#x958B;&#x767A;&#x306E;&#x305F;&#x3081;&#x306B;&#x91CD;&#x8981;&#x3067;&#x3059;&#x3002;&#x30DB;&#x30B9;&#x30DB;&#x30A4;&#x30CE;&#x30B7;&#x30C1;&#x30C9; &#x30AD;&#x30CA;&#x30FC;&#x30BC;&#x306E;&#x9175;&#x7D20;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x306F;&#x983B;&#x7E41;&#x306B;&#x6E96;&#x5099;&#x3092;&#x30EA;&#x30DD;&#x30BD;&#x30FC;&#x30E0; &#x30B5;&#x30A4;&#x30BA;&#x306E;&#x5909;&#x52D5;&#x304C;&#x51CD;&#x7D50;&#x4E7E;&#x71E5;&#x8102;&#x8CEA;&#x6E96;&#x5099;&#x304B;&#x3089;&#x8D85;&#x97F3;&#x6CE2;&#x51E6;&#x7406;&#x306B;&#x3088;&#x308B;&#x8102;&#x8CEA;&#x57FA;&#x677F;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x3001;PI5P4K&alpha; &#x306E;&#x5747;&#x4E00; 1536&#x5E74;&#x30A6;&#x30A7;&#x30EB; &#x30EB;&#x30B7;&#x30D5;&#x30A7;&#x30E9;&#x30FC;&#x30BC;&#x7D50;&#x5408;&#x767A;&#x5149;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x3092;&#x5831;&#x544A;&#x3057;&#x307E;&#x3059;&#x3002;&#x57FA;&#x677F;&#x306E;&#x6E96;&#x5099;&#x306F;&#x5C0F;&#x8AAC;&#x3067;&#x3042;&#x308A;&#x3001;DMSO &#x3092;&#x542B;&#x3093;&#x3067;&#x3044;&#x308B;&#x57FA;&#x677F;&#x30BD;&#x30EA;&#x30E5;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x9577;&#x3044;&#x30EA;&#x30DD;&#x30BD;&#x30FC;&#x30E0;&#x3092;&#x5FC5;&#x8981;&#x3068;&#x305B;&#x305A;&#x306E;&#x8FC5;&#x901F;&#x306A;&#x751F;&#x7523;&#x6E96;&#x5099;&#x306E;&#x30D7;&#x30ED;&#x30C8;&#x30B3;&#x30EB;&#x306F;&#x3001;&#x3053;&#x306E;&#x3088;&#x3046;&#x306B;&#x3053;&#x306E;&#x3053;&#x308C;&#x307E;&#x3067;&#x96E3;&#x3057;&#x3044;&#x30BF;&#x30A4;&#x30D7;&#x306E;&#x8A66;&#x91D1;&#x306E;&#x30B9;&#x30B1;&#x30FC;&#x30EB; &#x30A2;&#x30C3;&#x30D7;&#x3092;&#x6709;&#x52B9;&#x306B;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x3002;Z&#39;-&#x30D5;&#x30A1;&#x30AF;&#x30BF;&#x30FC;&#x5024;&#x306F; 0.7 &#x9AD8;&#x30B9;&#x30EB;&#x30FC;&#x30D7;&#x30C3;&#x30C8; &#x30B9;&#x30AF;&#x30EA;&#x30FC;&#x30CB;&#x30F3;&#x30B0;&#x7528;&#x306E;&#x9069;&#x5408;&#x6027;&#x3092;&#x793A;&#x3059;&#x3001;PI5P4K&alpha; &#x306E;&#x8A66;&#x91D1;&#x306E;&#x305F;&#x3081;&#x3088;&#x308A;&#x5927;&#x304D;&#x304B;&#x3063;&#x305F;&#x3002;Tyrphostin