DEVELOPMENT OF ALLOSTERIC PFKFB3 PHOSPHATASE MODULATORS

Cardiovascular disease is a severe health problem, especially in the Western world. Its primary cause is atherosclerosis, which is characterized by the arterial wall thickening. Modern therapeutic strategies have restricted efficacy and the mortality still remains high. Current research has supported the idea of targeting dysregulated endothelial cell (EC) metabolism as a novel therapeutic strategy. In the scope of this PhD research work, we aim to further explore the possibilities for an improved treatment of this life threatening disease. EC glycolytic flux is up-regulated during angiogenesis and it is controlled by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3), which is hence an innovative target for atherosclerosis therapy. PFKFB3 is a homodimeric bifunctional enzyme that has a very high kinase to phosphatase activity ratio. Its activity is controlled by the N-terminus autoregulatory domain in the kinase region. The main task of this research work was to explore the alternatives for PFKFB3 modulation apart from potentially problematic ATP kinase inhibition. Virtual screening was performed on the targeted allosteric binding site and here we present the synthesis and biological evaluation of the selected libraries of PFKFB3 allosteric phosphatase modulators deriving from two design strategies. In vitro activity measurement and binding assays were performed on the isolated recombinant enzyme. A phosphatase activity measurement method was developed in-house using LC-MS and the binding assay was performed using microscale thermophoresis. Three peptides (HM 20-22) were found to be able to bind PFKFB3 with a micromolar affinity. HM 21 and 22 were able modulate the PFKFB3 phosphatase activity and interestingly showed the same overall effect on the enzyme kinetics as in the case of a very potent ATP competitive inhibitor. The outcome of the research work presented here suggests that it is possible to use an alternative approach in blocking PFKFB3 activity without inhibiting the kinase. This intriguing discovery could have a significant impact on further research about this important metabolic target and the possibility of its use in atherosclerosis therapy.