DESIGN, SYNTHESIS AND IN VITRO EVALUATION OF PFKFB3 PHOSPHATASE ACTIVITY ALLOSTERIC MODULATORS

Cardiovascular disease is a severe health problem, especially in the Western world, and its primary cause is atherosclerosis, which is characterized by the arterial wall thickening. Modern therapeutic strategies have restricted efficacy and mortality still remains high^[1]. Current research has supported the idea of targeting disregulated endothelial cell (EC) metabolism as a novel therapeutic strategy^[2]. In the scope of MSCA Moglynet EJD, 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)^[3], 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^[4].

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 phosphatase modulators deriving from two design strategies. In vitro activity and binding assays were performed on the isolated recombinant enzyme. Phosphatase activity method was developed in-house using LC-MS instrument and binding assay was performed using Microscale thermophoresis.

^[1] J. Goveia, P. Stapor, P. Carmeliet, EMBO Mol Med 2016, 6, 1105–1120.

^[2] D. J. Rader, A. Daugherty, Nature 2008, 541, 904-913.

^[3] J. Chesney, R. Mitchell, F. Benigni, M. Bacher, L. Spiegel, Y. Al-Abed, J.H. Han, C. Metz, R. Bucala, Proc Natl Acad Sci U S A 1999, 96, 3047–3052.

^[4] S.G. Kim, N.P. Manes, M.R. El-Maghrabi, Y.H. Lee, J.Biol.Chem 2006, 281, 2939-2944.