The PFKFB3 Inhibitor AZ67 Inhibits Angiogenesis Independently of Glycolysis Inhibition

Besa Emini Veseli; Pieter Van Wielendaele; Mirela Delibegovic; Wim Martinet; Guido R. Y. De Meyer

doi:10.3390/ijms22115970

The PFKFB3 Inhibitor AZ67 Inhibits Angiogenesis Independently of Glycolysis Inhibition

Besa Emini Veseli, Pieter Van Wielendaele, Mirela Delibegovic, Wim Martinet, Guido R. Y. De Meyer^*

^*Corresponding author for this work

University of Antwerp

Research output: Contribution to journal › Article › peer-review

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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.

Original language	English
Article number	e5970
Number of pages	15
Journal	International Journal of Molecular Sciences
Volume	22
Issue number	11
DOIs	https://doi.org/10.3390/ijms22115970
Publication status	Published - 31 May 2021

Bibliographical note

Funding: This study was funded by the University of Antwerp (BOF project ID 29068, 40183 and iBOF project ID 21-053). Besa Emini Veseli is a PhD fellow of the Horizon 2020 program of the Eu-ropean Union - Marie Skłodowska-Curie Actions, Innovative Training Networks (ITN), Call: H2020-MSCA-ITN-2015, NUMBER—675527—MOGLYNET.

Data Availability Statement: The data presented in this study are available on request from the corresponding author.

Acknowledgments: The authors thank Mandy Vermont for excellent technical support and Anne-Marie Lambeir for scientific advice. The authors are grateful to Bronwen Martin for critical reading of the manuscript.

Keywords

AZ PFKFB3 67
PFKFB3
endothelial cells
angiogenesis

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title = "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.",

keywords = "AZ PFKFB3 67, PFKFB3, endothelial cells, angiogenesis",

author = "{Emini Veseli}, Besa and {Van Wielendaele}, Pieter and Mirela Delibegovic and Wim Martinet and {De Meyer}, {Guido R. Y.}",

note = "Funding: This study was funded by the University of Antwerp (BOF project ID 29068, 40183 and iBOF project ID 21-053). Besa Emini Veseli is a PhD fellow of the Horizon 2020 program of the Eu-ropean Union - Marie Sk{\l}odowska-Curie Actions, Innovative Training Networks (ITN), Call: H2020-MSCA-ITN-2015, NUMBER—675527—MOGLYNET. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: The authors thank Mandy Vermont for excellent technical support and Anne-Marie Lambeir for scientific advice. The authors are grateful to Bronwen Martin for critical reading of the manuscript.",

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AU - Emini Veseli, Besa

AU - Van Wielendaele, Pieter

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AU - Martinet, Wim

AU - De Meyer, Guido R. Y.

N1 - Funding: This study was funded by the University of Antwerp (BOF project ID 29068, 40183 and iBOF project ID 21-053). Besa Emini Veseli is a PhD fellow of the Horizon 2020 program of the Eu-ropean Union - Marie Skłodowska-Curie Actions, Innovative Training Networks (ITN), Call: H2020-MSCA-ITN-2015, NUMBER—675527—MOGLYNET. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: The authors thank Mandy Vermont for excellent technical support and Anne-Marie Lambeir for scientific advice. The authors are grateful to Bronwen Martin for critical reading of the manuscript.

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N2 - 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.

AB - 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.

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KW - endothelial cells

KW - angiogenesis

U2 - 10.3390/ijms22115970

DO - 10.3390/ijms22115970

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SN - 1422-0067

VL - 22

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

IS - 11

M1 - e5970

ER -

The PFKFB3 Inhibitor AZ67 Inhibits Angiogenesis Independently of Glycolysis Inhibition

Abstract

Bibliographical note

Keywords

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