A quantitative high-throughput screen for modulators of IL-6 signaling: a model for interrogating biological networks using chemical libraries.
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.
Johnson
Ronald L
RL
NIH Chemical Genomics Center, National Institutes of Health, Bethesda, MD 20892, USA. rjohnso2@mail.nih.gov
Huang
Ruili
R
Jadhav
Ajit
A
Southall
Noel
N
Wichterman
Jennifer
J
MacArthur
Ryan
R
Xia
Menghang
M
Bi
Kun
K
Printen
John
J
Austin
Christopher P
CP
Inglese
James
J
D000818:Animals; D002460:Cell Line; D019295:Computational Biology / Q000379:methods*; D030562:Databases, Protein; D005456:Fluorescent Dyes; D017930:Genes, Reporter; D015850:Interleukin-6 / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012189:Retrospective Studies; D015398:Signal Transduction; D054852:Small Molecule Libraries; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism; D000093242:Interleukin-6 Inhibitors
D005456:Fluorescent Dyes; D015850:Interleukin-6; D011993:Recombinant Fusion Proteins; D054852:Small Molecule Libraries; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases; D000093242:Interleukin-6 Inhibitors