AG-82 PI5P4K&alpha; &#x963B;&#x5BB3;&#x5264;&#x3068;&#x3057;&#x3066; PI5P &#x3078;&#x306E; &gamma;-(32) P-ATP &#x306E;&#x30EA;&#x30F3;&#x8EE2;&#x9001;&#x306E;&#x7A0B;&#x5EA6;&#x3092;&#x8A55;&#x4FA1;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x8B58;&#x5225;&#x3055;&#x308C;&#x3066;&#x3044;&#x305F;&#x3002;PI5P4K&alpha; &#x306B;&#x5BFE;&#x3059;&#x308B;&#x963B;&#x5BB3;&#x6D3B;&#x6027;&#x306F;&#x73FE;&#x5728;&#x5C0F;&#x578B;&#x8A66;&#x9A13;&#x3067;&#x78BA;&#x8A8D;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#xFF08;&#x79C1;&#x306F;-&#x9752;&#x6885;-AG-538) &#x304B;&#x3089;&#x306E;&#x30D1;&#x30A4;&#x30ED;&#x30C3;&#x30C8;&#x753B;&#x9762; - &#x9752;&#x6885; tyrphostin AG 538 &#x5225; tyrphostin &#x751F;&#x7406;&#x6D3B;&#x6027;&#x5316;&#x5408;&#x7269;&#x306E;&#x30E9;&#x30A4;&#x30D6;&#x30E9;&#x30EA;&#x306E; PI5P4K&alpha; &#x963B;&#x5BB3;&#x5264;&#x306E;&#x63A2;&#x7D22;&#x306E;&#x30AD;&#x30E3;&#x30F3;&#x30DA;&#x30FC;&#x30F3;&#x306E;&#x305F;&#x3081;&#x306E;&#x8A66;&#x91D1;&#x306E;&#x9069;&#x5408;&#x6027;&#x3092;&#x78BA;&#x8A8D;&#x3057;&#x3001;ic50 &#x5024; &#xFF08;50 1 &mu; m &#x3067;&#x306E; ATP &#x7AF6;&#x5408;&#x963B;&#x5BB3;&#x5264;&#x3068;&#x3057;&#x3066;&#x8B58;&#x5225;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x3053;&#x306E;&#x5747;&#x8CEA;&#x306A;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x4ED6;&#x8102;&#x8CEA;&#x30AD;&#x30CA;&#x30FC;&#x30BC;&#x306B;&#x9069;&#x7528;&#x3055;&#x308C;&#x3001;&#x9AD8;&#x30B9;&#x30EB;&#x30FC;&#x30D7;&#x30C3;&#x30C8; &#x30B9;&#x30AF;&#x30EA;&#x30FC;&#x30CB;&#x30F3;&#x30B0;&#x306E;&#x52AA;&#x529B;&#x3092;&#x6709;&#x52B9;&#x306B;&#x3057;&#x3066;&#x3053;&#x306E;&#x9175;&#x7D20;&#x306E;&#x30AF;&#x30E9;&#x30B9;&#x306E;&#x6F5C;&#x5728;&#x9867;&#x5BA2;&#x306E;&#x8B58;&#x5225;&#x3067;&#x52A9;&#x3051;&#x308B;&#x3079;&#x304D;&#x3067;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;

