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In silico methods have been used to identify five different classes of compounds as inhibitors of the essential Plasmodium falciparum enzyme lactate dehydrogenase (LDH). The molecules were assayed for in vitro antimalarial ac- tivity in both cell- and enzyme-based inhibition models. 5-Bromo-2-hydroxypyridine-3-carboxylic acid 19 is the most ac- tive with IC50 of 3.5 nM for chloroquine sensitive and 5 nM for resistant strains of Plasmodium falciparum, compared to 11 nM and 100 nM for the standard chloroquine. In LDH-enzyme inhibition assays the leading compounds are 5, 10, 18 and 19. Docking studies and the 3D-QSAR technique - CoRIA have been used to identify key binding elements between the molecules and residues in the LDH active site. A bifurcated salt bridge that associates the carboxylate group on the molecules with the guanidino group in the side chain of both Arg 109 and Arg 171 along with � -stack of the heterocycle with the pyridine ring of the cofactor NAD + , are the prime interactions. In silico ADME/toxicity studies also suggest these

OBJECTIVES Indolone-N-oxides are characterized by the presence of a highly reactive pharmacophore, the nitrone moiety (C=N(+)-O(-)), which undergoes oxidation-reduction reactions. The aims of the present study were to: (i) evaluate the in vitro activity of the parent compound, designated as compound 1, against 34 fresh clinical isolates of Plasmodium falciparum; (ii) compare the activity of compound 1 with that of chloroquine and dihydroartemisinin to assess the potential for cross-resistance; (iii) investigate drug interactions of indolone-N-oxides with standard antimalarials; and (iv) determine the stage-dependent activity of indolone-N-oxides. METHODS In vitro antimalarial activity was evaluated against clinical isolates collected from Cameroonian patients by the [(3)H]hypoxanthine incorporation assay. In vitro interactions between compound 1 or another analogue, compound 4, and established antimalarial drugs were assessed by the fixed ratio method. Stage specificity was evaluated by light microscopy using highly synchronized P. falciparum cultures. RESULTS The geometric mean 50% inhibitory concentration (IC(50)) of compound 1 was 48.6 nM. Its activity did not differ between the chloroquine-susceptible and the chloroquine-resistant isolates. There was no correlation between chloroquine and compound 1 responses (r = 0.015; P > 0.05), but the in vitro responses of compound 1 and dihydroartemisinin were significantly and positively correlated (r = 0.444; P

A series of 66 new indolone-N-oxide derivatives was synthesized with three different methods. Compounds were evaluated for in vitro activity against CQ-sensitive (3D7), CQ-resistant (FcB1), and CQ and pyrimethamine cross-resistant (K1) strains of Plasmodium falciparum (P.f.), as well as for cytotoxic concentration (CC(50)) on MCF7 and KB human tumor cell lines. Compound 26 (5-methoxy-indolone-N-oxide analogue) had the most potent antiplasmodial activity in vitro (3 nM on FcB1 and = 1.7 nM on 3D7) with a very satisfactory selectivity index (CC(50) MCF7/IC(50) FcB1: 14623; CC(50) KB/IC(50) 3D7: 198823). In in vivo experiments, compound 1 (dioxymethylene derivatives of the indolone-N-oxide) showed the best antiplasmodial activity against Plasmodium berghei, 62% inhibition of the parasitaemia at 30 mg/kg/day.

The immunological basis of the aberrant immune response in hyperreactive malarial splenomegaly (HMS) is poorly understood, but believed to be associated with polyclonal B cell activation by an unidentified malaria mitogen, leading to unregulated immunoglobulin and autoantibody production. HMS has been previously reported in Yanomami communities in the Upper Orinoco region of the Venezuelan Amazon. To investigate a possible association between antibody responses against Plasmodium falciparum and uninfected red blood cell (URBC) glycolipids and splenomegaly, a direct comparison of the parasite versus host anti-glycolipid antibody responses was made in an isolated community of this area. The anti-P. falciparum glycolipid (Pfglp) response was IgG3 dominated, whereas the uninfected red blood cell glycolipid (URBCglp) response showed a predominance of IgG1. The levels of IgG1 against Pfglp, and of IgG4 and IgM against URBCglp were significantly higher in women, while the anti-Pfglp or URBCglp IgM levels were inversely correlated with the degree of splenomegaly. Overall, these results suggest differential regulation of anti-parasite and autoreactive responses and that these responses may be linked to the development and evolution of HMS in this population exposed to endemic malaria. The high mortality rates associated with HMS point out that its early diagnosis together with the implementation of malaria control measures in these isolated Amerindian communities are a priority.

Artemisinin - the next generation: Efficacies of artemisone against the malaria parasite are substantially greater than those of the current artemisinin "gold standard", artesunate. Also, in contrast to most current artemisinins it displays low lipophilicity and negligible neuro- and cytotoxicity in in vitro and in vivo assays. Thus, the drug offers promise for use in artemisinin-based combination therapy. (Chemical Equation Presented). © 2006 Wiley-VCH Verlag GmbH & Co. KGaA.

Plasmodium falciparum lactate dehydrogenase (PfLDH) is essential for ATP generation. Based on structural differences within the active site between P. falciparum and human LDH, we have identified a series of heterocyclic azole-based inhibitors that selectively bind within the PfLDH but not the human LDH (hLDH) active site and showed anti-malarial activity in vitro and in vivo. Here we expand on an azole, OXD1, from this series and found that the anti-P. falciparum activity was retained against a panel of strains independently of their anti-malarial drug sensitivity profile. Trophozoites had relatively higher PfLDH enzyme activity and PfLDH-RNA expression levels than rings and were the most susceptible stages to OXD1 exposure. This is probably linked to their increased energy requirements and consistent with glycolysis being an essential metabolic pathway for parasite survival within the erythrocyte. Further structural elaboration of these azoles could lead to the identification of compounds that target P. falciparum through such a novel mechanism and with more potent anti-malarial activity.

Chemicals targeting the liver stage (LS) of the malaria parasite are useful for causal prophylaxis of malaria. In this study, four lichen metabolites, evernic acid (1); vulpic acid (2), psoromic acid (3), and, (+)-usnic acid (4), were evaluated against LS parasites of Plasmodium berghei. Inhibition Of P. falciparum blood Stage (BS) parasites was also assessed to determine stage specificity. Compound 4 displayed the highest LS activity and stage specificity (LS IC50 value 2.3 mu M, BS IC50 value 47.3 mu M). The compounds 1 - 3 inhibited one Or more enzymes (Pf FabI, PfFabG, and pfFabZ), from the Plasmodial fatty acid biosynthesis (FAS-II) pathway, a potential drug. target for LS activity. To determine species specificity and to clarify the mechanism of reported antibacterial effects, 1-4 were also evaluated against FabI homologues and Whole cells of various pathogens -(S. aureus, E. coli M. tuberculosis). Molecular modeling studies suggest that lichen acids act indirectly via binding to allosteric sites on the protein surface of the FAS-II enzymes. Potential. toxicity, of compounds was assessed in human hepatocyte and cancer cells (in vitro) as well as in a zebrafish model (in vivo):. This study indicates the therapeutic and prophylactic potential of lichen metabolites as antibacterial and antiplasmodial agents.

Malarial dihydrofolate reductase (DHFR) is the target of antifolate antimalarial drugs such as pyrimethamine and cycloguanil, the clinical efficacy of which have been compromised by resistance arising through mutations at various sites on the enzyme. Here, we describe the use of cocrystal structures with inhibitors and substrates, along with efficacy and pharmacokinetic profiling for the design, characterization, and preclinical development of a selective, highly efficacious, and orally available antimalarial drug candidate that potently inhibits both wild-type and clinically relevant mutated forms of Plasmodium falciparum (Pf) DHFR. Important structural characteristics of P218 include pyrimidine side-chain flexibility and a carboxylate group that makes charge-mediated hydrogen bonds with conserved Arg122 (PfDHFR-TS amino acid numbering). An analogous interaction of P218 with human DHFR is disfavored because of three species-dependent amino acid substitutions in the vicinity of the conserved Arg. Thus, P218 binds to the active site of PfDHFR in a substantially different fashion from the human enzyme, which is the basis for its high selectivity. Unlike pyrimethamine, P218 binds both wild-type and mutant PfDHFR in a slow-on/slow-off tight-binding mode, which prolongs the target residence time. P218, when bound to PfDHFR-TS, resides almost entirely within the envelope mapped out by the dihydrofolate substrate, which may make it less susceptible to resistance mutations. The high in vivo efficacy in a SCID mouse model of P. falciparum malaria, good oral bioavailability, favorable enzyme selectivity, and good safety characteristics of P218 make it a potential candidate for further development.

