Complete inhibition of anisomycin and UV radiation but not cytokine induced JNK and p38 activation by an aryl-substituted dihydropyrrolopyrazole quinoline and mixed lineage kinase 7 small interfering RNA.
Mixed lineage kinase 7 (MLK7) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the pro-apoptotic signaling pathways p38 and JNK. A library of potential kinase inhibitors was screened, and a series of dihydropyrrolopyrazole quinolines was identified as highly potent inhibitors of MLK7 in vitro catalytic activity. Of this series, an aryl-substituted dihydropyrrolopyrazole quinoline (DHP-2) demonstrated an IC50 of 70 nM for inhibition of pJNK formation in COS-7 cell MLK7/JNK co-transfection assays. In stimulated cells, DHP-2 at 200 nM or MLK7 small interfering RNA completely blocked anisomycin and UV induced but had no effect on interleukin-1beta or tumor necrosis factor-alpha-induced p38 and JNK activation. Additionally, the compound blocked anisomycin and UV-induced apoptosis in COS-7 cells. Heart tissue homogenates from MLK7 transgenic mice treated with DHP-2 at 30 mg/kg had reduced JNK and p38 activation with no apparent effect on ERK activation, demonstrating that this compound can be used to block MLK7-driven MAPK pathway activation in vivo. Taken together, these data demonstrate that MLK7 is the MAPKKK required for modulation of the stress-activated MAPKs downstream of anisomycin and UV stimulation and that DHP-2 can be used to block MLK7 pathway activation in cells as well as in vivo.
Wang
Xushan
X
Cardiovascular Discovery Research, Eli Lilly and Company, Indianapolis, Indiana 46285, USA.
Mader
Mary M
MM
Toth
John E
JE
Yu
Xiaohong
X
Jin
Najia
N
Campbell
Robert M
RM
Smallwood
Jeffrey K
JK
Christe
Michael E
ME
Chatterjee
Arindam
A
Goodson
Theodore
T
Vlahos
Chris J
CJ
Matter
William F
WF
Bloem
Laura J
LJ
D000818:Animals; D000841:Anisomycin / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000494:pharmacology; D017209:Apoptosis; D015153:Blotting, Western; D019556:COS Cells; D002384:Catalysis; D016207:Cytokines / Q000378:metabolism*; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D007375:Interleukin-1 / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D048670:MAP Kinase Kinase 4; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D008956:Models, Chemical; D009124:Muscle Proteins / Q000378:metabolism / Q000502:physiology*; D009206:Myocardium / Q000378:metabolism; D019384:Nucleic Acid Synthesis Inhibitors / Q000737:chemistry / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000502:physiology*; D011720:Pyrazoles / Q000737:chemistry / Q000494:pharmacology*; D011804:Quinolines / Q000737:chemistry / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D019076:Transgenes; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D014466:Ultraviolet Rays; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
D016207:Cytokines; C501857:DHP-2 compound; D004791:Enzyme Inhibitors; D007375:Interleukin-1; D009124:Muscle Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D011720:Pyrazoles; D011804:Quinolines; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; D000841:Anisomycin; C037219:quinoline; D005982:Glutathione Transferase; D017346:Protein Serine-Threonine Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D020930:MAP Kinase Kinase Kinases; C413047:MAP3K20 protein, human; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases