Elisabeth Davioud-Charvet

Glutathione reductase-catalyzed cascade of redox reactions to bioactivate potent antimalarial 1,4-naphthoquinones--a new strategy to combat malarial parasites.

Our work on targeting redox equilibria of malarial parasites propagating in red blood cells has led to the selection of six 1,4-naphthoquinones, which are active at nanomolar concentrations against the human pathogen Plasmodium falciparum in culture and against Plasmodium berghei in infected mice. With respect to safety, the compounds do not trigger hemolysis or other signs of toxicity in mice. Concerning the antimalarial mode of action, we propose that the lead benzyl naphthoquinones are initially oxidized at the benzylic chain to benzoyl naphthoquinones in a heme-catalyzed reaction within the digestive acidic vesicles of the parasite. The major putative benzoyl metabolites were then found to function as redox cyclers: (i) in their oxidized form, the benzoyl metabolites are reduced by NADPH in glutathione reductase-catalyzed reactions within the cytosols of infected red blood cells; (ii) in their reduced forms, these benzoyl metabolites can convert methemoglobin, the major nutrient of the parasite, to indigestible hemoglobin. Studies on a fluorinated suicide-substrate indicate as well that the glutathione reductase-catalyzed bioactivation of naphthoquinones is essential for the observed antimalarial activity. In conclusion, the antimalarial naphthoquinones are suggested to perturb the major redox equilibria of the targeted infected red blood cells, which might be removed by macrophages. This results in development arrest and death of the malaria parasite at the trophozoite stage.

Antimalarial versus cytotoxic properties of dual drugs derived from 4-aminoquinolines and Mannich bases: interaction with DNA.

The synthesis and biological evaluation of new organic and organometallic dual drugs designed as potential antimalarial agents are reported. A series of 4-aminoquinoline-based Mannich bases with variations in the aliphatic amino side chain were prepared via a three-steps synthesis. These compounds were also tested against chloroquine-susceptible and chloroquine-resistant strains of Plasmodium falciparum and assayed for their ability to inhibit the formation of beta-hematin in vitro using a colorimetric beta-hematin inhibition assay. Several compounds showed a marked antimalarial activity, with IC(50) and IC(90) values in the low nM range but also a high cytotoxicity against mammalian cells, in particular a highly drug-resistant glioblastoma cell line. The newly designed compounds revealed high DNA binding properties, especially for the GC-rich domains. Altogether, these dual drugs seem to be more appropriate to be developed as antiproliferative agents against mammalian cancer cells than Plasmodium parasites.

Antimalarial dual drugs based on potent inhibitors of glutathione reductase from Plasmodium falciparum.

Plasmodium parasites are exposed to higher fluxes of reactive oxygen species and need high activities of intracellular antioxidant systems providing a steady glutathione flux. As a future generation of dual drugs, 18 naphthoquinones and phenols (or their reduced forms) containing three different linkers between the 4-aminoquinoline core and the redox active component were synthesized. Their antimalarial effects have been characterized in parasite assays using chloroquine-sensitive and -resistant strains of Plasmodium, alone or in drug combination, and in the Plasmodium berghei rodent model. In particular, two tertiary amides 34 and 36 showed potent antimalarial activity in the low nanomolar range against CQ-resistant parasites. The ability to compete both for (Fe (III))protoporphyrin and for chloroquine transporter was determined. The data are consistent with the presence of a carrier for uptake of the short chloroquine analogue 2 but not for the potent antimalarial amide 34, suggesting a mode of action distinct from chloroquine mechanism.

New spermine and spermidine derivatives as potent inhibitors of Trypanosoma cruzi Trypanothione Reductase

Several spermine and spermidine derivatives containing 2-amino diphenylsulfide substituents were prepared and tested for their inhibiting effects on Trypanosoma cruzi trypanothione reductase. IC50 values were assessed between 0.3 and 3 microM. Compound 32 (Ki = 0.4 microM) is the most potent TR inhibitor described so far.

Trypanothione reductase inhibition/trypanocidal activity relationships in a 1,4-bis(3-aminopropyl)piperazine series

A series of symmetrically substituted 1,4-bis(3-aminopropyl)piperazines was synthesized and tested towards trypanothione reductase and for its in vitro trypanocidal potency. The most trypanocidal amongst them was found to be totally inactive towards the enzyme and thus constitutes a lead structure for the identification of new potential Trypanosoma cruzi target(s).

1,4-Naphthoquinones and Other NADPH-Dependent Glutathione Reductase- Catalyzed Redox Cyclers as Antimalarial Agents

The homodimeric flavoenzyme glutathione reductase catalyzes NADPH-dependent glutathione disulfide reduction. This reaction is important for keeping the redox homeostasis in human cells and in the human pathogen Plasmodium falciparum. Different types of NADPH-dependent disulfide reductase inhibitors were designed in various chemical series to evaluate the impact of each inhibition mode on the propagation of the parasites. Against malaria parasites in cultures the most potent and specific effects were observed for redox-active agents acting as subversive substrates for both glutathione reductases of the Plasmodium-infected red blood cells. In their oxidized form, these redox-active compounds are reduced by NADPH-dependent flavoenzyme-catalyzed reactions in the cytosol of infected erythrocytes. In their reduced forms, these compounds can reduce molecular oxygen to reactive oxygen species, or reduce oxidants like methemoglobin, the major nutrient of the parasite, to indigestible hemoglobin. Furthermore, studies on a fluorinated suicide-substrate of the human glutathione reductase indicate that the glutathione reductase-catalyzed bioactivation of 3-benzylnaphthoquinones to the corresponding reduced 3-benzoyl metabolites is essential for the observed antimalarial activity. In conclusion, the antimalarial lead naphthoquinones are suggested to perturb the major redox equilibria of the targeted cells. These effects result in developmental arrest of the parasite and contribute to the removal of the parasitized erythrocytes by macrophages.

