INHIBITION OF ANGIOGENESIS USING GLYCOLYSIS INHIBITORS: AN IN VITRO STUDY

Aberrant angiogenesis contributes to many pathophysiological conditions such as cancer and diabetes mellitus. Current anti-angiogenic therapies aim at targeting key angiogenic growth factors or their endothelial cell-expressed receptors including VEGF and VEGFRs. This strategy, however, often fails to render sustained responses with minimal increased survival rate in treatment of several cancers due to toxicity and drug resistance. A novel approach in the angiogenic field is by indirect and partial inhibition of glycolysis by targeting phosphofructokinase-fructose-2,6-bisphophatase 3 (PFKFB3). Neo-angiogenesis by endothelial cells (ECs) is regulated by metabolism: mainly glycolysis. The lead anti-glycolytic compound, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), was identified as a promising compound to reduce pathological angiogenesis and tumor growth. However, doubts has risen about its true mechanism of action. We confirm that 3PO partially reduced glycolysis, EC migration enough to impair vessel sprouting. Further, we demonstrate that these effects cannot be attributed to its’ suggested binding to the target PFKFB3. 3PO does not bind to PFKFB3 and does not inhibit PFKFB3 kinase activity. Finally, 3PO caused intracellular acidification caused by lactate accumulation in ECs. In another study we have observed a strong transcriptional regulation of these transporters by 3PO. For these reasons, we speculate that 3PO regulate activity of lactate transporters, monocarboxylate transporter 1 (MCT1) and MCT4. We next identified two selective PFKFB3 inhibitors, PA-1 and PA-2, with validated binding and potent inhibitory activity towards PFKFB3. We demonstrate that these inhibitors reduced glycolysis to similar levels as silencing of PFKFB3 or using 3PO. In turn, formation capillary-like structures was impaired by inhibiting EC proliferation and migration. PFKFB3-mediated inhibition of glycolysis resulted in inhibition of transcription and activity of matrix metalloproteases (MMP)-2 and MMP-9. Furthermore, blockade of PFKFB3 with PA compounds also acted on the VEGFA/VEGFR2 axis and reduced angiogenic activation in inflamed ECs. These insights offer promising opportunities to treat aberrant angiogenesis and vasculogenesis. Taken together, our research has provided two selective PFKFB3 inhibitors to suppress neo-angiogenesis. Derivatives of these compounds are being studied in preclinical atherosclerotic models characterized by intraplaque angiogenesis. Future research will provide insight into their potential to promote atherosclerotic plaque stability.