3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one and Disease-Models--Animal

The glycolytic rate in neurons is low in order to allow glucose to be metabolized through the pentose-phosphate pathway (PPP), which regenerates NADPH to preserve the glutathione redox status and survival. This is controlled by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3), the pro-glycolytic enzyme that forms fructose-2,6-bisphosphate, a powerful allosteric activator of 6-phosphofructo-1-kinase. In neurons, PFKFB3 protein is physiologically inactive due to its proteasomal degradation. However, upon an excitotoxic stimuli, PFKFB3 becomes stabilized to activate glycolysis, thus hampering PPP mediated protection of redox status leading to neurodegeneration. Here, we show that selective inhibition of PFKFB3 activity by the small molecule AZ67 prevents the NADPH oxidation, redox stress and apoptotic cell death caused by the activation of glycolysis triggered upon excitotoxic and oxygen-glucose deprivation/reoxygenation models in mouse primary neurons. Furthermore, in vivo administration of AZ67 to mice significantly alleviated the motor discoordination and brain infarct injury in the middle carotid artery occlusion ischemia/reperfusion model. These results show that pharmacological inhibition of PFKFB3 is a suitable neuroprotective therapeutic strategy in excitotoxic-related disorders such as stroke.

Topics: A549 Cells; Animals; Brain Ischemia; Cerebral Cortex; Disease Models, Animal; Enzyme Inhibitors; Fructosediphosphates; Gene Expression Regulation; Glutamic Acid; Glycolysis; Humans; Male; Mice; Neurons; Neuroprotective Agents; Pentose Phosphate Pathway; Phosphofructokinase-1; Phosphofructokinase-2; Primary Cell Culture; Proteasome Endopeptidase Complex; Proteolysis; Psychomotor Performance; Pyridines; Pyrrolidines; Reperfusion Injury

Cardiac dysfunction is a vital complication during endotoxemia (ETM). Accumulating evidence suggests that enhanced glycolytic metabolism promotes inflammatory and myocardial diseases. In this study, we performed deep mRNA sequencing analysis on the hearts of control and lipopolysaccharide (LPS)-challenged mice (40 mg/kg, i.p.) and identified that the glycolytic enzyme, 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase 3 (PFKFB3) might play an indispensable role in ETM-induced cardiac damage. Quantitative real-time PCR validated the transcriptional upregulation of PFKFB3 in the myocardium of LPS-challenged mice and immunoblotting and immunostaining assays confirmed that LPS stimulation markedly increased the expression of PFKFB3 at the protein level both in vivo and in vitro. The potent antagonist 3-(3pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) was used to block PFKFB3 activity in vivo (50 mg/kg, i.p.) and in vitro (10 μM). Echocardiographic analysis and TUNEL staining showed that 3PO significantly alleviated LPS-induced cardiac dysfunction and apoptotic injury in vivo. 3PO also suppressed the LPS-induced secretion of tumor necrosis factor-α, interleukin (IL)-1β, IL-6 and lactate in the serum, in addition to lactate in the myocardium. PFKFB3 inhibition also diminished the nuclear translocation and phosphorylation of transcription factor nuclear factor-κB (NF-κB) in both adult cardiomyocytes and HL-1 cells. Furthermore, immunoblotting analysis showed that 3PO inhibited LPS-induced apoptotic induction in cardiomyocytes. Taken together, these findings demonstrate that PFKFB3 participates in LPS-induced cardiac dysfunction via mediating inflammatory and apoptotic signaling pathway.

Topics: Animals; Anti-Inflammatory Agents; Apoptosis; Cell Line; Disease Models, Animal; Endotoxemia; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Heart Diseases; Inflammation Mediators; Lipopolysaccharides; Male; Mice, Inbred C57BL; Myocytes, Cardiac; Phosphofructokinase-2; Pyridines; Signal Transduction

Strategies targeting pathological angiogenesis have focused primarily on blocking vascular endothelial growth factor (VEGF), but resistance and insufficient efficacy limit their success, mandating alternative antiangiogenic strategies. We recently provided genetic evidence that the glycolytic activator phosphofructokinase-2/fructose-2,6-bisphosphatase 3 (PFKFB3) promotes vessel formation but did not explore the antiangiogenic therapeutic potential of PFKFB3 blockade. Here, we show that blockade of PFKFB3 by the small molecule 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) reduced vessel sprouting in endothelial cell (EC) spheroids, zebrafish embryos, and the postnatal mouse retina by inhibiting EC proliferation and migration. 3PO also suppressed vascular hyperbranching induced by inhibition of Notch or VEGF receptor 1 (VEGFR1) and amplified the antiangiogenic effect of VEGF blockade. Although 3PO reduced glycolysis only partially and transiently in vivo, this sufficed to decrease pathological neovascularization in ocular and inflammatory models. These insights may offer therapeutic antiangiogenic opportunities.

Topics: Angiogenesis Inhibitors; Animals; Cell Movement; Cell Proliferation; Disease Models, Animal; Gene Expression Regulation; Glycolysis; Human Umbilical Vein Endothelial Cells; Humans; Mice; Mice, Inbred C57BL; Neovascularization, Pathologic; Neovascularization, Physiologic; Phosphofructokinase-2; Pyridines; Retinal Vessels; Vascular Endothelial Growth Factor Receptor-1; Zebrafish