Potent and specific inhibitors of trypanothione reductase from Trypanosoma cruzi: bis(2-aminodiphenylsulfides) for fluorescent labeling studies

In order to optimise the activity of bis(2-aminodiphenylsulfides) upon trypanothione reductase (TR) from Trypanosoma cruzi, a new series of bis(2-aminodiphenylsulfides) possessing three side chains was synthesized. Various moieties were introduced at the end of the third side chain, including acridinyl or biotinyl moieties for fluorescent labeling studies. TR inhibition was improved: the most potent inhibitor (IC50 = 200 nM) was selective towards TR versus human glutathione reductase and corresponded to a single myristyl group. Compounds were also tested in vitro upon Trypanosoma cruzi and Leishmania infantum amastigotes, upon-Trapanosoma brucei trypomastigotes, and for their cytotoxicity upon human MRC-5 cells. In the presence of serum, acridine derivative was no longer detectable in mass spectrometry and its antitrypanosomal activity no longer observed. This transformation might explain the absence of correlation between the potent TR inhibition and the in vitro and in vivo antiparasitic activity with both of the first generation of 2-aminodiphenylsulfides.

Parallel synthesis of a library of 1,4-naphthoquinones and automated screening of potential inhibitors of trypanothione reductase from Trypanosoma cruzi

Solid- and solution-phase parallel syntheses of 1,4-naphthoquinones (1,4-NQ) are described. A library of 1360 amides was constructed from the combination of 12 newly synthesised 1,4-NQ carboxylic acid and 120 amines, and was screened for inhibition of trypanothione reductase (TR) from Trypanosoma cruzi. The most active hits from a primary screening were re-synthesised and confirmed. This approach proves that it is possible to design potent and highly specific TcTR inhibitors deriving from menadione, juglone and plumbagin.

Evidence for the co-existence of glutathione reductase and trypanothione reductase in the non-trypanosomatid Euglenozoa: Euglena gracilis Z.

Two NADPH‐dependent disulfide reductases, glutathione reductase and trypanothione reductase, were shown to be present in Euglena gracilis, purified to homogeneity and characterized. The glutathione reductase (M r 50 kDa) displays a high specificity towards glutathione disulfide with a K M of 54 μM. The amino acid sequences of two peptides derived from the trypanothione reductase (M r 54 kDa) show a high level of identity (81% and 64%) with sequences of trypanothione reductases from trypanosomatids. The trypanothione reductase is able to efficiently reduce trypanothione disulfide (K M 30.5 μM) and glutathionylspermidine disulfide (K M 90.6 μM) but not glutathione disulfide, nor Escherichia coli thioredoxin disulfide, nor 5,5′‐dithiobis(2‐nitrobenzoate) (DTNB). These results demonstrate for the first time (i) the existence of trypanothione reductase in a non‐trypanosomatid organism and (ii) the co‐existence of trypanothione reductase and glutathione reductase in E. gracilis.

A Sugar‐Modified Phosphole Gold Complex with Antiproliferative Properties Acting as a Thioredoxin Reductase Inhibitor in MCF‐7 Cells

TrxR possesses a Cys SeCys catalytic pair in the more flexible C-terminal domain, in addition to the N-terminal Cys XXXX Cys active site. Trx contains a Cys pair which, in a reduced state, is essential for reducing disulfide bridges critical for protein activity. TrxR and Trx form an important redox system involved in multiple physiological processes. Reduced thioredoxin can provide reducing equivalents to a number of proteins including ribonucleotide reductase, which is involved in DNA synthesis and consequently in cell proliferation. The TrxR/Trx system is also involved in the cellular protection from or repair following oxidative stress, and in the regeneration of antioxidants such as vitamin C, ubiquinone, and lipoic acid. Additionally, the TrxR/Trx system modulates the activity of transcription factors involved in various signaling pathways related to apoptosis and cellular response to growth factors. TrxR and Trx have been shown to be overexpressed in several tumor types, and increased levels are known to regulate both cancer cell growth and survival. Furthermore, overexpression appears to be correlated with chemotherapeutic resistance, aggressive tumor types and poor prognosis. Taken together, these observations present the TrxR/Trx system as a target of interest for the development of new anticancer agents. Several TrxR inhibitors have already been identified and have shown growth inhibition against tumor cells (for a recent review, see ref. [6]). Among those, metal–ligand complexes are of particular interest because of their combined abilities to irreversibly target the dithiol/selenol catalytic pair essential for TrxR activity and bind DNA. Nine compounds have been evaluated in the present study for their cytotoxic activities against the human breast cancer MCF-7 cell line using a flow cytometric analysis with 2’, 7’-dichlorodihydrofluorescein diacetate (H2DCFDA) and propidium iodide (PI) staining. These compounds were selected or designed with the aim of targeting disulfide reductase activity known to be overexpressed in cancer. Analogues 1 and 2 (Figure 1) are metal-free phosphole ligands and derivative 3 has shown slight cytotoxicity in B16 melanoma and P388 leukemia cells. Phosphine-coordinated gold(I) thiosugar complexes such as auranofin (4) was developed for the treatment of rheumatoid arthritis, a disorder associated with TrxR overexpression, and reported to inhibit the purified TrxR from human placenta. Phosphole-containing gold and platinum complexes 5, 6, 7 and GoPI (8, {1-phenyl-2,5-di(2-pyridyl)phosphole}AuCl) are irreversible inhibitors of purified human GR and TrxR. 15]