OBJECTIVES: Curcumin is a natural plant product with antimalarial activity and immunomodulatory properties. In this study we aimed to investigate its effects on CD36 expression and CD36-mediated Plasmodium falciparum phagocytosis as well as the role played by reactive oxygen species (ROS) and the peroxisome proliferator-activated receptor ? retinoid X receptor (PPAR?-RXR) in these processes. METHODS: In vitro antimalarial activity was evaluated by the [(3)H]hypoxanthine assay. ROS production and surface CD36 in human monocyte/macrophages were measured by flow cytometry. PPAR? and CD36 mRNA expression was determined by the QuantiGene Plex(®) assay and RT-qPCR. Nuclear PPAR? activation was analysed by a DNA-binding ELISA while nuclear erythroid-related factor 2 (Nrf2) expression was analysed by western blotting. P. falciparum phagocytosis was assessed by light microscopy. RESULTS: Curcumin's antimalarial activity was confirmed and did not differ between drug-susceptible and -resistant P. falciparum strains. Curcumin increased monocyte ROS production and expression of PPAR? and CD36 at the mRNA and protein levels. Although PPAR? activation was blocked by the PPAR? antagonist GW9662, CD36 expression and CD36-mediated P. falciparum phagocytosis were only inhibited by N-acetylcysteine (NAC), suggesting a PPAR?-independent CD36 expression pathway. We then identified seven putative Nrf2 antioxidant response elements on the CD36 gene promoter and showed that NAC inhibited curcumin-induced Nrf2 protein expression. CONCLUSIONS: CD36 expression and CD36-mediated P. falciparum phagocytosis by curcumin are dependent on ROS production and probably involve the Nrf2 pathway. The dual immunomodulatory and antimalarial mechanisms of curcumin action may mean that curcumin has potential as an adjuvant treatment limiting the risk of recrudescence following standard antimalarial therapy.

The clinical manifestations of cerebral malaria (CM) are well correlated with underlying major pathophysiological events occurring during an acute malaria infection, the most important of which, is the adherence of parasitized erythrocytes to endothelial cells ultimately leading to sequestration and obstruction of brain capillaries. The consequent reduction in blood flow, leads to cerebral hypoxia, localized inflammation and release of neurotoxic molecules and inflammatory cytokines by the endothelium. The pharmacological regulation of these immunopathological processes by immunomodulatory molecules may potentially benefit the management of this severe complication. Adjunctive therapy of CM patients with an appropriate immunomodulatory compound possessing even moderate anti-malarial activity with the capacity to down regulate excess production of proinflammatory cytokines and expression of adhesion molecules, could potentially reverse cytoadherence, improve survival and prevent neurological sequelae. Current major drug discovery programmes are mainly focused on novel parasite targets and mechanisms of action. However, the discovery of compounds targeting the host remains a largely unexplored but attractive area of drug discovery research for the treatment of CM. This review discusses the properties of the plant immune-modifier curcumin and its potential as an adjunctive therapy for the management of this complication.

3-Azido-, 3-amino- and 3-(1,2,3-triazol-1-yl)-β-lactams were synthesized and evaluated for their antiplasmodial activity against four strains of Plasmodium falciparum and KB cells for their cytotoxicity profiles. The presence of a cyclohexyl substituent at N-1 and a phenyl group on the triazole ring markedly improved the activity profiles of triazole-tethered β-lactam exhibiting IC(50) values of 1.13, 1.21 and 1.00 μM against 3D7, K1 and W2 strains respectively.

