Nicoletta Basilico

A combinatorial approach to 2,4,6-trisubstituted triazines with potent antimalarial activity: combining conventional synthesis and microwave-assistance.

Malaria nowadays remains one of the world’s greatest public health problems. It is responsible for two million deaths per year, mostly African children under five years old, particularly affecting peoples in developing countries. Among the four malaria species that infect humans, the parasite P. falciparum is universally considered the most aggressive. Particularly impressive is its ability in mutating forms in response to administered antiplasmodial treatment, rapidly giving rise to adaptation and resistance. Hence, it is extremely urgent to find an effective combination of antimalarial drugs, not only to improve the efficacy of the therapy, but also to prevent further development of resistance. The discovery of the great potential of artemisinin has significantly encouraged the search in this area of medicinal chemistry. However, artemisinin and its active derivatives are ideal for rapid parasite clearance and clinical recovery, but they need to be combined with longer-acting drugs to prevent recrudescence. Continuous efforts in the search for new drugs together with modeling and analytical investigations on the plasmodia mechanism of invasion have highlighted two possible important targets. The dihydrofolate reductase (DHFR) of P. falciparum is one of the few well-defined targets in antimalarial therapy. This enzyme catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate. DHFR is considered the target of cycloguanil 1 and of other antifolates in use for anti-

A Plasmodium falciparum screening assay for anti-gametocyte drugs based on parasite lactate dehydrogenase detection

OBJECTIVES Plasmodium gametocytes, responsible for malaria parasite transmission from humans to mosquitoes, represent a crucial target for new antimalarial drugs to achieve malaria elimination/eradication. We developed a novel colorimetric screening method for anti-gametocyte compounds based on the parasite lactate dehydrogenase (pLDH) assay, already standardized for asexual stages, to measure gametocyte viability and drug susceptibility. METHODS Gametocytogenesis of 3D7 and NF54 Plasmodium falciparum strains was induced in vitro and asexual parasites were depleted with N-acetylglucosamine. Gametocytes were treated with dihydroartemisinin, epoxomicin, methylene blue, primaquine, puromycin or chloroquine in 96-well plates and the pLDH activity was evaluated using a modified Makler protocol. Mosquito infectivity was measured by the standard membrane feeding assay (SMFA). RESULTS A linear correlation was found between gametocytaemia determined by Giemsa staining and pLDH activity. A concentration-dependent reduction in pLDH activity was observed after 72 h of drug treatment, whereas an additional 72 h of incubation without drugs was required to obtain complete inhibition of gametocyte viability. SMFA on treated and control gametocytes confirmed that a reduction in pLDH activity translates into reduced oocyst development in the mosquito vector. CONCLUSIONS The gametocyte pLDH assay is fast, easy to perform, cheap and reproducible and is suitable for screening novel transmission-blocking compounds, which does not require parasite transgenic lines.

Ellagitannins of the fruit rind of pomegranate (Punica granatum) antagonize in vitro the host inflammatory response mechanisms involved in the onset of malaria

BackgroundThe sun-dried rind of the immature fruit of pomegranate (Punica granatum) is presently used as a herbal formulation (OMARIA, Orissa Malaria Research Indigenous Attempt) in Orissa, India, for the therapy and prophylaxis of malaria. The pathogenesis of cerebral malaria, a complication of the infection by Plasmodium falciparum, is an inflammatory cytokine-driven disease associated to an up-regulation and activity of metalloproteinase-9 and to the increase of TNF production. The in vitro anti-plasmodial activity of Punica granatum (Pg) was recently described. The aim of the present study was to explore whether the anti-malarial effect of OMARIA could also be sustained via other mechanisms among those associated to the host immune response.MethodsFrom the methanolic extract of the fruit rind, a fraction enriched in tannins (Pg-FET) was prepared. MMP-9 secretion and expression were evaluated in THP-1 cells stimulated with haemozoin or TNF. The assays were conducted in the presence of the Pg-FET and its chemical constituents ellagic acid and punicalagin. The effect of urolithins, the ellagitannin metabolites formed by human intestinal microflora, was also investigated.ResultsPg-FET and its constituents inhibited the secretion of MMP-9 induced by haemozoin or TNF. The effect occurred at transcriptional level since MMP-9 mRNA levels were lower in the presence of the tested compounds. Urolithins as well inhibited MMP-9 secretion and expression. Pg-FET and pure compounds also inhibited MMP-9 promoter activity and NF-kB-driven transcription.ConclusionsThe beneficial effect of the fruit rind of Punica granatum for the treatment of malarial disease may be attributed to the anti-parasitic activity and the inhibition of the pro-inflammatory mechanisms involved in the onset of cerebral malaria.

