Targeting PFKFB3 alleviates cerebral ischemia-reperfusion injury in mice

Abstract

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.

The PFKFB3 Inhibitor AZ67 Inhibits Angiogenesis Independently of Glycolysis Inhibition

“…In their efforts to design potent and selective inhibitors of metabolic kinase PFKFB3, Boyd et al, reported a series of inhibitors with high selectivity over the related PFKFB3 isoform. Among these, AZ67 was the focus of our study, as it has a high selectivity for PFKFB3 compared to other isoforms, it decreases F-2,6-P 2 levels, and it inhibits PFKFB3 kinase activity—showing good activity in the cell assays, albeit with no apparent effects on lactate production [ 33 , 36 ]. This latter effect, later also confirmed in astrocytes [ 36 ], was an interesting point when confirming angiogenesis reduction upon PFKFB3 blockage with no impact on the glycolysis pathway.…”

“…Among these, AZ67 was the focus of our study, as it has a high selectivity for PFKFB3 compared to other isoforms, it decreases F-2,6-P 2 levels, and it inhibits PFKFB3 kinase activity—showing good activity in the cell assays, albeit with no apparent effects on lactate production [ 33 , 36 ]. This latter effect, later also confirmed in astrocytes [ 36 ], was an interesting point when confirming angiogenesis reduction upon PFKFB3 blockage with no impact on the glycolysis pathway. This was important as other groups have reported that PFKFB3-driven glycolysis directly modulates vessel sprouting [ 11 ].…”

“…Our group has clearly reported that there is no binding between 3PO and the PFKFB3 protein [ 35 ]. PFK15 has shown a much higher IC 50 towards the PFKFB3 enzyme compared to what has been reported (IC 50 > 1000 µM versus 0.2 µM), while PFK158 has no effect on PFKFB3 enzymatic activity [ 36 ]. All in all, reported data on PFKFB3 inhibition rely on compounds with low specificity so that additional off targets could not be ruled out.…”

The role of omega-3 fatty acids in preventing glucocorticoid-induced reduction in human hippocampal neurogenesis and increase in apoptosis

“…Among these pathways of relevance are "Sirtuin Signaling Pathway", "fibroblast growth factor (FGF) Signaling" and "Interferon (IFN) Signaling". The gene 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) and FGF Receptor 3 (FGFR3) respectively belong to the first and second pathway, and are known to positively regulate cell generation and proliferation 48,49 . Whereas the gene interferon induced transmembrane protein 3 (1-8U) (IFITM3) belongs to the last signaling pathway and it is involved in the innate inflammatory response and known to suppress cell proliferation 50 .…”

Mitochondrial collapse links PFKFB3-promoted glycolysis with CLN7/MFSD8 neuronal ceroid lipofuscinosis pathogenesis

“…We undertook this by inhibiting PFKFB3 activity using the highly selective, rationally designed 34 compound AZ67. Incubation of Cln7 Δ ex2 neurons with AZ67 at a concentration that inhibits PFKFB3 activity without compromising survival 35 , prevented the increase in F-2,6-P2 ( Fig. 4a ) and glycolysis ( Fig.…”