&#x30DB;&#x30B9;&#x30D5;&#x30A1;&#x30C1;&#x30B8;&#x30EB;&#x30A4;&#x30CE;&#x30B7;&#x30C8;&#x30FC;&#x30EB; 5-&#x30EA;&#x30F3;&#x9178; 4 &#x30AD;&#x30CA;&#x30FC;&#x30BC;&#x5C0F;&#x8AAC;&#x3001;&#x6025;&#x901F;&#x306A;&#x57FA;&#x677F;&#x306E;&#x6E96;&#x5099;&#x306E;&#x305F;&#x3081;&#x306E;&#x5747;&#x4E00;&#x3001;&#x9AD8;&#x30B9;&#x30EB;&#x30FC;&#x30D7;&#x30C3;&#x30C8; &#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x3002;

Phosphoinositide kinases&#xB294; &#xB2E4;&#xC591; &#xD55C; &#xC138;&#xD3EC; &#xAE30;&#xB2A5;&#xC744; &#xC870;&#xC808; &#xD558; &#xACE0; &#xB2F9;&#xB1E8;&#xBCD1;&#xACFC; &#xC554; &#xAC19;&#xC740; &#xC9C8;&#xBCD1;&#xC5D0; &#xB300; &#xD55C; &#xCE58;&#xB8CC; &#xAC1C;&#xBC1C;&#xC5D0; &#xB300; &#xD55C; &#xC911;&#xC694; &#xD55C; &#xBAA9;&#xD45C;. &#xC9C0;&#xC9C8; &#xAE30;&#xD310;&#xC758; &#xC900;&#xBE44;&#xB294; &#xAC15;&#xB825; &#xD558; &#xACE0; miniaturizable &#xC9C0;&#xC9C8; &#xB2C8; &#xBD84;&#xC11D; &#xACB0;&#xACFC; &#xAC1C;&#xBC1C;&#xC5D0; &#xB300; &#xD55C; &#xACB0;&#xC815;&#xC801; &#xC774;&#xB2E4;. Phosphoinositide kinases &#xC704;&#xD55C; &#xD6A8;&#xC18C; &#xBD84;&#xC11D; &#xC2E4;&#xD5D8; &#xC900;&#xBE44; &#xC900;&#xBE44; liposome &#xD06C;&#xAE30;&#xC5D0; &#xBCC0;&#xD654;&#xC5D0;&#xB294; sonication, &#xB3D9;&#xACB0; &#xAC74;&#xC870; &#xB41C; &#xC9C0;&#xC9C8; &#xC900;&#xBE44;&#xC5D0;&#xC11C; &#xC900;&#xBE44;&#xD55C; &#xC9C0;&#xC9C8; &#xAE30;&#xD310; &#xC0AC;&#xC6A9; &#xD569;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30;&#xC5D0;, &#xC6B0;&#xB9AC;&#xB294; pi5p4k&alpha;&#xC5D0; &#xB300; &#xD55C; &#xADE0;&#xC9C8; 1536-&#xC798; &#xB8E8;&#xC2DC;&#xD398;&#xB77C;&#xC544;&#xC81C; &#xACB0;&#xD569; &#xC0DD;&#xBB3C; &#xBC1C;&#xAD11; &#xBD84;&#xC11D; &#xACB0;&#xACFC; &#xBCF4;&#xACE0;&#xD569;&#xB2C8;&#xB2E4;. &#xAE30;&#xD310; &#xC900;&#xBE44; &#xC18C;&#xC124; &#xC774;&#xBA70; &#xC900;&#xBE44; &#xD504;&#xB85C;&#xD1A0;&#xCF5C;, &#xC804;&#xD1B5;&#xC801;&#xC73C;&#xB85C; &#xC5B4;&#xB824;&#xC6B4; &#xC774;&#xC640; &#xAC19;&#xC740; &#xBD84;&#xC11D; &#xACB0;&#xACFC; &#xB300; &#xD55C; &#xC2A4;&#xCF00;&#xC77C; &#xC5C5; &#xD65C;&#xC131;&#xD654; &#xAE34; liposome&#xC758; &#xD544;&#xC694;&#xC131; &#xC5C6;&#xC774; DMSO&#xB97C; &#xD3EC;&#xD568; &#xD558;&#xB294; &#xAE30;&#xD310; &#xC194;&#xB8E8;&#xC158;&#xC758; &#xC2E0;&#xC18D;&#xD55C; &#xC0DD;&#xC0B0;&#xC744; &#xD5C8;&#xC6A9; &#xD569;&#xB2C8;&#xB2E4;. Z&#39;-&#xBE44;&#xC728; &#xAC12; 