Abstract
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.
Highlights
The mitogen-activated protein kinases (MAPK)1 are a highly conserved family of signal transduction molecules that trans
glutathione Stransferase (GST)-Mixed lineage kinase 7 (MLK7) was purified to homogeneity and a filter binding assay for MLK7 catalytic function with myelin basic protein (MBP) as substrate was developed
A similar experiment was performed to determine the effect of. This screen for inhibitors of MLK7 identified 7-aryl-substituted dihydropyrrolopyrazole quinolines as potent competitive inhibitors of MLK7 catalytic activity in vitro and in cell-based assays. Using this compound along with MLK7 small interfering RNA (siRNA) gene silencing, we demonstrated that the MLK7 catalytic domain functions as the mitogen-activated protein kinase kinase kinase (MAPKKK) required for Jun N-terminal kinase (JNK) and p38 signal transduction in response to anisomycin and UV radiation and has some effect on pathway activation in response to hyperosmotic stress
Summary
Compounds and Reagents—The DHP compounds were prepared as reported by Sawyer et al [23] and Toth and co-workers [24] with the key synthetic intermediate being DHP bromide (7-bromo-(2-dihydro4H-pyrrolo[1,2-b]pyrazol-3-yl)-quinoline). Transfection of cells was described previously with the exception that transfections were carried out in 96-well plates and medium was removed after 6 h of transfection and replaced with DMEM, 0.5% FBS [25]. Incubation was continued for 2 h at 37 °C, at which time medium was removed and pJNK levels in the cells were determined using phosphospecific JNK antibodies formatted either as the BioPlex pJNK assay kit (Bio-Rad) according to the manufacturer’s protocol or as a cellular ELISA. Plates were incubated with 0.6% H2O2 in PBS supplemented with 0.1% Tween 20 (PBST) for 20 min at room temperature with shaking followed by four washes in PBST. Blocking was performed at room temperature for 1.5 h with 10% FBS in PBST, and the plates were rinsed once with PBST and incubated with 1:500 dilution of pJNK antibody (Cell Signaling) at 4 °C overnight. The use of mice was approved by the Institutional Animal Care and Use Committees of the American Association for Accreditation of Laboratory animal Care-accredited institutions (Eli Lilly and Company)
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