A quantitative high-throughput screen for modulators of IL-6 signaling: a model for interrogating biological networks using chemical libraries.

Methods Development

Abstract

Small molecule modulators are critical for dissecting and understanding signaling pathways at the molecular level. Interleukin 6 (IL-6) is a cytokine that signals via the JAK-STAT pathway and is implicated in cancer and inflammation. To identify modulators of this pathway, we screened a chemical collection against an IL-6 responsive cell line stably expressing a beta-lactamase reporter gene fused to a sis-inducible element (SIE-bla cells). This assay was optimized for a 1536-well microplate format and screened against 11 693 small molecules using quantitative high-throughput screening (qHTS), a method that assays a chemical library at multiple concentrations to generate titration-response profiles for each compound. The qHTS recovered 564 actives with well-fit curves that clustered into 32 distinct chemical series of 13 activators and 19 inhibitors. A retrospective analysis of the qHTS data indicated that single concentration data at 1.5 and 7.7 microM scored 35 and 71% of qHTS actives, respectively, as inactive and were therefore false negatives. Following counter screens to identify fluorescent and non-selective series, we found four activator and one inhibitor series that modulated SIE-bla cells but did not show similar activity in reporter gene assays induced by EGF and hypoxia. Small molecules within these series will make useful tool compounds to investigate IL-6 signaling mediated by JAK-STAT activation.

Authors

Johnson, Ronald L; Huang, Ruili; Jadhav, Ajit; Southall, Noel; Kouznetsova, Jennifer; MacArthur, Ryan; Xia, Menghang; Bi, Kun; Printen, John; Austin, Christopher; Inglese, James;

Keywords

  • Animals
  • Cell Line
  • Computational Biology/ methods
  • Databases, Protein
  • Fluorescent Dyes
  • Genes, Reporter
  • Interleukin-6/ antagonists & inhibitors
  • Interleukin-6/ metabolism
  • Mice
  • Models, Biological
  • Recombinant Fusion Proteins/ genetics
  • Recombinant Fusion Proteins/ metabolism
  • Reproducibility of Results
  • Retrospective Studies
  • Signal Transduction
  • Small Molecule Libraries
  • beta-Lactamase Inhibitors
  • beta-Lactamases/ genetics
  • beta-Lactamases/ metabolism

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