0.7 &#xB192;&#xC740; &#xCC98;&#xB9AC;&#xB7C9; &#xAC80;&#xC5F4; &#xC751;&#xC6A9; &#xD504;&#xB85C;&#xADF8;&#xB7A8;&#xC5D0; &#xB300; &#xD55C; &#xC801;&#xD569;&#xC131;&#xC744; &#xB098;&#xD0C0;&#xB0B4;&#xB294; PI5P4K&alpha; &#xBD84;&#xC11D; &#xACB0;&#xACFC; &#xB300; &#xD55C; &#xBCF4;&#xB2E4; &#xD070; &#xD588;&#xB2E4;. Tyrphostin AG-82 PI5P;&#xC5D0; &gamma;-(32) P-ATP&#xC758; &#xC778; &#xC591;&#xB3C4;&#xC758; &#xC815;&#xB3C4; &#xD3C9;&#xAC00; &#xD558; &#xC5EC; pi5p4k&alpha;&#xC758; &#xC5B5;&#xC81C;&#xC81C;&#xB85C; &#xBC1C;&#xACAC; &#xD588;&#xB2E4; pi5p4k&alpha;&#xC5D0; &#xB300; &#xD55C; &#xADF8;&#xAC83;&#xC758; &#xAE08;&#xC9C0; &#xD65C;&#xB3D9;&#xC73C;&#xB85C; &#xD604;&#xC7AC; &#xC18C;&#xD615;&#xB41C; &#xBD84;&#xC11D; &#xACB0;&#xACFC;&#xC5D0;&#xC11C; &#xD655;&#xC778; &#xB410;&#xB2E4;. (&#xB09C;-&#xC624;&#xBA54;-AG-538), &#xC0DD;&#xB9AC; &#xD65C;&#xC131; &#xD654;&#xD569;&#xBB3C;, &#xB2E4;&#xB978; tyrphostin-&#xC624;&#xBA54; tyrphostin AG 538 &#xB77C;&#xC774;&#xBE0C;&#xB7EC;&#xB9AC;&#xC758; &#xD30C;&#xC77C;&#xB7FF; &#xD654;&#xBA74;&#xC5D0;&#xC11C; &#xC5B5;&#xC81C;&#xC81C; &#xAC80;&#xC0C9; &#xCEA0;&#xD398;&#xC778;&#xC5D0; &#xB300; &#xD55C; &#xBD84;&#xC11D; &#xACB0;&#xACFC;&#xC758; &#xC801;&#xD569;&#xC131;&#xC744; &#xAE0D;&#xC815; &#xD558;&#xB294; 1 &micro; M&#xC758; IC(50)&#xC640; pi5p4k&alpha;&#xC758; ATP &#xACBD;&#xC7C1;&#xC801; &#xC5B5;&#xC81C;&#xC81C;&#xB85C; &#xD655;&#xC778; &#xB418;&#xC5C8;&#xB2E4;. &#xC774; &#xB3D9; &#xC9C8; &#xBD84;&#xC11D; &#xACB0;&#xACFC; &#xB2E4;&#xB978; &#xC9C0;&#xC9C8; kinases&#xC5D0; &#xC801;&#xC6A9; &#xB420; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xBC0F; &#xB192;&#xC740; &#xCC98;&#xB9AC;&#xB7C9; &#xAC80;&#xC5F4; &#xB178;&#xB825; &#xD568;&#xC73C;&#xB85C;&#xC368; &#xD6A8;&#xC18C;&#xC758;&#xC774; &#xD074;&#xB798;&#xC2A4;&#xC5D0; &#xB300; &#xD55C; &#xB9AC;&#xB4DC;&#xC758; &#xC2DD;&#xBCC4;&#xC5D0; &#xB3C4;&#xC6C0;&#xC774; &#xB429;&#xB2C8;&#xB2E4;.

