The PFKFB3 Inhibitor AZ67 Inhibits Angiogenesis Independently of Glycolysis Inhibition

Abstract

Angiogenesis is the process of new blood vessel formation. In this complex orchestrated growth, many factors are included. Lately, focus has shifted to endothelial cell metabolism, particularly to the PFKFB3 protein, a key regulatory enzyme of the glycolytic pathway. A variety of inhibitors of this important target have been studied, and a plethora of biological effects related to the process of angiogenesis have been reported. However, recent studies have disputed their mechanism of action, questioning whether all the effects are indeed due to PFKFB3 inhibition. Remarkably, the most well-studied inhibitor, 3PO, does not bind to PFKFB3, raising questions about this target. In our study, we aimed to elucidate the effects of PFKFB3 inhibition in angiogenesis by using the small molecule AZ67. We used isothermal titration calorimetry and confirmed binding to PFKFB3. In vitro, AZ67 did not decrease lactate production in endothelial cells (ECs), nor ATP levels, but exhibited good inhibitory efficacy in the tube-formation assay. Surprisingly, this was independent of EC migratory and proliferative abilities, as this was not diminished upon treatment. Strikingly however, even the lowest dose of AZ67 demonstrated significant inhibition of angiogenesis in vivo. To our knowledge, this is the first study to demonstrate that the process of angiogenesis can be disrupted by targeting PFKFB3 independently of glycolysis inhibition.

Combination of 3PO analog PFK15 and siPFKL efficiently suppresses the migration, colony formation ability, and PFK‐1 activity of triple‐negative breast cancers by reducing the glycolysis

“…48 In a recent publication by Veseli et al, authors identified that AZ67 inhibitor is highly selective to PFKFB3, and inhibits the angiogenesis process independent of glycolysis inhibition. 49 In the present manuscript, we also identified similar findings, where we show that AZ67 has no effect on the expression of PFKP and PFKL, and on extracellular lactate production, unlike PFK15, thereby suggesting their independent mechanism of action in TNBC cells. However, our results also clearly demonstrate the ability of PFK15 to modulate PFK-1 F I G U R E 8 Schematic diagram of the proposed anticancer mechanism of combination treatment (PFK15 + siPFKL) in triplenegative breast cancers (TNBCs).…”

“…Herein, we also like to discuss an ongoing debate among research fraternity over the specificity of 3PO inhibitors like PFK15, as it has been shown that PFK15 have much higher IC 50 toward PFKFB3 enzyme 48 . In a recent publication by Veseli et al, authors identified that AZ67 inhibitor is highly selective to PFKFB3, and inhibits the angiogenesis process independent of glycolysis inhibition 49 . In the present manuscript, we also identified similar findings, where we show that AZ67 has no effect on the expression of PFKP and PFKL, and on extracellular lactate production, unlike PFK15, thereby suggesting their independent mechanism of action in TNBC cells.…”

Gene‐dosage effect of Pfkfb3 on monocyte/macrophage biology in atherosclerosis

“…Second, a dose‐dependent effect of PFKFB3 inhibition might exist. Third, the PFKFB3 inhibitors may have “off‐target” effects, that have already been demonstrated (Emini Veseli et al, 2021; Wik et al, 2020). Further investigations are needed to verify these possibilities.…”

Upper glycolytic components contribute differently in controlling retinal vascular endothelial cellular behavior: Implications for endothelial-related retinal diseases

“…This involvement occurs by facilitating the expression of the pro-apoptotic transcription factor FOXO3A and its downstream genes, including p21, p27, and FAS [ 29 ]. Moreover, PFKFB3 has shown to promote pathological endothelial cell proliferation and vessel sprouting through filopodia/lamellipodia formation and directional migration [ 30 ]. Additionally, PFKFB3 activation has been linked to the impairment of endothelial barrier function within the tumor microenvironment by promoting VE-cadherin endocytosis in endothelial cells and reducing the adhesive properties of pericytes through downregulating N-cadherin expression [ 31 ].…”