Inhibition assay of beta-hematin formation initiated by lecithin for screening new antimalarial drugs

Measurement of heme crystallization provides a tool for screening new antimalarial drugs. Current assays for heme crystallization have employed initiators such as thermo, histidine-rich proteins, and lipids extracted from parasites and infected plasma. These initiators are unnatural or require laborious steps to prepare. In this study, we used a commercially available lipid, lecithin, a kind of phospholipid containing about 50% unsaturated fatty acids, as an initiator for heme crystal (beta-hematin) formation. We demonstrated that the inhibition of lecithin-based beta-hematin formation by antimalarial drugs is highly correlated with the preformed beta-hematin-based method. In addition, the lecithin-based assay is sensitive and convenient for large-scale screening of new novel antimalarials. We also indicated that dimethyl sulfoxide is an ideal solvent for preparation of heme stock solution, which is stable and can be used for 1 month.     

Experimental conditions for testing the inhibitory activity of chloroquine on the formation of beta-hematin

Some antimalarial drugs act by inhibiting the process of ferriprotoporphyrin IX polymerization which protects the parasite against the noxious effect of this product of host cell hemoglobin digestion. As the quest for new drugs with a similar mode of action continues, high-throughput screening methods are needed. We demonstrate herein that such a recently described screening technique (Basilico et al., J. Antimicrob. Chemother. 42, 55-60, 1998) is considerably disturbed by certain ions. Thus, at the assay's pH 2.6, the phosphate ions are responsible for the inhibitory activity of chloroquine phosphate, rather than chloroquine itself. Using a combination of solubility tests and Fourier transform infrared spectrometry we also show that two alternative methods using higher pH's are also prone to salt effects and demonstrate that these can be minimized by extensive washing of the product with DMSO.     

In vitro evaluations of antimalarial drugs and their relevance to clinical outcomes

Plasmodium falciparum resistance to the former first-line antimalarials chloroquine and sulfadoxine/pyrimethamine has reached critically high levels in many malaria-endemic regions. This has spurred the introduction of several new artemisinin-based combination therapies (ACTs) that display excellent potency in treating drug-resistant malaria. Monitoring for the emergence of drug resistant P. falciparum is important for maximising the clinically effective lifespan of ACTs. Here, we provide a commentary on the article by Kaddouri et al., published in this issue of the International Journal of Parasitology, which documents the levels of susceptibility to ACT drugs and chloroquine in P. falciparum isolates from Mali. These authors report that some isolates approached a proposed in vitro threshold of resistance to monodesethyl-amodiaquine (the principal effective metabolite of amodiaquine, an important ACT partner drug), and establish baseline levels of susceptibility to the ACT drugs dihydroartemisinin and lumefantrine. The majority of clinical isolates manifested in vitro resistance to chloroquine. The authors also show good concordance between field-based assays employing a non-radioactive lactate dehydrogenase-based method of determining in vitro drug IC₅₀ values and the well-established [³H]hypoxanthine-based radioactive method. This work illustrates a good example of drug resistance surveillance, whose global coordination is being championed by the World Antimalarial Resistance Network. Our current opinion also more generally discusses the complexities inherent to conducting in vitro investigations with P. falciparum patient isolates and correlating these findings with treatment outcome data.     

An in vitro assay system for the identification of potential antimalarial drugs

Current models for antimalarial drug screening generally measure the survival of drug-treated rodents infected with Plasmodium berghei. Modifications of existing continuous culture methods for P. falciparum allow the rapid, accurate and economical determination of drug effects directly against the human pathogen. Parasite cultures can be maintained in RPMI 1640 medium supplemented with human or rabbit serum or with hypoxanthine-supplemented bovine serum. The antiparasite effects of four drugs, chloroquine, chloramphenicol, clindamycin, and halofuginone, are identical in these sera; drugs can be screened routinely against P. falciparum grown in bovine serum supplemented with hypoxanthine. Drug effects may be rapidly and accurately determined by monitoring the incorporation of 3H-hypoxanthine into parasite nucleic acids. Results obtained with this technique are highly correlated with those derived from visual counting of parasites in thin blood films. Compounds with antimalarial activity in culture may be further screened by measuring the effects of serum obtained from drug-treated rabbits on parasites in culture. The advantages of this system over models currently used for antimalarial screening are discussed.     

A colorimetric high-throughput beta-hematin inhibition screening assay for use in the search for antimalarial compounds

Antimalarial drugs such as chloroquine are believed to act by inhibiting hemozoin formation in the food vacuole of the malaria parasite. We have developed a new assay for measuring and detecting inhibition of synthetic hemozoin (beta-hematin) formation. Aqueous pyridine (5% v/v, pH 7.5) forms a low-spin complex with hematin but not with beta-hematin. Its absorbance obeys Beer's law, making it useful for quantitating hematin concentration in hematin/beta-hematin mixtures, allowing compounds to be investigated for inhibition of beta-hematin formation. The assay is rapid (60 min incubation) and requires no centrifugation. The beta-hematin inhibition data show good agreement with alternative assay methods reported by four laboratories. The assay was adapted for high-throughput colorimetric screening, allowing visual identification of beta-hematin inhibitors. In this mode, the assay successfully detected all 18 beta-hematin inhibitors in a set of 47 compounds tested, with no false positive results. The quantitative in vitro antimalarial activities of a set of 13 aminoquinolines and quinoline methanols were found to correlate significantly with beta-hematin inhibition values determined using the assay.     

Standardization of parameters for the mycobacillin synthetase activity

An effective method of preparation involving sonication was developed for cell-free mycobacillin synthetase fromBacillus subtilis. The enzyme showed optimum activity at a buffer concentration of 50 mM (Tris-HCl) and pH 7.5. ATP and Mg²⁺ which were essential for synthesis showed an optimum requirement at a ratio of 1∶1. The synthetase was markedly inhibited by ADP whereas AMP was without any effect. ATP or ATP-generating system could not be replaced by GTP, UTP or CTP. Co²⁺ and Mn²⁺ could to some extent substitute Mg²⁺. Mercapto reagents inhibited the antibiotic synthesis. Exogenous addition of pantothenic acid had no effect.     

In vitro schizontocidal activity of standard antimalarial drugs on chloroquine-sensitive and chloroquine-resistant isolates of Plasmodium falciparum

The expanding foci of multiple drug resistant malaria and emergence of different strains requires the reassessment of antimalarial activity with various drugs. In vitro response of a chloroquine sensitive and a chloroquine resistant isolate of P. falciparum to a group of 6 quinine derived and 3 artemisinin derived standard drugs has been screened, to evaluate schizontocidal activity of the drugs. In a conventional test system the IC50s were derived from the log dose response curves and evaluated by a rigorous statistical interpretation. Analysis by Tukey's test was significant for the quinine related drugs (Q < or = 0.01) and excludes the statistical significance of artemisinin related drugs in these isolates. The dose-responses of these two isolates vary with quinine derivatives, with some overlap at lower doses for the sensitive isolate than for the resistant one which manifests at higher doses.     

A physicochemical and immunologic comparison of the cell growth inhibitory activity of human lymphotoxins and interferons in vitro.

The physicochemical, immunologic, and biologic relationships between humam lymphotoxins (LT) and interferons (IF) present in supernatant fluids from lectin-stimulated peripheral blood lymphocytes (PBL) and a continuous B-lymphoblastoid cell line (PGLC-33h) were analyzed. LT activity obtained from lectin-activated PBL could not be resolved from IF activity by gel filtration chromatography. LT activity eluted in multiple peaks of activity at 70 to 90,000, and 40 to 50,000 m.w., characteristic of alpha and beta LT, respectively. IF activity in these supernatant fluids eluted as a broad band between 35 and 80,000 m.w., also suggestive of molecular heterogeneity. In contrast, this m.w. heterogeneity was not observed in LT and IF activities obtained from the PGLC-33h cell line. LT and IF eluted as separate peaks of activity at 90,000 and 25,000 m.w., respectively. In addition, acid and heat lability of PGLC-33h IF suggested similarity to type II IF. Immunologic studies, with a rabbit anti-alpha class serum that neutralized LT activity from both PBL and PGLC-33h, did not affect IF activity from either of these sources. Supernatant fluids from PGLC-33h cultures were also capable of inhibiting the proliferation of HeLa cells in vitro. The growth inhibitory activity was attributed to LT- and IF-like molecules. This evidence suggests that although cytotoxic and anti-viral activities were due to separate molecules, LT and IF have overlapping biologic activities in their ability to inhibit the proliferation of cells in vitro.     

Improvement of both plasmepsin inhibitory activity and antimalarial activity by 2-aminoethylamino substitution

We attached 2-aminoethylamino groups to allophenylnorstatine-containing plasmepsin (Plm) inhibitors and investigated SAR of the methyl or ethyl substitutions on the amino groups. Unexpectedly, compounds 22 (KNI-10743) and 25 (KNI-10742) exhibited extremely potent Plm II inhibitory activities (K_i <0.1 nM). Moreover, among our peptidomimetic Plm inhibitors, we identified the compounds with the highest antimalarial activity using a SYBR Green I-based fluorescence assay.     

Fitness costs of resistance to antimalarial drugs

It has been recently reported that the prevalence of mutations associated with chloroquine resistance declined during the dry season. Fitness costs of drug resistance were suggested to be responsible for reduced survival of mutant parasites, and only parasites surviving chronic infections were transmitted at the onset of the rainy season. This implies that during seasonal transmission, significant changes can occur in allele frequency over the course of months, rather than years. The practical consequences of these findings for monitoring dynamics of drug-resistance markers are: (i) in areas of seasonal transmission, the sampling date matters; (ii) fluctuations in mutation frequencies might be explained by seasonality; and (iii) a much-awaited experimental determination of fitness costs of drug resistance becomes within reach.     

Superinfection and the evolution of resistance to antimalarial drugs

A major issue in the control of malaria is the evolution of drug resistance. Ecological theory has demonstrated that pathogen superinfection and the resulting within-host competition influences the evolution of specific traits. Individuals infected with Plasmodium falciparum are consistently infected by multiple parasites; however, while this probably alters the dynamics of resistance evolution, there are few robust mathematical models examining this issue. We developed a general theory for modelling the evolution of resistance with host superinfection and examine: (i) the effect of transmission intensity on the rate of resistance evolution; (ii) the importance of different biological costs of resistance; and (iii) the best measure of the frequency of resistance. We find that within-host competition retards the ability and slows the rate at which drug-resistant parasites invade, particularly as the transmission rate increases. We also find that biological costs of resistance that reduce transmission are less important than reductions in the duration of drug-resistant infections. Lastly, we find that random sampling of the population for resistant parasites is likely to significantly underestimate the frequency of resistance. Considering superinfection in mathematical models of antimalarial drug resistance may thus be important for generating accurate predictions of interventions to contain resistance.     

Cell tracking and velocimetric parameters analysis as an approach to assess activity of mussel (Mytilus edulis) hemocytes in vitro

Hemocytes constitute the key element of innate immunity in bivalves, being responsible for secretion of antimicrobial peptides and release of zymogens from the prophenoloxidase system within the hemolymph compartment, reactive oxygen species production and phagocytosis. Hemocytes are found (and collected) as cells in suspension in circulating hemolymph. Hemocytes are adherent cells as well, infiltrating tissues and migrating to infected areas. In the present study, we applied an approach based on fluorescent staining and nuclei-tracking to determine migration velocity of hemocytes from the blue mussel, Mytilus edulis, in culture. Freshly collected hemocytes attached to substrate and start to move spontaneously in few minutes. Two main hemocyte morphologies can be observed: small star-shaped cells which were less motile and spread granular cells with faster migrations. Cell-tracking was combined to MTT mitochondria metabolic rate measurements in order to monitor global cell population activity over 4 days of culture. A transient peak of cell activity was recorded after 24–48 h of culture, corresponding to a speed up of cell migration. Videomicroscopy and cell tracking techniques provide new tools to characterize activity of mussel immunocytes in culture. Our analysis of hemocyte migration reveals that motility is very sensitive to cell environmental factors.     

Synthesis and antimalarial activity of urenyl Bis-chalcone in vitro and in vivo

Some novel derivatives of Bis-chalcone were synthesized and characterized by their physical and spectral data. All the synthesized compounds were subsequently screened for in vitro globin hydrolysis, β-hematin formation, and murine Plasmodium berghei, using chloroquine as the reference drug. Most of the synthesized compounds exhibited mild to moderate susceptibilities toward the parasite in comparison with the standard. The most active antimalarial compound was 1,1-Bis-[(3′,4′-N-(urenylphenyl)-3-(3″,4″,5″-trimethoxyphenyl)]-2-propen-1-one 5, with a percentage of inhibition of heme polymerization of 87.05?±?0.77, and this compound increased the survival time after infection, reduce the parasitemia and delay the progression of malaria.     

Identification of antimalarial leads with dual falcipain-2 and falcipain-3 inhibitory activity

Falcipains (FPs), cysteine proteases in the malarial parasite, are emerging as the promising antimalarial drug targets. In order to identify novel FP inhibitors, we generated a pharmacophore derived from the reported co-crystal structures of inhibitors of Plasmodium falciparum Falcipain-3 to screen the ZINC library. Further, the filters were applied for dock score, drug-like characters, and clustering of similar structures. Sixteen molecules were purchased and subject to in vitro enzyme (FP-2 and FP-3) inhibition assays. Two compounds showed in vitro inhibition of FP-2 and FP-3 at low µM concentration. The selectivity of the inhibitors can be explained based on the predicted interactions of the molecule in the active site. Further, the inhibitors were evaluated in a functional assay and were found to induce morphological changes in line with their mode of action arresting Plasmodium development. Compound 15 was most potent inhibitor identified in this study.     

Evidence for in vitro and in vivo activity of the antimalarial pyronaridine against Schistosoma

BackgroundSchistosomiasis is highly prevalent in Africa. Praziquantel is effective against adult schistosomes but leaves prepatent stages unaffected—which is a limit to patient management and elimination. Given the large-scale use of praziquantel, development of drug resistance by Schistosoma is feared. Antimalarials are promising drugs for alternative treatment strategies of Schistosoma infections. Development of drugs with activity against both malaria and schistosomiasis is particularly appealing as schistosome infections often occur concomitantly with malaria parasites in sub-Saharan Africa. Therefore, antiplasmodial compounds were progressively tested against Schistosoma in vitro, in mice, and in a clinical study.ResultsAmongst 16 drugs and 1 control tested, pyronaridine, methylene blue and 5 other antimalarials were highly active in vitro against larval stage schistosomula with a 50% inhibitory concentration below 10 μM. Both drugs were lethal to ex vivo adult worms tested at 30 μM with methylene blue also active at 10 μM. Pyronaridine treatment of mice infected with S. mansoni at the prepatent stage reduced worm burden by 82% and cured 7 out of 12 animals, however in mice adult stages remained viable. In contrast, methylene blue inhibited adult worms by 60% but cure was not achieved. In an observational pilot trial in Gabon in children, the antimalarial drug combination pyronaridine-artesunate (Pyramax) reduced S. haematobium egg excretion from 10/10 ml urine to 0/10 ml urine, and 3 out of 4 children were cured.ConclusionPyronaridine and methylene blue warrant further investigation as candidates for schistosomiasis treatment. Both compounds are approved for human use and evidence for their potential as antischistosomal compounds can be obtained directly from clinical testing. Particularly, pyronaridine-artesunate, already available as an antimalarial drug, calls for further clinical evaluation.Trial registrationClinicalTrials.gov Identifier {"type":"clinical-trial","attrs":{"text":"NCT03201770","term_id":"NCT03201770"}}NCT03201770.     

Mechanisms of resistance of Plasmodium falciparum to antimalarial drugs

Chemotherapy and chemoprophylaxis are the principal means of combating malaria parasite infections in the human host. In the last 75 years, since the introduction of synthetic antimalarials, only a small number of compounds have been found suitable for clinical usage, and this limited armoury is now greatly compromised by the spread of drug-resistant parasite strains. Our current knowledge of the molecular mechanisms underlying resistance in the lethal species Plasmodium falciparum is reviewed here.     

Design, synthesis and in vitro antimalarial activity of an acylhydrazone library

A library of acylhydrazone iron chelators was synthesized and tested for its ability to inhibit the growth of a chloroquine-resistant strain of Plasmodium falciparum. Some of these new compounds are significantly more active than desferrioxamine DFO, the iron chelator in widespread clinical use and also than the most effective chelators.     

In vitro chloroquine resistance modulation study on fresh isolates of Brazilian Plasmodium falciparum: intrinsic antimalarial activity of phenothiazine drugs

Phenothiazine drugs - fluphenazine, chlorpromazine, methotrimeprazine and trifluoperazine - were evaluated as modulating agents against Brazilian chloroquine-resistant fresh isolates of Plasmodium falciparum. Aiming to simulate therapeutic schedules, chloroquine was employed at the concentration used for sensitive falciparum malaria treatment and anti-psychotic therapeutic concentrations of the phenothiazine drugs were adopted in two-fold serial dilutions. The in vitro microtechnique for drug susceptibility was employed. Unlike earlier reported data, the phenothiazine modulating effect was not observed. However, all the drugs demonstrated intrinsic antiplasmodial activity in concentrations lower than those described in the literature. In addition, IC50 estimates have been shown to be inferior to the usual anti-psychotic therapeutic concentrations. Statistical analysis also suggested an increase in the parasitaemia rate or, even, a predominant antiparasitic effect of phenothiazine over chloroquine when used in combination.