Phosphatidylinositol 5-&#xC778;&#xC0B0; &#xC5FC; 4-&#xB2C8; &#xC18C;&#xC124;, &#xAE09;&#xC18D; &#xD55C; &#xAE30;&#xD310; &#xC900;&#xBE44;&#xC5D0; &#xB300; &#xD55C; &#xB3D9;&#xC885;, &#xB192;&#xC740; &#xCC98;&#xB9AC;&#xB7C9; &#xC2DC;&#xD5D8;.

Fosfoinositol quinasas regulan diversas funciones celulares y son objetivos importantes para el desarrollo terap&eacute;utico para enfermedades como la diabetes y el c&aacute;ncer. Preparaci&oacute;n del sustrato l&iacute;pidos es crucial para el desarrollo de un ensayo de la quinasa de l&iacute;pidos robusto y miniaturizable. Ensayos enzim&aacute;ticos para Fosfoinositol quinasas utilizan sustratos l&iacute;pidos preparados de preparaciones de l&iacute;pidos liofilizado por sonicaci&oacute;n, que resultan en la variabilidad en el tama&ntilde;o de liposomas de la preparaci&oacute;n para la preparaci&oacute;n. Adjunto, Divulgamos un ensayo homog&eacute;neo 1536-bien acoplado luciferasa bioluminiscencia para PI5P4K&alpha;. La preparaci&oacute;n del sustrato es novela y permite la r&aacute;pida producci&oacute;n de una soluci&oacute;n de sustrato que contiene DMSO sin necesidad de largo liposoma protocolos de preparaci&oacute;n, lo que permite el escalado de este tipo tradicionalmente dif&iacute;cil de ensayo. El Z&#39;-factor valor fue superior a 0.7 para el ensayo de PI5P4K&alpha;, indicando su conveniencia para aplicaciones de alto rendimiento de cribado. Tyrphostin AG-82 hab&iacute;a sido identificado como un inhibidor de la PI5P4K&alpha; por evaluar el grado de transferencia phospho de &gamma;-(32) P-ATP a PI5P; su actividad inhibitoria frente a PI5P4K&alpha; fue confirmada en el presente ensayo miniaturizado. Desde una pantalla piloto de una biblioteca de compuestos bioactivos, otro tyrphostin, tyrphostin OMe AG-538 (I-OMe-AG-538), fue identificado como un inhibidor competitivo ATP de PI5P4K&alpha; con un IC(50) de 1 &micro;M, afirmando la idoneidad del ensayo para campa&ntilde;as de descubrimiento del inhibidor. Este ensayo homog&eacute;neo puede aplicarse a otras quinasas de l&iacute;pidos y debe ayudar en la identificaci&oacute;n de cables para esta clase de enzimas habilitando los esfuerzos de proyecci&oacute;n de alto rendimiento.

Un an&aacute;lisis homog&eacute;neo, de alto rendimiento para 5-fosfato Fosfatidilinositol 4-quinasa con una preparaci&oacute;n del sustrato de la novela, r&aacute;pido.

The cytochrome P450 (CYP) family contains 57 enzymes in humans. The activity of CYPs against xenobiotics is a primary consideration in drug optimization efforts. Here we describe a series of bioluminescent assays that enable the rapid profiling of CYP activity against compound collections. The assays employ a coupled-enzyme format where firefly luciferase is used to measure CYP enzyme activity through metabolism of pro-luciferase substrates.

Bioluminescent assays for cytochrome P450 enzymes.

Pilot testing of an assay intended for high-throughput screening (HTS) with small compound sets is a necessary but often time-consuming step in the validation of an assay protocol. When the initial testing concentration is less than optimal, this can involve iterative testing at different concentrations to further evaluate the pilot outcome, which can be even more time-consuming. Quantitative HTS (qHTS) enables flexible and rapid collection of assay performance statistics, hits at different concentrations, and concentration-response curves in a single experiment. Here we describe the qHTS process for pilot testing in which eight-point concentration-response curves are produced using an interplate asymmetric dilution protocol in which the first four concentrations are used to represent the range of typical HTS screening concentrations and the last four concentrations are added for robust curve fitting to determine potency/efficacy values. We also describe how these data can be analyzed to predict the frequency of false-positives, false-negatives, hit rates, and confirmation rates for the HTS process as a function of screening concentration. By taking into account the compound pharmacology, this pilot-testing paradigm enables rapid assessment of the assay performance and choosing the optimal concentration for the large-scale HTS in one experiment. 

