Enzymology & RedOx equilibrium

      A major contribution of our laboratory that serves as a corner stone to our various projects is the understanding that the targeted disulfide reductase (PfGR, and probably other disulfide reductases) turns over with a negative cooperativity towards NADPH binding. The half-site reactivity, an extreme case of negative cooperativity, was supposed to render the enzyme insensitive to environmental changes and was thought to provide a way to insulate some enzymes from changes in the medium, thus allowing a constant enzymatic activity despite large fluctuations in the metabolite concentration. It is precisely this property that we exploited in the drug bioactivation of the benzylNQ into the redox-active benzoylNQ in the parasite. While most laboratories in this field have not reached the objectives of developing antiparasitic drugs acting by reversible inhibition of disulfide reductases, the success of our strategy is based on the findings that we can use the half-site reactivity of the target to produce continuously a reduced product (and generated biometabolites) acting as poisons for the parasites whatever the metabolic fluctuations in the cell.
      All new compounds are tested in various mechanistic assays established in our laboratory, from enzymology to physicochemistry. They include GR substrates in GR assays, evaluation of the inhibition type and parameters (reversible and uncompetitive, irreversible and suicide), metHb redox-cycling coupled with the NADPH-GR system, inhibition of the β-hematin formation. These studies include the production and the purification of both GR from human erythrocytes and P. falciparum, as recombinant proteins overexpressed by E. coli in cultures. The screening for antimalarial activity allowed us to select a series of potent antimalarial compounds active both in vitro and in vivo in P. berghei-infected mice (intraperitoneally and per os). This parasitology investigation is performed through joined collaborations (Heidelberg, London, Antwerp), and with a PhD student, Katharina Ehrhardt, in co-tutelle between Strasbourg and Heidelberg universities.

Redox-Cycling Activity of Methemoglobin(Fe3+) into Oxyhemoglobin(Fe2+)

      Reversibility of 1,4-NQ redox reactions allows these substrates to efficiently oxidize intracellular reductants (reductases) and subsequently to reduce relevant plasmodial oxidants, including O2 and Fe(III) from various sources (FePPIX, metHb). These substrates concomitantly target insoluble heme polymers produced by the parasites and trigger their dissociation into isolated monomers (FeIIIPPIX), which in their turn are efficiently reduced into deleterious Fentonian ferrous FeIIPPIX species. To gain a better understanding of the mode of action of our redox cyclers, spectrophotometric binding titrations and assays (hematin polymerization and metHb reduction assays) were achieved. These experiments were performed to evaluate the potential of the substrates (and their putative metabolites) to interact with hematin p-p dimer (the predominant FePPIX species at pH 7.5), which is generally recognized to be an important condition to kill parasites. The ability of our 1,4-NQ to efficiently reduce metHb in a coupled GR/NADPH assay as well as to inhibit hematin polymerization has been also examined. Two series of compounds, benzylNQ and benzoylNQ series provided important information on the mechanism of action of our antiparasitic leads identified in the malaria and schistosomiasis projects. Methylene Blue, an antimalarial drug of renewed interest and use, was also revisited in this context. All the compounds possess a high affinity (KD in the µM range) for hematin Π-Π dimer comparable to those measured for the major antimalarial drugs. Even though all of the tested substrates interact with hematin, only a very few of them displayed a potent inhibitory activity in the hematin polymerization assay. We highlighted a direct correlation between ability to reduce metHb and to inhibit hematin polymerization. Besides, pre-reduction of the benzoylNQ by GR/NADPH was confirmed to be a critical step in a direct assay with metHb and only the isolated and pure benzoyl-dihydroNQ is the key species capable to reduce metHb.