Ferroquine as an oxidative shock antimalarial.

Over the course of the development of the antimalarial ferroquine, we have developed many ideas about its specific mechanism of action. Those ideas have enabled us to propose several experiments to control the validity of these hypotheses concerning differences between ferroquine and chloroquine and their respective mechanisms of action. We have now established an ultimate theory reconciling the hydrogen bond and the redox mechanisms hypotheses of ferroquine and fitting a wider range of published experimental results.

The ferroquine antimalarial conundrum: redox activation and reinvasion inhibition.

Metal health: Ferroquine is a ferrocene-based analogue of the antimalarial drug chloroquine. In addition to the primary mechanism of quinoline action, fluorescent probe studies in infected red blood cells show another mechanism is at work. It is based on the production of HO(·) in the acidic and oxidizing environment of the digestive vacuole of the malaria parasite and implies that, with ferroquine, reinvasion can be inhibited.

Deciphering the Resistance-Counteracting Functions of Ferroquine in Plasmodium falciparum-Infected Erythrocytes.

The aminoquinoline chloroquine (CQ) has been widely used for treating malaria since World War II. Resistance to CQ began to spread around 1957 and is now found in all malarious areas of the world. CQ resistance is caused by multiple mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT). These mutations result in an increased efflux of CQ from the acidic digestive vacuole (DV) to the cytosol of the parasite. This year, we proposed a strategy to locate and quantify the aminoquinolines in situ within infected red blood cells (iRBCs) using synchrotron based X-ray nanoprobe fluorescence. Direct measurements of unlabeled CQ and ferroquine (FQ) (a ferrocene-CQ conjugate, extremely active against CQ-resistant strains) enabled us to evidence fundamentally different transport mechanisms from the cytosol to the DV between CQ and FQ in the CQ-susceptible strain HB3. These results inspired the present study of the localization of CQ and FQ in the CQ-resistant strain W2. The introduction of the ferrocene core in the lateral side chain of CQ has an important consequence: the transporter is unable to efflux FQ from the DV. We also found that resistant parasites treated by FQ accumulate a sulfur-containing compound, credibly glutathion, in their DV.

The antimalarial ferroquine: role of the metal and intramolecular hydrogen bond in activity and resistance.

Inhibition of hemozoin biocrystallization is considered the main mechanism of action of 4-aminoquinoline antimalarials including chloroquine (CQ) but cannot fully explain the activity of ferroquine (FQ) which has been related to redox properties and intramolecular hydrogen bonding. Analogues of FQ, methylferroquine (Me-FQ), ruthenoquine (RQ), and methylruthenoquine (Me-RQ), were prepared. Combination of physicochemical and molecular modeling methods showed that FQ and RQ favor intramolecular hydrogen bonding between the 4-aminoquinoline NH group and the terminal amino group in the absence of water, suggesting that this structure may enhance its passage through the membrane. This was further supported by the use of Me-FQ and Me-RQ where the intramolecular hydrogen bond cannot be formed. Docking studies suggest that FQ can interact specifically with the {0,0,1} and {1,0,0} faces of hemozoin, blocking crystal growth. With respect to the structure-activity relationship, the antimalarial activity on 15 different P. falciparum strains showed that the activity of FQ and RQ were correlated with each other but not with CQ, confirming lack of cross resistance. Conversely, Me-FQ and Me-RQ showed significant cross-resistance with CQ. Mutations or copy number of pfcrt, pfmrp, pfmdr1, pfmdr2, or pfnhe-1 did not exhibit significant correlations with the IC(50) of FQ or RQ. We next showed that FQ and Me-FQ were able to generate hydroxyl radicals, whereas RQ and me-RQ did not. Ultrastructural studies revealed that FQ and Me-FQ but not RQ or Me-RQ break down the parasite digestive vacuole membrane, which could be related to the ability of the former to generate hydroxyl radicals.

Synthesis and antimalarial activities of rhenium bioorganometallics based on the 4-aminoquinoline structure.

A bioorganometallic approach to malaria therapy led to the discovery of ferroquine (FQ, SSR97193). To assess the importance of the electronic properties of the ferrocenyl group, cyclopentadienyltricarbonylrhenium analogues related to FQ, were synthesized. The reaction of [N-(7-chloro-4-quinolinyl)-1,2-ethanodiamine] with the cyrhetrenylaldehyde complexes (η(5)-C(5)H(4)CHO)Re(CO)(3) and [η(5)-1,2-C(5)H(3)(CH(2)OH)(CHO)]Re(CO)(3) produces the corresponding imine derivatives [η(5)-1,2-C(5)H(3)(R)(CHN-CH(2)CH(2)NH-QN)]Re(CO)(3) R=H 3a; R=CH(2)OH 3b; QN=N-(7-Cl-4-quinolinyl). Reduction of 3a and 3b with sodium borohydride in methanol yields quantitatively the amine complexes [η(5)-1,2-C(5)H(3)(R)(CH(2)-NH-CH(2)CH(2)NH-QN)]Re(CO)(3) R=H 4a; R=CH(2)OH 4b. To establish the role of the cyrethrenyl moiety in the antimalarial activity of this series, purely organic parent compounds were also synthesized and tested. Evaluation of antimalarial activity measured in vitro against the CQ-resistant strains (W2) and the CQ-susceptible strain (3D7) of Plasmodium falciparum indicates that these cyrhetrene conjugates are less active compared to their ferrocene and organic analogues. These data suggest an original mode-of-action of FQ and ferrocenyl analogues in relationship with the redox pharmacophore.