Application of Titration-Based Screening for the Rapid Pilot Testing of High-Throughput Assays.

Two mutant forms (R132H and R132C) of isocitrate dehydrogenase 1 (IDH1) have been associated with a number of cancers including glioblastoma and acute myeloid leukemia. These mutations confer a neomorphic activity of 2-hydroxyglutarate (2-HG) production, and 2-HG has previously been implicated as an oncometabolite. Inhibitors of mutant IDH1 can potentially be used to treat these diseases. In this study, we investigated the mechanism of action of a newly discovered inhibitor, ML309, using biochemical, cellular, and biophysical approaches. Substrate binding and product inhibition studies helped to further elucidate the IDH1 R132H catalytic cycle. This rapidly equilibrating inhibitor is active in both biochemical and cellular assays. The (+) isomer is active (IC50 = 68 nm), whereas the (-) isomer is over 400-fold less active (IC50 = 29 μm) for IDH1 R132H inhibition. IDH1 R132C was similarly inhibited by (+)-ML309. WT IDH1 was largely unaffected by (+)-ML309 (IC50 >36 μm). Kinetic analyses combined with microscale thermophoresis and surface plasmon resonance indicate that this reversible inhibitor binds to IDH1 R132H competitively with respect to α-ketoglutarate and uncompetitively with respect to NADPH. A reaction scheme for IDH1 R132H inhibition by ML309 is proposed in which ML309 binds to IDH1 R132H after formation of the IDH1 R132H NADPH complex. ML309 was also able to inhibit 2-HG production in a glioblastoma cell line (IC50 = 250 nm) and had minimal cytotoxicity. In the presence of racemic ML309, 2-HG levels drop rapidly. This drop was sustained until 48 h, at which point the compound was washed out and 2-HG levels recovered. 

Biochemical, cellular, and biophysical characterization of a potent inhibitor of mutant isocitrate dehydrogenase IDH1.

The wealth of bioactivity information now available on low-molecular weight compounds has enabled a paradigm shift in chemical biology and early phase drug discovery efforts. Traditionally chemical libraries have been most commonly employed in screening approaches where a bioassay is used to characterize a chemical library in a random search for active samples. However, robust curating of bioassay data, establishment of ontologies enabling mining of large chemical biology datasets, and a wealth of public chemical biology information has made possible the establishment of highly annotated compound collections. Such annotated chemical libraries can now be used to build a pathway/target hypothesis and have led to a new view where chemical libraries are used to characterize a bioassay. In this article we discuss the types of compounds in these annotated libraries composed of tools, probes, and drugs. As well, we provide rationale and a few examples for how such libraries can enable phenotypic/forward chemical genomic approaches. As with any approach, there are several pitfalls that need to be considered and we also outline some strategies to avoid these. 

Composition and applications of focus libraries to phenotypic assays.

The calcium-activated chloride channel ANO1 regulates multiple physiological processes. However, little is known about the mechanism of channel gating and regulation of ANO1 activity. Using a high-throughput, random mutagenesis-based variomics screen, we generated and functionally characterized ∼6000 ANO1 mutants and identified novel mutations that affected channel activity, intracellular trafficking, or localization of ANO1. Mutations such as S741T increased ANO1 calcium sensitivity and rendered ANO1 calcium gating voltage-independent, demonstrating a critical role of the re-entrant loop in coupling calcium and voltage sensitivity of ANO1 and hence in regulating ANO1 activation. Our data present the first unbiased and comprehensive study of the structure-function relationship of ANO1. The novel ANO1 mutants reported have diverse functional characteristics, providing new tools to study ANO1 function in biological systems, paving the path for a better understanding of the function of ANO1 and its role in health and diseases. 