Tumour necrosis factor-alpha (TNF-alpha) is an endogenous mediator of shock and inflammation. Many of the life-threatening and severe pathologies associated with complicated and cerebral malaria are thought to result from the overproduction of this cytokine in response to agents of parasite origin. The identification and characterization of these agents may therefore provide the molecular basis for a detailed understanding of the disease process. Recently it has been shown that glycosylphosphatidylinositols are a novel class of glycolipid toxin produced by the parasite, which substitute for the endogenous inositolglycan-based signal transduction pathways of the host. Glycosylphosphatidylinositol stimulates high levels of TNF-alpha and interleukin-1 production by macrophages and induces hypoglycaemia through an insulin-mimetic activity, and may therefore contribute to the cerebral syndrome and other malarial pathophysiology. That monoclonal antibodies to parasite-derived glycosylphosphatidylinositol can neutralize the toxic activities of whole parasite extracts is also demonstrated here. These findings suggest a central role for glycosylphosphatidylinositol of parasite origin in the aetiology of severe malaria and suggest novel approaches for the immunotherapy or immunoprophylaxis of disease.

Chemical investigation of the methanol extract from the leaves of Arrabidaea patellifera, a Bignoniaceae from Panama, afforded mangiferin, isomangiferin, and six new derivatives (3′-O-p-hydroxybenzoylmangiferin, 3′-O-trans-coumaroylmangiferin, 6′-O-trans-coumaroylmangiferin, 3′-O-trans-cinnamoylmangiferin, 3′-O-trans-caffeoylmangiferin, and 3′-O-benzoylmangiferin). All these compounds had antioxidant and radical-scavenging activities, and four of them were relatively active in vitro against Plasmodium falciparum. The structures were determined by spectrometric and chemical methods, including 1D and 2D NMR experiments and MS analysis.

The aim of this study was to identify new compounds active against Plasmodium falciparum based on our previous research carried out on 3-phenyl-quinoxaline-2-carbonitrile 1,4-di-N-oxide derivatives. Twelve compounds were synthesized and evaluated for antimalarial activity. Eight of them showed an IC(50) less than 1 microM against the 3D7 strain. Derivative 1 demonstrated high potency (IC(50)= 0.63 microM) and good selectivity (SI=10.35), thereby becoming a new lead-compound.

To improve practical, accurate diagnosis of malaria in the Amazon rainforest of Venezuela, two rapid diagnostic tests (RDT) (OptiMAL-IT) and FalciVax) and a laboratory light microscope, used in the field with a battery-operated head lamp as an external light source, were evaluated against the standard laboratory microscope procedure for malaria detection. One hundred and thirty-six Yanomami patients were studied for the presence of malaria parasites. Thirty-three patients (24%) were positive for malaria (Plasmodium falciparum, P. vivax, P. malariae). Twenty-one (64%) of the positive patients had

As a continuation of our research and with the aim of obtaining new antimalarial agents, new series of 3-phenylquinoxaline 1,4-di-N-oxide derivatives have been synthesized following the classical Beirut reaction. Antiplasmodial activity was evaluated in vitro against Plasmodium falciparum by the incorporation of [3H]-hypoxanthine. Cytotoxicity was tested in KB cells by AlamarBlue assay. Twenty-one of the 60 compounds that were assayed against 3D7 (CQ-sensitive) showed enough activity to be also evaluated against K1 (CQ-resistant) strain. Ten of them were shown to be more active than chloroquine in the resistant strain. The most interesting compounds are 7-(methyl or methoxy)-3-(4′-fluoro or chloro)phenylquinoxaline-2-carbonitrile 1,4-di-N-oxides because of their low IC50 and their high SI shown for the K1 strain, making them valid new leads.