Artemisinin Antimalarials Do Not Inhibit Hemozoin Formation

The mechanism of action of artemisinin antimalarials may be ascribed to C-centered radicals that alkylate biomolecules or to the peroxide moiety, which inhibits a specific, as yet undefined, target (4). It is also proposed that artemisinins kill the parasite through inhibition of hemozoin formation, thereby allowing buildup of toxic heme monomer (1, 2, 5). Artemisinin and dihydroartemisinin may be unstable under aqueous conditions used for hemozoin studies; ring-opened products (3) may therefore bind to the heme and inhibit hemozoin formation. 10-Deoxodihydroartemisin (Fig. ​(Fig.1)1) has no oxygen at C-10 and is less able to undergo ring opening under aqueous conditions. Therefore, even though it is an active antimalarial it may not interfere with hemozoin formation. To test this, dihydroartemisinin and 10-deoxodihydroartemisinin were screened for inhibition of β-hematin (hemozoin) formation by using both the HPIA (heme polymerization inhibitory activity) assay (2) (hematin in acetic acid at pH 2.7, 37°C, 18 h) and the BHIA (β-hematin inhibitory activity) assay (hemin in dimethyl sulfoxide-acetate buffer at pH 5.0, 37°C, 18 h). The first identifies ligands that bind axially with the protoporphyrin iron, and the second identifies ligands undergoing π-π interactions with hematin (6). FIG. 1. Structures of artemisinin, dihydroartemisinin, and 10-deoxodihydroartemisinin. Dihydroartemisinin showed a dose-dependent inhibition in the HPIA assay but not in the BHIA assay. The 10-deoxy compound was inactive in both assays (Fig. ​(Fig.2).2). Thus, peroxidic antimalarials do not interfere with hemozoin formation in the parasite and are differentiated from quinoline antimalarials in that they cannot bind via π-π interactions with the heme molecule. This is also evident in their failure to inhibit β-hematin formation in the BHIA assay (6). It is uncertain if dihydroartemisinin undergoes ring opening under the HPIA assay conditions. In this case, it cannot be excluded that in the HPIA assay (pH 2.7), the ring-opened form of dihydroartemisinin will form an axial ligand with the porphyrin iron, thus inhibiting β-hematin formation. 10-Deoxodihydroartemisinin cannot easily undergo ring opening and therefore cannot bind porphyrin—it has no effect on β-hematin formation, yet it displays potent antimalarial activity. FIG. 2. Results of in vitro assays of inhibition of β-hematin formation. DHA, dihydroartemisinin. Thus, inhibition of β-hematin formation in the HPIA assay by artemisinin (2) and dihydroartemisinin (this letter), but not by 10-deoxodihydroartemisinin, reflects a reactivity that is not related to their antimalarial action. Furthermore, binding of artemisinins with Fe(III)PPIX is not necessary for antimalarial activity. These data also confirm that the HPIA and BHIA assays are useful for distinguishing compounds forming π-π interactions with heme from those forming axial ligands.

Design, synthesis, and structure-activity relationship studies of 4-quinolinyl- and 9-acrydinylhydrazones as potent antimalarial agents.

Malaria is a major health problem in poverty-stricken regions where new antiparasitic drugs are urgently required at an affordable price. We report herein the design, synthesis, and biological investigation of novel antimalarial agents with low potential to develop resistance and structurally based on a highly conjugated scaffold. Starting from a new hit, the designed modifications were performed hypothesizing a specific interaction with free heme and generation of radical intermediates. This approach provided antimalarials with improved potency against chloroquine-resistant plasmodia over known drugs. A number of structure-activity relationship (SAR) trends were identified and among the analogues synthesized, the pyrrolidinylmethylarylidene and the imidazole derivatives 5r, 5t, and 8b were found as the most potent antimalarial agents of the new series. The mechanism of action of the novel compounds was investigated and their in vivo activity was assessed.

Combining 4-aminoquinoline- and clotrimazole-based pharmacophores toward innovative and potent hybrid antimalarials.