Variomics screen identifies the re-entrant loop of the calcium-activated chloride channel ANO1 that facilitates channel activation.

Assays in which the detection of a biological phenomenon is coupled to the production of bioluminescence by luciferase have gained widespread use. As firefly luciferases (FLuc) and kinases share a common substrate (ATP), coupling of a kinase to FLuc allows for the amount of ATP remaining following a kinase reaction to be assessed by quantitating the amount of luminescence produced. Alternatively, the amount of ADP produced by the kinase reaction can be coupled to FLuc through a two-step process. This chapter describes the bioluminescent assays that were developed for three classes of kinases (lipid, protein, and metabolic kinases) and miniaturized to 1536-well format, enabling their use for quantitative high-throughput (qHTS) of small-molecule libraries. 

Bioluminescence Methods for Assaying Kinases in Quantitative High-Throughput Screening (qHTS) Format Applied to Yes1 Tyrosine Kinase, Glucokinase, and PI5P4Kα Lipid Kinase.

Luciferase-based reporter assays are powerful tools for monitoring gene expression in cells because of their ultrasensitive detection capacity and wide dynamic range. Here we describe the characterization and use of a luciferase reporter enzyme derived from the marine copepod Metridia luciferase family, referred to as TurboLuc luciferase (TurboLuc). To develop TurboLuc, the wild-type luciferase was modified to decrease its size, increase brightness, slow luminescent signal decay, and provide for efficient intracellular expression. To determine the enzyme susceptibility to compound inhibition and judge the suitability of using of TurboLuc as a reporter in screening assays, purified TurboLuc enzyme was screened for inhibitors using two different compound libraries. No inhibitors of this enzyme were identified in a library representative of typical diverse low molecular weight (LMW) compounds using a purified TurboLuc enzyme assay supporting that such libraries will show very low interference with this enzyme. We were able to identify a few inhibitors from a purified natural product library which can serve as useful tools to validate assays using TurboLuc. In addition to the inhibitor profile for TurboLuc we describe the use of this reporter in cells employing miniaturized assay volumes within 1536-well plates. TurboLuc luciferase is the smallest luciferase reporter enzyme described to date (16 kDa), shows bright luminescence and low interference by LMW compounds, and therefore should provide an ideal reporter in assays applied to high-throughput screening.

Article	Year
Comparison of compound administration methods in biochemical assays: effects on apparent compound potency using either assay-ready compound plates or pin tool-delivered compounds. Journal of biomolecular screening\| PubMed ID: 22904199	2013
A homogeneous, high-throughput assay for phosphatidylinositol 5-phosphate 4-kinase with a novel, rapid substrate preparation. PloS one\| PubMed ID: 23326584	2013
Bioluminescent assays for cytochrome P450 enzymes. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 23475663	2013
Application of Titration-Based Screening for the Rapid Pilot Testing of High-Throughput Assays. Journal of biomolecular screening\| PubMed ID: 24246376	2014
Biochemical, cellular, and biophysical characterization of a potent inhibitor of mutant isocitrate dehydrogenase IDH1. The Journal of biological chemistry\| PubMed ID: 24668804	2014
Composition and applications of focus libraries to phenotypic assays. Frontiers in pharmacology\| PubMed ID: 25104937	2014
Variomics screen identifies the re-entrant loop of the calcium-activated chloride channel ANO1 that facilitates channel activation. The Journal of biological chemistry\| PubMed ID: 25425649	2015
Bioluminescence Methods for Assaying Kinases in Quantitative High-Throughput Screening (qHTS) Format Applied to Yes1 Tyrosine Kinase, Glucokinase, and PI5P4Kα Lipid Kinase. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 26501901	2016
Characterization and Use of TurboLuc Luciferase as a Reporter for High-Throughput Assays. Biochemistry\| PubMed ID: 29641191	2018

Douglas S. Auld

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