Objectives: The in vitro and in vivo efficacy and drug‐drug interactions of the novel semi-synthetic endoperoxide artemisone with standard antimalarials were investigated in order to provide the basis for the selection of the best partner drug. Methods: Antimalarial activity and drug interactions were evaluated in vitro against Plasmodium falciparum by the incorporation of [ 3 H]hypoxanthine. In vivo efficacy and drug interactions were assessed using the standard 4-day Peters’ test. Results: Artemisone was 10 times more potent than artesunate in vitro against a panel of 12 P. falciparum strains, independent of their susceptibility profile to antimalarial drugs, and consistently 4 to 10 times more potent than artesunate in rodent models against drug-susceptible and primaquine- or sulfadoxine/pyrimethamine-resistant Plasmodium berghei lines and chloroquine- or artemisinin-resistant lines of Plasmodium yoelii. Slight antagonistic trends were found between artemisone and chloroquine, amodiaquine, tafenoquine, atovaquone or pyrimethamine and additive to slight synergistic trends with artemisone and mefloquine, lumefantrine or quinine. Various degrees of synergy were observed in vivo between artemisone and mefloquine, chloroquine or clindamycin. Conclusions: These results confirm the increased efficacy of artemisone over artesunate against multidrug-resistant P. falciparum and provide the basis for the selection of potential partner drugs for future deployment in areas of multidrug-resistant malaria. Artemisone represents an important addition to the repertoire of artemisinin combination therapies currently in use, as it has enhanced antimalarial activity, improved bioavailability and stability over current endoperoxides.

Plasmodium falciparum causes one of the deadliest forms of malaria and resistance to the currently available drugs makes it imperative to develop new, safe and potent drugs. Parasites such as P. falciparum are unable to synthesise purines de novo and to this end often have multiple purine uptake and salvage systems. With this in mind, we have designed and synthesised libraries of purine analogues as potential anti-malarial agents. Herein, we report three compounds with promising activity against the highly chloroquine-resistant VS1 P. falciparum namely: N(6)-hydroxyadenine (1c), 2-amino-N(6)-aminoadenosine (2b) and 2-amino-N(6)-amino-N(6)-methyladenosine (4b).

Gossypol is a di-sesquiterpene natural-product in the form of a functionalised binaphthyl and is isolated from cotton plants. The compound has long been known to exhibit anti-malarial and other biological activities. Previous studies have indicated that compounds of this type target Plasmodium falciparum lactate dehydrogenase (pfLDH), an essential enzyme for energy generation within the parasite. In this study, we report that simple naphthalene-based compounds, the core of the gossypol structure, exhibit weak inhibition of the parasite lactate dehydrogenase. Crystal structures of the complexes formed by binding of these naphthalene-based compounds to their target enzyme have been used to delineate the molecular features likely to form the gossypol binding site. Two modes of binding are observed: one overlapping the pyruvate but not the co-factor site, the other bridging the binding sites for the co-factor nicontinamide group and pyruvate substrate. This latter site encompasses molecular features unique to Plasmodium forms of LDH and is likely to represent the mode of binding for gossypol derivatives that show selectivity for the parasite enzymes. We also report a substrate analogue that unexpectedly binds within the adenine pocket of the co-factor groove. Although these core pharmacophore-like molecules only exhibit low levels of inhibitory activity, these molecular snapshots provide a rational basis for renewed structure-based development of naphthalene-based compounds as anti-malarial agents.

Glycosylphosphatidyl-inositols (GPIs) are vital major glycoconjugates in intraerythrocytic stages of Plasmodium. Here, we report on the biosynthesis and the characterization of GPIs synthesized by the murine malarial parasite P. yoelii yoelii YM. Parasitized erythrocytes were labeled in vivo and in vitro with either radioactive nucleotide sugar precursors, ethanolamine or glucosamine. The pathway leading to the formation of GPI precursors was found to resemble that described for P. falciparum; however, in P. yoelii, the formation of an additional hydrophilic precursor containing an acid-labile modification was detected. The data suggest that this modification is linked to the fourth mannose attached to the trimannosyl backbone in an alpha1-2 linkage. The modification was susceptible to hydrofluoric acid (HF), but not to nitrous acid (HNO(2)). Data obtained from size-exclusion chromatography on Bio-Gel P4, and Mono Q analysis of the fragments generated by HNO(2) deamination suggest that the modification is due to the presence of an additional ethanolamine linked to the fourth mannose via a phosphodiester bond.