Antimalarial agents structurally based on novel pharmacophores, synthesized by low-cost synthetic procedures and characterized by low potential for developing resistance are urgently needed. Recently, we developed an innovative class of antimalarials based on a polyaromatic pharmacophore. Hybridizing the 4-aminoquinoline or the 9-aminoacridine system of known antimalarials with the clotrimazole-like pharmacophore, characterized by a polyarylmethyl group, we describe herein the development of a unique class (4a-l and 5a-c) of antimalarials selectively interacting with free heme and interfering with Plasmodium falciparum (Pf) heme metabolism. Combination of the polyarylmethyl system, able to form and stabilize radical intermediates, with the iron-complexing and conjugation-mediated electron transfer properties of the 4(9)-aminoquinoline(acridine) system led to potent antimalarials in vitro against chloroquine sensitive and resistant Pf strains. Among the compounds synthesized, 4g was active in vivo against P. chabaudi and P. berghei after oral administration and, possessing promising pharmacokinetic properties, it is a candidate for further preclinical development.

Non‐iron porphyrins inhibit β‐haematin (malaria pigment) polymerisation

Infrared spectroscopy was used to evaluate the effect of non‐iron porphyrins (protoporphyrin IX and haematoporphyrin) on haematin polymerisation to β‐haematin at acidic pH. Both molecules effectively inhibited the reaction, with haematoporphyrin 6 times as active as protoporphyrin IX. We postulated that the interaction between the π electron system of porphyrin rings leads to the formation of π–π adducts, which inhibit polymer elongation in the same way as antimalarial drugs (e.g., chloroquine); the presence of hydroxyl groups able to bind haem iron enhances activity.

4-Aminoquinoline quinolizidinyl- and quinolizidinylalkyl-derivatives with antimalarial activity

A set of quinolizidinyl and quinolizidinylalkyl derivatives of 4-amino-7-chloroquinoline and of 9-amino-6-chloro-2-methoxyacridine were prepared and tested in vitro against CQ-sensitive (D-10) and CQ-resistant (W-2) strains of Plasmodium falciparum. All compounds but one exerted significant antimalarial activity. Some of the quinolizidine derivatives were from 5 to 10 times more active than chloroquine on the CQ-resistant strain. No toxicity against mammalian cells was observed.

Macrophage preconditioning with synthetic malaria pigment reduces cytokine production via heme iron-dependent oxidative stress.

Hemozoin (malaria pigment), a polymer of hematin (ferri-protoporphyrin IX) derived from hemoglobin ingested by intraerythrocytic plasmodia, modulates cytokine production by phagocytes. Mouse peritoneal macrophages (PM) fed with synthetic β-hematin (BH), structurally identical to native hemozoin, no longer produce tumor necrosis factor α (TNFα) and nitric oxide (NO) in response to lipopolysaccharide (LPS). Impairment of NO synthesis is due to inhibition of inducible nitric oxide synthase (iNOS) production. BH-mediated inhibition of PM functions cannot be ascribed to iron release from BH because neither prevention by iron chelators nor down-regulation of iron-regulatory protein activity was detected. Inhibition appears to be related to pigment-induced oxidative stress because (a) thiol compounds partially restored PM functions, (b) heme oxygenase (HO-1) and catalase mRNA levels were up-regulated, and (c) free radicals production increased in BH-treated cells. The antioxidant defenses of the cells determine the response to BH: microglia cells, which show a lower extent of induction of HO-1 and catalase mRNAs and lower accumulation of oxygen radicals, are less sensitive to the inhibitory effect of BH on cytokine production. Results indicate that BH is resistant to degradation by HO-1 and that heme-iron mediated oxidative stress may contribute to malaria-induced immunosuppression. This study may help correlate the different clinical manifestations of malaria, ranging from uncomplicated to severe disease, with dysregulation of phagocyte functions and promote better therapeutic strategies to counteract the effects of hemozoin accumulation.

Antiplasmodial Triterpenoids from the Fruits of Neem, Azadirachta indica

Eight known and two new triterpenoid derivatives, neemfruitins A (9) and B (10), have been isolated from the fruits of neem, Azadirachta indica, a traditional antimalarial plant used by Asian and African populations. In vitro antiplasmodial tests evidenced a significant activity of the known gedunin and azadirone and the new neemfruitin A and provided useful information about the structure-antimalarial activity relationships in the limonoid class.