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Development of a time-resolved fluorescence resonance energy transfer ultra-high throughput screening assay targeting SYK and FCER1G interaction.
Du, Yuhong; Wang, Dongxue; Katis, Vittorio L; Zoeller, Elizabeth L; Qui, Min; Levey, Allan I; Gileadi, Opher; Fu, Haian.
Afiliación
  • Du Y; Department of Pharmacology and Chemical Biology, Emory University School of Medicine, Atlanta, Georgia, USA; Emory Chemical Biology Discovery Center, Emory University School of Medicine, Atlanta, Georgia, USA. Electronic address: dyuhong@emory.edu.
  • Wang D; Department of Pharmacology and Chemical Biology, Emory University School of Medicine, Atlanta, Georgia, USA; Emory Chemical Biology Discovery Center, Emory University School of Medicine, Atlanta, Georgia, USA.
  • Katis VL; Alzheimer's Research UK Oxford Drug Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine Research Building, University of Oxford, Oxford, UK.
  • Zoeller EL; Department of Pharmacology and Chemical Biology, Emory University School of Medicine, Atlanta, Georgia, USA.
  • Qui M; Emory Chemical Biology Discovery Center, Emory University School of Medicine, Atlanta, Georgia, USA.
  • Levey AI; Department of Neurology, Emory Goizueta Alzheimer's Disease Research Center, Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, GA 30322, USA.
  • Gileadi O; Alzheimer's Research UK Oxford Drug Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine Research Building, University of Oxford, Oxford, UK.
  • Fu H; Department of Pharmacology and Chemical Biology, Emory University School of Medicine, Atlanta, Georgia, USA; Emory Chemical Biology Discovery Center, Emory University School of Medicine, Atlanta, Georgia, USA.
SLAS Discov ; 29(6): 100177, 2024 Sep.
Article en En | MEDLINE | ID: mdl-39154664
ABSTRACT
The spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) interaction has a major role in the normal innate and adaptive immune responses, but dysregulation of this interaction is implicated in several human diseases, including autoimmune disorders, hematological malignancies, and Alzheimer's Disease. Development of small molecule chemical probes could aid in studying this pathway both in normal and aberrant contexts. Herein, we describe the miniaturization of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay to measure the interaction between SYK and FCER1G in a 1536-well ultrahigh throughput screening (uHTS) format. The assay utilizes the His-SH2 domains of SYK, which are indirectly labeled with anti-His-terbium to serve as a TR-FRET donor and a FITC-conjugated phosphorylated ITAM domain peptide of FCER1G to serve as an acceptor. We have optimized the assay into a 384-well HTS format and further miniaturized the assay into a 1536-well uHTS format. Robust assay performance has been achieved with a Z' factor > 0.8 and signal-to-background (S/B) ratio > 15. The utilization of this uHTS TR-FRET assay for compound screening has been validated by a pilot screening of 2,036 FDA-approved and bioactive compounds library. Several primary hits have been identified from the pilot uHTS. One compound, hematoxylin, was confirmed to disrupt the SYK/FECR1G interaction in an orthogonal protein-protein interaction assay. Thus, our optimized and miniaturized uHTS assay could be applied to future scaling up of a screening campaign to identify small molecule inhibitors targeting the SYK and FCER1G interaction.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Transferencia Resonante de Energía de Fluorescencia / Ensayos Analíticos de Alto Rendimiento / Quinasa Syk Límite: Humans Idioma: En Revista: SLAS Discov Año: 2024 Tipo del documento: Article Pais de publicación: Estados Unidos
Texto completo
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Imprimir
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Transferencia Resonante de Energía de Fluorescencia / Ensayos Analíticos de Alto Rendimiento / Quinasa Syk Límite: Humans Idioma: En Revista: SLAS Discov Año: 2024 Tipo del documento: Article Pais de publicación: Estados Unidos