Free and protein-bound glycosylphosphatidylinositols (GPIs) of the blood stages of the rodent malarial parasite Plasmodium chabaudi chabaudi AS were identified and characterized. TLC analysis of material extracted by organic solvents from metabolically labelled parasites revealed a distinct set of glycolipids. These glycolipids were identified as GPIs by specific chemical and enzymic treatments and by structural analysis of their glycan and hydrophobic parts. These analyses revealed that P.c.chabaudi AS synthesizes a set of GPI-biosynthesis intermediates and two potential GPI-anchor precursors exhibiting the following structures: ethanolamine-phosphate [(alpha1-2)mannose]mannose (alpha 1-2) mannose (alpha 1-6) mannose (alpha 1-4) glucosamine - (acyl) inositol-phosphate-diacylglycerol (P.ch. alpha) and ethanolamine-phosphate - mannose (alpha 1-2) mannose (alpha 1-6) mannose (alpha 1-4) glucosamine-(acyl)inositol-phosphate-diacylglycerol (P.ch. beta). One of these GPI-anchor precursors (P.ch. alpha) possesses the same carbohydrate structure as the GPI membrane anchor of merozoite surface protein-1 from P.c.chabaudi AS.

A new region with human onchocerciasis is reported in the Unturán mountains, South Venezuela, affecting Yanomamö populations not surveyed in previous studies conducted in the Venezuelan-Brazilian border area. Its distribution probably extends towards the Upper Toototobi endemic area in Brazil. The age-standardized prevalence of Onchocerca volvulus microfilariae (mf) (67%), the prevalence of infection in those agedor = 20 years (86%), and the community microfilarial load (CMFL) (24 mf/mg), are consistent with hyperendemic transmission. Both prevalence and mean intensity increased monotonically with age without reaching a plateau, the highest values being recorded in theor = 45 years age class (respectively, 95% and 42 [geometric mean of Williams] or 172 [arithmetic mean] mf/mg). The degree of parasite overdispersion (measured by the variance/mean ratio) also increased with host age. The CMFL value, the presence of sclerosing keratitis, hanging groin, and severe skin lesions, indicated that the infection poses an important public health problem in the region.

Hydatid disease in tropical areas poses a serious diagnostic problem due to the high frequence of cross-reactivity with other endemic helminthic infections. The enzyme-linked-immunosorbent assay (ELISA) and the double diffusion arc 5 showed respectively a sensitivity of 73% and 57% and a specificity of 84-95% and 100%. However, the specificity of ELISA was greatly increased by using ovine serum and phosphorylcholine in the diluent buffer. The hydatic antigen obtained from ovine cyst fluid showed three main protein bands of 64,58 and 30 KDa using SDS PAGE and immunoblotting. Sera from patients with onchocerciasis, cysticercosis, toxocariasis and Strongyloides infection cross-reacted with the 64 and 58 KDa bands by immunoblotting. However, none of the analyzed sera recognized the 30 KDa band, that seems to be specific in this assay. The immunoblotting showed a sensitivity of 80% and a specificity of 100% when used to recognize the 30 KDa band. La Hidatidosis en áreas tropicales representa un serio problema diagnóstico por la alta frecuencia de reactividad cruzada con otras infecciones helmínticas. Las técnicas de inmunoensayo enzimático (ELISA) y doble difusión arco 5 (DD5) mostraron una sensibilidad de 73 y 57 % y una especificidad de 84-95% y 100%, respectivamente. La especificidad en la técnica de ELISA, fue mejorada sustancialmente al emplear como diluyente de los sueros una solución buffer conteniendo suero ovino normal y fosforilcolina. En líquido obtenido de hidátides de Echinococcus granulosus de origen ovino, se demostraron tres bandas de origen proteico de 64, 58 y 30 KDa de peso molecular, empleando SDS e inmunoblotting. Sueros de pacientes con estrongiloidiasis, oncocer-cosis, toxocariasis y cisticercosis reaccionaron con las bandas de 64 y 58 KDa en inmunoblotting. Sin embargo ninguno de los sueros reconoció la banda de 30 KD, la cual parece ser específica para este ensayo. La técnica de inmunobloting empleada para poner en evidencia la banda de 30 KDa con fines diagnósticos, exhibió una sensibilidad de 80% y una especificidad de 100%.