Plasmodium falciparum dynein light chain 1 interacts with actin/myosin during blood stage development.

Dynein light chain 1 (LC1), a member of the leucine-rich repeat protein family, has been shown to be engaged in controlling flagellar motility in Chlamydomonas reinhardtii and Trypanosoma brucei via its interaction with the dynein gamma heavy chain. In Plasmodium falciparum, we have identified the LC1 ortholog, designated Pfdlc1. Negative attempts to disrupt the dlc1 gene by reverse genetic approaches in both P. falciparum and P. berghei suggest either its essentiality for parasite survival or the inaccessibility of its locus. Expression studies revealed high levels of DLC1 protein in late trophozoites and schizonts, pointing to an unexpected role of this protein in blood-stage parasites as they do not have flagella. Interactions studies and co-immunoprecipitation experiments revealed that PfDLC1 was able to bind to P. falciparum myosin A and actin 1. The PfDLC1 interacting domains present in P. falciparum myosin A and actin 1 were mapped to sequences containing SDIE and/or EEMKT motifs present in the upper 50-kDa segment of the myosin A head domain and in the subdomain IV of actin 1, respectively. Detection of PfDLC1 by fluorescence tagging and immunofluorescence staining using specific antibodies showed a cytoplasmic location similar to actin and immunofluorescence studies showed a co-localization of PfDLC1 and myosin A. Taken together, these findings suggest that PfDLC1 might play an important role in P. falciparum erythrocytic stages by its interaction with myosin A and actin 1, known to be essential for parasite development.

Enhancement of the antimalarial activity of ciprofloxacin using a double prodrug/bioorganometallic approach.

The derivatization of the fluoroquinolone ciprofloxacin greatly increases its antimalarial activity by combining bioorganometallic chemistry and the prodrug approach. Two new achiral compounds 2 and 4 were found to be 10- to 100-fold more active than ciprofloxacin against Plasmodium falciparum chloroquine-susceptible and chloroquine-resistant strains. These achiral derivatives killed parasites more rapidly than did ciprofloxacin. Compounds 2 and 4 were revealed to be promising leads, creating a new family of antimalarial agents.

Systematic structural studies of iron superoxide dismutases from human parasites and a statistical coupling analysis of metal binding specificity.

Superoxide dismutases (SODs) are a crucial class of enzymes in the combat against intracellular free radical damage. They eliminate superoxide radicals by converting them into hydrogen peroxide and oxygen. In spite of their very different life cycles and infection strategies, the human parasites Plasmodium falciparum, Trypanosoma cruzi and Trypanosoma brucei are known to be sensitive to oxidative stress. Thus the parasite Fe-SODs have become attractive targets for novel drug development. Here we report the crystal structures of FeSODs from the trypanosomes T. brucei at 2.0 A and T. cruzi at 1.9 A resolution, and that from P. falciparum at a higher resolution (2.0 A) to that previously reported. The homodimeric enzymes are compared to the related human MnSOD with particular attention to structural aspects which are relevant for drug design. Although the structures possess a very similar overall fold, differences between the enzymes at the entrance to the channel which leads to the active site could be identified. These lead to a slightly broader and more positively charged cavity in the parasite enzymes. Furthermore, a statistical coupling analysis (SCA) for the whole Fe/MnSOD family reveals different patterns of residue coupling for Mn and Fe SODs, as well as for the dimeric and tetrameric states. In both cases, the statistically coupled residues lie adjacent to the conserved core surrounding the metal center and may be expected to be responsible for its fine tuning, leading to metal ion specificity.

Antibody response in children infected with Giardia intestinalis before and after treatment with Secnidazole.

We examined 364 school children for intestinal parasites in a sub-urban zone of Caracas, Venezuela. Giardia intestinalis was the most prevalent parasite in stool samples from 34 children. Levels of IgA and IgG antibodies to G. intestinalis were assessed by enzyme-linked immunosorbent assay and Western blot before and after treatment with secnidazole. All patients were cured with a reduction of IgA antibody levels in 26 of 34 children and a reduction in IgG-specific antibody levels in 18 of 34 children. Serum of infected patients reacted with proteins of 14 kD to 137 kD. Some patients did not show a change in IgA serum reactivity for parasite proteins by Western blot after treatment. Seventeen children showed reduction of the reactivity or disappearance of protein reactivity (mainly the 14-kD, 122-kD, and 137-kD proteins). Antibody response was not related to clinical status, but quantitative and qualitative serum antibody response against G. intestinalis infection could be used to assess levels of new protein markers that decrease or disappear with successful chemotherapy.

Ferroquine, an ingenious antimalarial drug: thoughts on the mechanism of action.

Ferroquine (FQ or SR97193) is a novel antimalarial drug candidate, currently in development at Sanofi-Aventis. In contrast to conventional drugs, FQ is the first organometallic drug: a ferrocenyl group covalently flanked by a 4-aminoquinoline and a basic alkylamine. FQ is able to overcome the CQ resistance problem, an important limit to the control of Plasmodium falciparum, the principal causative agent of malaria. After fifteen years of effort, it is now possible to propose a multifactorial mechanism of action of FQ by its capacity to target lipids, to inhibit the formation of hemozoin and to generate reactive oxygen species.