The study of enzyme kinetics, and of cell metabolism, is central for the understanding of cellular function and metabolic diseases. In case of genetic disorders, the capacity of the cellular enzymes to adapt often determines the severity of the disease. Over five hundred human diseases are due to metabolic defects. Analyses of metabolic pathways and their interconnections represent an innovative way of developing strategies to identify new therapeutic targets and molecules. An impressive recent example was demonstrated in the case of Multiple Sclerosis, where the sole use of biotin, a cofactor involved in several key enzymes has led to clinical improvement. A detailed account of metabolic flows in the cell is of primary importance in order to understand and circumvent metabolic defects. Several pathways of the cell metabolic machinery have been modeled by numerical simulations. This allowed one to anticipate the effect of certain drugs on the control of metabolic flows and pathways. These predictions have not yet been confronted with experimental evidence of such pathways, even in such a basic case as Glycolysis. In this project, we will use Dissolution Dynamic Nuclear Polarisation (DDNP) techniques to study the oxidative stage of the Pentose Phosphate Pathway (PPP) in detail. This stage includes a cascade of three enzymes glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconolactonase (6PGL) and 6-phosphogluconate dehydrogenase (6PGDH) to produce NADPH. NADPH fuels macromolecular and fatty acid biosyntheses, and is a crucial antioxidant. PPP activity is modulated by numerous factors and its alterations dramatically impairs cell functions. These facts make an in-depth understanding of the individual steps of the PPP, and a characterization of its overall kinetics and coupling to other metabolic pathways, particularly relevant. We will investigate each enzyme of the oxidative stage of the PPP one at a time, and also through a global approach; in vitro and in cell suspensions (CHO, Jurkat; Trypanosoma brucei, the causative agent of the Sleeping Sickness) in order to achieve near physiological conditions where interactions with other metabolic pathways are present. This is made possible through Dissolution Dynamic Nuclear Polarization techniques that provide unprecedented NMR signal intensity enhancements, up to 4-5 orders of magnitude. D-DNP can be applied to various nuclear spins. This dramatic sensitivity enhancement allows NMR monitoring of enzyme kinetics with unique time resolution, smaller than a second. PPP enzyme and pathway kinetics will be assessed through observation of its metabolites. Alterations caused by specific inhibitors will be studied through these experimental setups. Acronyme du projet : ENZYPOL
We plan to use and develop a D-DNP methodology that gives access to quantitative analyses of time-resolved enzyme kinetics, something impossible by traditional methods of enzymology, thereby giving access to the kinetic rate constants of the various enzymes. 6PGL from Trypanosoma brucei may represent a therapeutic target for T. brucei. This was suggested by the fact that the PPP is present not only in the cytosol, but also in the protozoon glycosome, which is absent in human cells. This could be used to increase selectivity towards T. brucei. Moreover, the lactone toxicity towards the cell, which 6PGL prevents, is likely to increase the efficiency of such a target. These represent all the more interesting perspectives, since the rather limited therapeutic panoply against this widespread Sleeping Sickness is of modest efficacy and of high toxicity. Our strategy is supported by recent work discussing G6PDH and 6PGDH as therapeutic targets. These elements represent additional incentives to our studies and the present research proposal. The first 6PGL inhibitor, synthesized by Partner 3, will be studied, and combination of known trypanocidal drugs with PPP inhibitors will be investigated on various strains of T. brucei by in-cell DDNP.
Project ID: ANR-17-CE11-0016