Nuclear export inhibitors and kinase inhibitors identified using a MAPK-activated protein kinase 2 redistribution screen.
Redistribution (BioImage) A/S, Søborg, Denmark) is a novel high-throughput screening technology that monitors translocation of specific protein components of intracellular signaling pathways within intact mammalian cells, using green fluorescent protein as a tag. A single Redistribution assay can be used to identify multiple classes of compounds that act at, or upstream of, the level of the protein target used in the primary screening assay. Such compounds may include both conventional and allosteric enzyme inhibitors, as well as protein-protein interaction modulators. We have developed a series of Redistribution assays to discover and characterize compounds that inhibit tumor necrosis factor-alpha biosynthesis via modulation of the p38 mitogen-activated protein kinase (MAPK) pathway. A primary assay was designed to identify low-molecular-weight compounds that inhibit the activation-dependent nuclear export of the p38 kinase substrate MAPK-activated protein kinase 2 (MK2). Hits from the primary screen were categorized, using secondary assays, either as direct inhibitors of MK2 nuclear export, or as inhibitors of the upstream p38 MAPK pathway. Activity profiles are presented for a nuclear export inhibitor, and a compound that structurally and functionally resembles a known p38 kinase inhibitor. These results demonstrate the utility of Redistribution technology as a pathway screening method for the identification of diverse and novel compounds that are active within therapeutically important signaling pathways.
Almholt
Dorthe L C
DL
BioImage A/S, Søborg, Denmark.
Loechel
Frosty
F
Nielsen
Søren J
SJ
Krog-Jensen
Christian
C
Terry
Robert
R
Bjørn
Sara P
SP
Pedersen
Hans C
HC
Praestegaard
Morten
M
Møller
Søren
S
Heide
Morten
M
Pagliaro
Len
L
Mason
Anthony J
AJ
Butcher
Steven
S
Dahl
Søren W
SW
D021581:Active Transport, Cell Nucleus / Q000187:drug effects*; D000465:Algorithms; D002460:Cell Line; D004396:Coloring Agents; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007202:Indicators and Reagents; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics* / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D010078:Oxazines; D010770:Phosphotransferases / Q000037:antagonists & inhibitors*; D010957:Plasmids / Q000235:genetics; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D014966:Xanthenes; D048051:p38 Mitogen-Activated Protein Kinases
D004396:Coloring Agents; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D010078:Oxazines; D014409:Tumor Necrosis Factor-alpha; D014966:Xanthenes; C005843:resazurin; D010770:Phosphotransferases; D019950:Mitogen-Activated Protein Kinase 1; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases