Effect of calpain inhibitors on the invasion of human erythrocytes by the parasite Plasmodium flaciparum

17 different proteinase inhibitors were screened for their effect on the erythrocyte invasion by the malaria parasite Plasmodium flaciparum. The effect was tested when the inhibitors were present in the culture medium and when they were trapped into erythrocyte ghosts. A very strong inhibition of invasion was observed in the presence of calpain inhibitors, with IC₅₀ in the order of 10^?7 M. Chymostatin, leupeptin, leupeptin, pepstatin A and bestatin also caused inhibition of the invasion, but with IC₅₀ in the order of 10^?5 M. The results suggest that participation of various proteinases in the process and point to the possibility of a calpain-mediated proteolytic event. This study may explain previous observations on the role of calcium in the invasion of the human erythrocyte by Plasmodium flaciparum.     

Impairment of the Plasmodium falciparum erythrocytic cycle induced by angiotensin peptides

Plasmodium falciparum causes the most serious complications of malaria and is a public health problem worldwide with over 2 million deaths each year. The erythrocyte invasion mechanisms by Plasmodium sp. have been well described, however the physiological aspects involving host components in this process are still poorly understood. Here, we provide evidence for the role of renin-angiotensin system (RAS) components in reducing erythrocyte invasion by P. falciparum. Angiotensin II (Ang II) reduced erythrocyte invasion in an enriched schizont culture of P. falciparum in a dose-dependent manner. Using mass spectroscopy, we showed that Ang II was metabolized by erythrocytes to Ang IV and Ang-(1-7). Parasite infection decreased Ang-(1-7) and completely abolished Ang IV formation. Similar to Ang II, Ang-(1-7) decreased the level of infection in an A779 (specific antagonist of Ang-(1-7) receptor, MAS)-sensitive manner. 10(-7) M PD123319, an AT(2) receptor antagonist, partially reversed the effects of Ang-(1-7) and Ang II. However, 10(-6) M losartan, an antagonist of the AT(1) receptor, had no effect. Gs protein is a crucial player in the Plasmodium falciparum blood cycle and angiotensin peptides can modulate protein kinase A (PKA) activity; 10(-8) M Ang II or 10(-8) M Ang-(1-7) inhibited this activity in erythrocytes by 60% and this effect was reversed by 10(-7) M A779. 10(-6) M dibutyryl-cAMP increased the level of infection and 10(-7) M PKA inhibitor decreased the level of infection by 30%. These results indicate that the effect of Ang-(1-7) on P. falciparum blood stage involves a MAS-mediated PKA inhibition. Our results indicate a crucial role for Ang II conversion into Ang-(1-7) in controlling the erythrocytic cycle of the malaria parasite, adding new functions to peptides initially described to be involved in the regulation of vascular tonus.     

Synthesis, biological evaluation and mechanistic studies of totarol amino alcohol derivatives as potential antimalarial agents

Herein we report on the semisynthesis and biological evaluation of β-amino alcohol derivatives of the natural product totarol and other simple aromatic systems. All β-amino alcohol derivatives of totarol exhibited higher antiplasmodial activity than totarol [IC(50): 11.69 μM (K1, chloroquine and multi-drug resistant strain), and 11.78 μM (D10, chloroquine sensitive strain)]-12e was the most active [IC(50): 0.63 μM (K1), and 0.61 μM (D10)]. The phenyl and naphthyl β-amino alcohol derivatives were much less active than their corresponding totarol equivalents. The majority of the β-amino alcohol derivatives of totarol were more active against K1 than the D10 strains of Plasmodium falciparum, a trend similar to the inverse relationship observed with the established aryl-amino alcohol antimalarial mefloquine. Selected compounds were shown to affect erythrocyte morphology, inhibit erythrocyte invasion and trigger CQ accumulation.     

Methylenebissantin: a rare methylene-bridged bisflavonoid from Dodonaea viscosa which inhibits Plasmodium falciparum enoyl-ACP reductase

A new methylene-bridged bisflavonoid, methylenebissantin (1), and nine known compounds, including flavonoids (2-5), diterpenoids (6 and 7), and phenol derivatives (8-10) were isolated from the aerial parts of Dodonaea viscosa Jacq. The structure elucidation was based on spectroscopic data analyses. The isolated compounds were evaluated for the inhibition of Plasmodium falciparum enoyl-ACP reductase (PfENR). Methylenebissantin (1) exhibited a moderate inhibition (IC(50) 91.13 μM) against PfENR.     

Selective antimalarial activity of tetrandrine against chloroquine resistant Plasmodium falciparum

Antimalarial activity of tetrandrine was studied using a continuous in vitro culture of Plasmodium falciparum. Experimental results showed that tetrandrine has potent antimalarial effect on both chloroquine sensitive and resistant strains of Plasmodium falciparum. Interestingly, tetrandrine is about three times more potent against the chloroquine resistant strain than it is against the sensitive strain based on their IC50 values, which were 5.09 x 10(-7) M for the sensitive strain and 1.51 x 10(-7) M for the resistant strain. In addition, reversal experiments revealed that tetrandrine cannot reverse chloroquine-resistance, although it has verapamil-like, calcium-channel-blocker activity.     

New benzimidazole derivatives as antiplasmodial agents and plasmepsin inhibitors: synthesis and analysis of structure-activity relationships

The newly synthesized benzimidazole compounds were suggested to be inhibitors of Plasmodium falciparum plasmepsin II and human cathepsin D by virtual screening of an internal library of synthetic compounds. This was confirmed by enzyme inhibition studies that gave IC(50) values in the low micromolar range (2-48μM). Ligand docking studies with plasmepsin II predicted binding of benzimidazole compounds at the center of the extended substrate-binding cleft. According to the plausible mode of binding, the pyridine ring of benzimidazole compounds interacted with S1' subsite residues whereas the acetophenone moiety was in contact with S1-S3 subsites of plasmepsin II active center. The benzimidazole derivatives were evaluated for capacity to inhibit the growth of intraerythrocytic P. falciparum in culture. Four benzimidazole compounds inhibited parasite growth at ?3μM. The most active compound 10, 1-(4-phenylphenyl)-2[2-(pyridinyl-2-yl)-1,3-benzdiazol-1-yl]ethanone showed an IC(50) of 160nM. The substitution of a phenyl group and a chlorine atom at the para position of the acetophenone moiety were shown to be crucial for antiplasmodial activity.     

Plasmepsin II inhibition and antiplasmodial activity of Primaquine-Statine 'double-drugs'

Statine-based inhibitors of Plasmepsin II (PLMII) coupled with Primaquine have been designed using the 'double-drug' approach. The IC50 values for PLMII inhibition ranged from 0.59 to 400 nM and the best IC50 value for inhibition of Plasmodium falciparum growth in vitro was 0.4 microM, which represent a remarkable improvement compared to other statine-based PLMII inhibitors.     

Design, synthesis and anti-plasmodial evaluation in vitro of new 4-aminoquinoline isatin derivatives

A new class of 4-aminoquinoline derivatives based on the natural product isatin scaffold were designed and synthesized for biological evaluation against three strains of the malaria parasite Plasmodium falciparum. These derivatives showed anti-plasmodial IC(50) values in the ranges of 1.3-0.079 and 2.0-0.050muM against a chloroquine-sensitive (D10) and two resistant (K1 and W2) strains of P. falciparum, respectively. In order to determine potential targets for this class of compounds in P. falciparum, selected compounds were also tested against the parasitic cysteine protease falcipain-2. In terms of further development of this class of isatin derivatives, two of the compounds based on a flexible alkyl chain linker and a thiosemicarbazone moiety warrant further investigation as potential anti-plasmodial leads. These two derivatives showed good in vitro activity against K1 and W2 with IC(50) values of 51 and 54nM, respectively, while retaining potency against the D10 strain with IC(50) values of 79 and 95nM, respectively. Generally speaking, the inhibitory potency of all compounds in the series against the parasites did not strongly correlate with inhibitory potency against falcipain-2 for selected compounds tested, which at best was weak to moderate, suggesting other mechanisms of inhibition may also be involved or compounds may be selectively taken up by Plasmodium falciparum.     

Purine-less death in Plasmodium falciparum induced by immucillin-H, a transition state analogue of purine nucleoside phosphorylase.

Plasmodium falciparum is responsible for the majority of life-threatening cases of malaria. Plasmodia species cannot synthesize purines de novo, whereas mammalian cells obtain purines from de novo synthesis or by purine salvage. Hypoxanthine is proposed to be the major source of purines for P. falciparum growth. It is produced from inosine phosphorolysis by purine nucleoside phosphorylase (PNP). Immucillins are powerful transition state analogue inhibitors of mammalian PNP and also inhibit P. falciparum PNP as illustrated in the accompanying article (Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Kim, K., and Schramm, V. L. (2002) J. Biol. Chem. 277, 3219-3225). This work tests the hypothesis that erythrocyte and P. falciparum PNP are essential elements for growth and survival of the parasite in culture. Immucillin-H reduces the incorporation of inosine but not hypoxanthine into nucleic acids of P. falciparum and kills P. falciparum cultured in human erythrocytes with an IC(50) of 35 nm. Growth inhibition by Imm-H is reversed by the addition of hypoxanthine but not inosine, demonstrating the metabolic block at PNP. The concentration of Imm-H required for inhibition of parasite growth varies as a function of culture hematocrit, reflecting stoichiometric titration of human erythrocyte PNP by the inhibitor. Human and P. falciparum PNPs demonstrate different specificity for inhibition by immucillins, with the 2'-deoxy analogues showing marked preference for the human enzyme. The IC(50) values for immucillin analogue toxicity to P. falciparum cultures indicate that inhibition of PNP in both the erythrocytes and the parasite is necessary to induce a purine-less death.     

Toxic effect of isolated glycophorin A on the in vitro growth of Plasmodium falciparum

We have examined the inhibitory potencies of glycophorin A, a mixture of glycophorins B and C, chymotryptic fragments of GpA, desialylated GpA, alkaliborohydride treated GpA, and the O-linked tetrasaccharide isolated from GpA on the invasion of human red blood cells by synchronous Plasmodium falciparum (strain FCB). 50% inhibition of invasion, as measured by 3H-hypoxanthine incorporation into parasites, was achieved at 14 and 155 microM for GpA and GpA-CH1, respectively. We have noticed, however, that isolated GpA exhibits a toxic effect on the intraerythrocytic growth of the parasite whereas the chymotryptic fragment (amino acid residues 1-64 of GpA) does not. Thus the inhibitory potency of isolated GpA during erythrocyte invasion by the merozoite should be regarded as the result of both an inhibitory and a toxic effect. The inhibitory effect should be attributed to the carbohydrate-rich outer portion of GpA carrying clusters of neuraminic acid. The toxic effect should be attributed to the hydrophobic region of GpA which might be capable of inserting into the membrane of free merozoites and/or erythrocytes. Our data suggest that results previously obtained with glycoprotein inhibitors carrying hydrophobic portions may have to be questioned.     

Synthesis and biological evaluation of phenolic Mannich bases of benzaldehyde and (thio)semicarbazone derivatives against the cysteine protease falcipain-2 and a chloroquine resistant strain of Plasmodium falciparum

A targeted series of phenolic Mannich bases of benzaldehyde and (thio)semicarbazone derivatives were synthesized and evaluated in vitro against the malarial cysteine protease falcipain-2 and a chloroquine resistant strain (W2) of Plasmodium falciparum. A novel series of 4-aminoquinoline semicarbazones were the most effective inhibitors of falcipain-2 (most potent inhibitor had IC(50)=0.63microM) while a bisquinoline semicarbazone compound 8f was the most potent antimalarial compound with an IC(50) of 0.07microM against W2. Compound 8f also weakly inhibited falcipain-2, with an IC(50) of 3.16microM, although its principal antiparasitic activity did not appear to be due to inhibition of this enzyme.     

Inhibition of p-aminobenzoate and folate syntheses in plants and apicomplexan parasites by natural product rubreserine

Glutamine amidotransferase/aminodeoxychorismate synthase (GAT-ADCS) is a bifunctional enzyme involved in the synthesis of p-aminobenzoate, a central component part of folate cofactors. GAT-ADCS is found in eukaryotic organisms autonomous for folate biosynthesis, such as plants or parasites of the phylum Apicomplexa. Based on an automated screening to search for new inhibitors of folate biosynthesis, we found that rubreserine was able to inhibit the glutamine amidotransferase activity of the plant GAT-ADCS with an apparent IC(50) of about 8 μM. The growth rates of Arabidopsis thaliana, Toxoplasma gondii, and Plasmodium falciparum were inhibited by rubreserine with respective IC(50) values of 65, 20, and 1 μM. The correlation between folate biosynthesis and growth inhibition was studied with Arabidopsis and Toxoplasma. In both organisms, the folate content was decreased by 40-50% in the presence of rubreserine. In both organisms, the addition of p-aminobenzoate or 5-formyltetrahydrofolate in the external medium restored the growth for inhibitor concentrations up to the IC(50) value, indicating that, within this range of concentrations, rubreserine was specific for folate biosynthesis. Rubreserine appeared to be more efficient than sulfonamides, antifolate drugs known to inhibit the invasion and proliferation of T. gondii in human fibroblasts. Altogether, these results validate the use of the bifunctional GAT-ADCS as an efficient drug target in eukaryotic cells and indicate that the chemical structure of rubreserine presents interesting anti-parasitic (toxoplasmosis, malaria) potential.     

Carbonic anhydrase inhibitors: inhibition of Plasmodium falciparum carbonic anhydrase with aromatic/heterocyclic sulfonamides-in vitro and in vivo studies

A library of aromatic/heterocyclic sulfonamides possessing a large diversity of scaffolds has been assayed for inhibition of the carbonic anhydrase (CA, EC 4.2.1.1) from the malaria parasite Plasmodium falciparum (pfCA). Low micromolar and submicromolar in vitro inhibitors were detected, whereas several compounds showed ex vivo anti-P. falciparum activity, in cell cultures. One derivative, that is, 4-(3,4-dichlorophenylureido)thioureido-benzenesulfonamide was an effective in vitro pfCA inhibitor (K(I) of 0.18 microM), inhibited the ex vivo growth of P. falciparum with an IC(50) of 1 microM, and was also effective as an antimalarial agent in mice infected with P. berghei, an animal model of human malaria infection, with an ID(50) of 10 mg/kg (chloroquine as standard showed an ID(50) of 5 mg/kg). By inhibiting the first step of pyrimidine nucleotide biosyntheses, that is, the CA-mediated carbamoylphosphate biosynthesis, sulfonamide inhibitors of the protozoan CAs may have potential for the development of novel therapies of human malaria.     

Synthesis and exploration of novel curcumin analogues as anti-malarial agents

Curcumin, a major yellow pigment and active component of turmeric, has been shown to possess anti-inflammatory and anti-cancer activities. Recent studies have indicated that curcumin inhibits chloroquine-sensitive (CQ-S) and chloroquine-resistant (CQ-R) Plasmodium falciparum growth in culture with an IC(50) of approximately 3.25 microM (MIC=13.2 microM) and IC(50) 4.21 microM (MIC=14.4 microM), respectively. In order to expand their potential as anti-malarials a series of novel curcumin derivatives were synthesized and evaluated for their ability to inhibit P. falciparum growth in culture. Several curcumin analogues examined show more effective inhibition of P. falciparum growth than curcumin. The most potent curcumin compounds 3, 6, and 11 were inhibitory for CQ-S P. falciparum at IC(50) of 0.48, 0.87, 0.92 microM and CQ-R P. falciparum at IC(50) of 0.45 microM, 0.89, 0.75 microM, respectively. Pyrazole analogue of curcumin (3) exhibited sevenfold higher anti-malarial potency against CQ-S and ninefold higher anti-malarial potency against CQ-R. Curcumin analogues described here represent a novel class of highly selective P. falciparum inhibitors and promising candidates for the design of novel anti-malarial agents.     

Erythrocyte membrane modifying agents and the inhibition of Plasmodium falciparum growth: structure-activity relationships for betulinic acid analogues

The natural triterpene betulinic acid and its analogues (betulinic aldehyde, lupeol, betulin, methyl betulinate and betulinic acid amide) caused concentration-dependent alterations of erythrocyte membrane shape towards stomatocytes or echinocytes according to their hydrogen bonding properties. Thus, the analogues with a functional group having a capacity of donating a hydrogen bond (COOH, CH(2)OH, CONH(2)) caused formation of echinocytes, whereas those lacking this ability (CH(3), CHO, COOCH(3)) induced formation of stomatocytes. Both kinds of erythrocyte alterations were prohibitive with respect to Plasmodium falciparum invasion and growth; all compounds were inhibitory with IC(50) values in the range 7-28 microM, and the growth inhibition correlated well with the extent of membrane curvature changes assessed by transmission electron microscopy. Erythrocytes pre-loaded with betulinic acid or its analogues and extensively washed in order to remove excess of the chemicals could not serve as hosts for P. falciparum parasites. Betulinic acid and congeners can be responsible for in vitro antiplasmodial activity of plant extracts, as shown for Zataria multiflora Boiss. (Labiatae) and Zizyphus vulgaris Lam. (Rhamnaceae). The activity is evidently due to the incorporation of the compounds into the lipid bilayer of erythrocytes, and may be caused by modifications of cholesterol-rich membrane rafts, recently shown to play an important role in parasite vacuolization. The established link between erythrocyte membrane modifications and antiplasmodial activity may provide a novel target for potential antimalarial drugs.     

Wright (a + b-) human erythrocytes and Plasmodium falciparum malaria

We find Wr(a + b-) erythrocytes of donor M. Fr., which appear to carry a rare glycophorin A variant, to be fully susceptible to invasion by nine isolates of Plasmodium falciparum. Thus we fail to confirm the previous publication on the refractoriness of these erythrocytes. In addition the serum of donor M. Fr., which is known to contain anti-Wrb directed against an epitope located on glycophorin A in close proximity to the erythrocyte membrane, was not found to inhibit P. falciparum invasion of blood group O Rh- red blood cells. Despite this, different lines of evidence still indicate that glycophorin A is one of the receptors for erythrocyte invasion by P. falciparum. The Wrb epitope, however, does not appear to represent a distinct receptor site, which is in contrast to previous suggestions.     

Involvement of spectrin and ATP in infection of resealed erythrocyte ghosts by the human malarial parasite, Plasmodium falciparum

Resealed erythrocyte ghosts were prepared under different experimental conditions and were tested in vitro for susceptibility to infection with the human malarial parasite, Plasmodium falciparum. Resealed ghosts, prepared by dialyzing erythrocytes in narrow membrane tubing against low ionic strength buffer that was supplemented with magnesium ATP, were as susceptible to parasite infection as were normal erythrocytes. There was a direct correlation between intraerythrocytic ATP content and susceptibility to parasite infection. Neither MgCl2 nor sodium ATP could be substituted for magnesium ATP in maintaining high intraerythrocytic ATP concentration. When resealed ghosts were loaded with antispectrin IgG, malaria merozoite invasion was inhibited. At an average intracellular antispectrin IgG concentration of 3.5 micrograms/10(8) cells, there was a 35% inhibition of parasite invasion. This inhibition was due to spectrin crosslinking within the resealed ghosts, since the monovalent, Fab' fragments of antispectrin IgG had no inhibitory effect on invasion. These results indicate that the cytoskeleton plays a role in the complex process of merozoite entry into the host erythrocyte.     

Combined Action of Inhibitors of S-Adenosylmethionine Decarboxylase with an Antimalarial Drug, Chloroquine, on Plasmodium falciparum

ABSTRACT. Methylglyoxal bis (guanylhydrazone), (MGBG) a potent competitive inhibitor of S-adenosyl-L-methionine decarboxylase activity, Berenil, a trypanocidal agent and chloroquine, the commonly used antimalarial resulted in a dose dependent inhibition of Plasmodium falciparum in vitro. The IC₅₀ values of MGBG, Berenil and chloroquine were 224 μM, 40 μM and 42 nM respectively. Parasites treated with different concentrations of MGBG or Berenil were arrested at the trophozoite stage of the erythrocytic cycle. The combined action of chloroquine (10 nM) with either Berenil (0.1 mM) or MGBG (0.1 mM) on P. falciparum growth showed an additive inhibitory effect. The effect of these inhibitors alone and in combination on polyamine biosynthesis is also reported.     

Anti-malarial effect of histone deacetylation inhibitors and mammalian tumour cytodifferentiating agents

The histones of Plasmodium falciparum represent a potential new target for anti-malarial compounds. A naturally occurring compound, apicidin, has recently been shown to inhibit the in vitro growth of P. falciparum. Apicidin was shown to hyperacetylate histones, suggesting that its mode of action is through histone deacetylase inhibition. We have tested the ability of known histone deacetylase inhibitors, mammalian tumour suppressor compounds, and cytodifferentiating agents to inhibit the in vitro growth of a drug sensitive and resistant strain of P. falciparum. Seven of the tested compounds had microM IC50 values, and trichostatin A, a histone deacetylation inhibitor and cytodifferentiating agent, was active at low nM concentrations. One compound, suberic acid bisdimethylamide, which selectively arrests tumour cells as opposed to normal mammalian cells, had an in vivo cytostatic effect against the acute murine malaria Plasmodium berghei, and one round of treatment with the compound failed to select for resistant mutations. These results suggest a promising role for histone deacetylase inhibitors and cytodifferentiating agents as antimalarial drug candidates.     

Synthesis and antimalarial and antituberculosis activities of a series of natural and unnatural 4-methoxy-6-styryl-pyran-2-ones, dihydro analogues and photo-dimers

Previous studies have identified the 3,6-dialkyl-4-hydroxy-pyran-2-one marine microbial metabolites pseudopyronines A and B to be modest growth inhibitors of Mycobacterium tuberculosis and a range of tropical diseases including Plasmodium falciparum and Leishmania donovani. In an effort to expand the structure-activity relationship of this compound class towards infectious diseases, a library of natural product and natural product-like 4-methoxy-6-styryl-pyran-2-ones and a subset of catalytically reduced examples were synthesized. In addition, the photochemical reactivity of several of the 4-methoxy-6-styryl-pyran-2-ones were investigated yielding head-to-head and head-to-tail cyclobutane dimers as well as examples of asymmetric aniba-dimer A-type dimers. All compounds were evaluated for cytotoxicity and activity against M. tuberculosis, P. falciparum, L. donovani, Trypanosoma brucei rhodesiense and Trypanosoma cruzi. Of the styryl-pyranones, natural product 3 and non-natural styrene and naphthalene substituted examples 13, 18, 21, 22 and 23 exhibited antimalarial activity (IC(50) <10 μM) with selectivity indices (SI) >10. Δ(7) Dihydro analogues were typically less active or lacked selectivity. Head-to-head and head-to-tail photodimers 5 and 34 exhibited moderate IC(50)s of 2.3 to 17 μM towards several of the parasitic organisms, while the aniba-dimer-type asymmetric dimers 31 and 33 were identified as being moderately active towards P. falciparum (IC(50) 1.5 and 1.7 μM) with good selectivity (SI ~80). The 4-tert-butyl aniba-dimer A analogue 33 also exhibited activity towards L. donovani (IC(50) 4.5 μM), suggesting further elaboration of this latter scaffold could lead to the identification of new leads for the dual treatment of malaria and leishmaniasis.