Tuning PFKFB3 Bisphosphatase Activity Through Allosteric Interference

The human inducible phospho-fructokinase bisphosphatase isoform 3, PFKFB3, is a crucial regulatory node in the cellular metabolism. The enzyme is an important modulator regulating the intracellular fructose-2,6-bisphosphate level. PFKFB3 is a bifunctional enzyme with an exceptionally high kinase to phosphatase ratio around 740:1. Its kinase activity can be directly inhibited by small molecules acting directly on the kinase active site. On the other hand, here we propose an innovative and indirect strategy for the modulation of PFKFB3 activity, achieved through allosteric bisphosphatase activation. A library of small peptides targeting an allosteric site was discovered and synthesized. The binding affinity was evaluated by microscale thermophoresis (MST). Furthermore, a LC-MS/MS analytical method for assessing the bisphosphatase activity of PFKFB3 was developed. The new method was applied for measuring the activation on bisphosphatase activity with the PFKFB3-binding peptides. The molecular mechanical connection between the newly discovered allosteric site to the bisphosphatase activity was also investigated using both experimental and computational methods

Novel MMP-inhibiting peptides for stabilizing atherosclerotic plaques

Aim: Matrix-degrading metalloproteases (MMPs) play an essential role in the atherogenic process, from the initial lesion to plaque rupture. A growing body of evidence show that inhibition of MMP activity or rebalancing the MMP/TIMP (tissue inhibitor of MMP) equilibrium has a potential therapeutic strategy for atherosclerosis. We studied the in vitro effects of newly designed peptides on MMP activity in endothelial cells and macrophages. Methods: Eight peptides were designed in silico and synthesized using the solid phase synthesis and characterized by circular dichroism and Dynamic Light Scattering. The mouse macrophage cell line J774A.1 and the human endothelial cell line EA.hy926 were treated with the peptides (10 \ub5\uf06dM and 10 \ub5\uf06dM) for 24 hours. Then, conditioned medium was collected and MMP-2, and MMP-9 activity was determined by gelatin gel zymography. Results: Analysis of the zymograms showed that upon 10 \ub5M treatment in endothelial cells, three out of eight peptides reduced the activity of proMMP-9 and proMMP-2 by more than 60 and 40 percent, respectively. Whereas, upon treatment at 100 \ub5M the proMMP-9 levels were less or not reduced. These could be consequent to aggregation of the peptide at a concentration higher than 50 \ub5\uf06dM. In macrophages, two of these peptides reduced the activity of proMMP-2 by more than 40 percent, but had no effect on proMMP-9 activity. The peptides, did not show any effects on MMP mRNA levels. Conclusions: These data show that these peptides show a promising inhibitory effect on MMP-9 and MMP-2 activity in endothelial cells and macrophages

Computer aided design and NMR characterization of an oligopeptide targeting the Ebola virus VP24 protein

The Ebola viral Protein 24 (VP24) inhibits interferon signaling through its interaction with the human protein Karyopherin, thus impairing the immune response of the host against the infection and increasing its rate of diffusion into the organism and its lethality. This makes VP24 a potential pharmacological target, as the inhibition of its interaction with Karyopherin could reduce Ebola virulence. In this work, we carried out an atomic level study of the network of interactions between VP24 and Karyopherin using molecular dynamics and computational alanine scanning. Modeling the VP24-Karyopherin complex allowed us to identify the amino acid residues responsible for protein-protein binding and led to the identification of a nonapeptide with VP24 binding potential. Subsequently, the ability of this peptide to actually bind VP24 in solution has been assayed using Saturation Transfer Difference NMR and Circular Dichroism. Experimental and molecular modeling data concerning the VP24-peptide complex have been compared and putative peptide binding sites and modes are discussed

Oxidative stress in pregnancy and fertility pathologies

Oxidative stress designates the state of imbalance between reactive oxygen species (ROS) production and antioxidant levels. In a healthy placenta, there is an increase in ROS production, due to formation of new tissues and inherent metabolism, but this is balanced by higher levels of antioxidants. However, this balance is lost in some situations, with a consequent increase in oxidative stress levels. Oxidative stress has been implicated in several placental disorders and pregnancy pathologies. The present review intends to summarize what is known about the relationship between oxidative stress and well-known pregnancy disorders

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