Plasmodium Falciparum 3D7 Research Articles

ChemInform Abstract: Diverted Total Synthesis of Falcitidin Acyl Tetrapeptides as New Antimalarial Leads.

AbstractThe total syntheses of antimalarial agent Falcitidin (I) as well as related N‐acyl analogues (II) are outlined and the efficiency of the latter compounds against chloroquine‐sensitive Plasmodium falciparum 3D7 is tested.

In vitro antitrypanosomal and antiplasmodial activities of crude extracts and essential oils of Ocimum gratissimum Linn from Benin and influence of vegetative stage

Ethnopharmacological relevanceDifferent parts of Ocimum gratissimum Linn are largely used in folk medicine for the treatment of many diseases, some of which related to parasitical infections as fevers and headaches. In order to validate their use and to clarify the plant part which possesses the best antiparasitic properties, we decided to evaluate the in vitro antiplasmodial and antitrypanosomal activities of essential oils and crude extracts from leaves, stems and seeds of Ocimum gratissimum as well as their cytotoxicity. Materials and methodsThe essential oils and ethanol crude extracts of leaves and stems of Ocimum gratissimum from Benin, were obtained in pre and full flowering stages. Seeds obtained only in full flowering stage, were also extracted. The oils were isolated by hydrodistillation and analyzed by GC/MS and GC/FID. Extracts and essential oils were tested in vitro against Trypanosoma brucei brucei and Plasmodium falciparum. Cytotoxicity was evaluated in vitro against Chinese Hamster Ovary (CHO) cells and the human non cancer fibroblast cell line (WI38) through MTT assay to evaluate the selectivity and toxicity was assessed against Artemia salina Leach. ResultsThe essential oils and non-volatile crude extracts of Ocimum gratissimum were more active on Trypanosoma brucei brucei than on Plasmodium falciparum (3D7). This activity varies according to the vegetative stage (pre and full flowering) and the plant part (seeds, stems and leaves) extracted. The best growth inhibition of Trypanosoma brucei brucei was observed with ethanol crude extracts of leaves (IC50=1.66±0.48μg/mL) and seeds (IC50=1.29±0.42μg/mL) in full flowering stage with good selectivity (SI>10). The chemical composition of the essential oil from aerial parts (47 compounds), characterized by the presence as main constituents of p-cymene, thymol, γ-terpinene, β-myrcene and α-thujene, depends on the vegetative stage. The oil contained some minor compounds such as myrcene (IC50=2.24±0.27μg/mL), citronellal (IC50=2.76±1.55μg/mL), limonene (IC50=4.24±2.27μg/mL), with good antitrypanosomal activities. These oils and crude extracts were not toxic against Artemia salina Leach and had a low cytotoxicity except leaves and seeds ethanol extracts obtained in full flowering which showed toxicity against CHO and WI38 cells. ConclusionsOur study shows that ethanol crude extracts of leaves and seeds of Ocimum gratissimum in full flowering stage can be a good source of antitrypanosomal agents. This is the first report about the relation between the plant part extracted, the vegetative stage of the plant, the antitrypanosomal and antiplasmodial activities and the cytotoxicity of essential oils and non-volatile extracts of Ocimum gratissimum from Benin.

Development of diaminoquinazoline histone lysine methyltransferase inhibitors as potent blood-stage antimalarial compounds.

Modulating epigenetic mechanisms in malarial parasites is an emerging avenue for the discovery of novel antimalarial drugs. Previously we demonstrated the potent in vitro and in vivo antimalarial activity of (1-benzyl-4-piperidyl)[6,7-dimethoxy-2-(4-methyl-1,4-diazepin-1-yl)-4-quinazolinyl]amine (BIX01294; 1), a known human G9a inhibitor, together with its dose-dependent effects on histone methylation in the malarial parasite. This work describes our initial medicinal chemistry efforts to optimise the diaminoquinazoline chemotype for antimalarial activity. A variety of analogues were designed by substituting the 2 and 4 positions of the quinazoline core, and these molecules were tested against Plasmodium falciparum (3D7 strain). Several analogues with IC50 values as low as 18.5 nM and with low mammalian cell toxicity (HepG2) were identified. Certain pharmacophoric features required for antimalarial activity were found to be analogous to the previously published SAR of these analogues for G9a inhibition, thereby suggesting potential similarities between the malarial and human HKMT targets of this chemotype. Physiochemical, in vitro activity, and in vitro metabolism studies were also performed for a select set of potent analogues to evaluate their potential as antimalarial leads.

Identification of novel phenyl butenonyl C-glycosides with ureidyl and sulfonamidyl moieties as antimalarial agents.

A new series of C-linked phenyl butenonyl glycosides bearing ureidyl(thioureidyl) and sulfonamidyl moieties in the phenyl rings were designed, synthesized, and evaluated for their in vitro antimalarial activities against Plasmodium falciparum 3D7 (CQ sensitive) and K1 (CQ resistant) strains. Among all the compounds screened the C-linked phenyl butenonyl glycosides bearing sulfonamidyl moiety (5a) and ureidyl moiety in the phenyl ring (7d and 8c) showed promising antimalarial activities against both 3D7 and K1 strains with IC50 values in micromolar range and low cytotoxicity offering new HITS for further exploration.

Antimalarial activity of Evolvulus alsinoids Linn.-an in vitro Plasmodium falciparum specific lactate dehydrogenase enzyme inhibition assay

ObjectiveTo investigate the effect of standardized methanol extract of Evolvulus alsinoids Linn. (E. alsinoids) on Plasmodium falciparum specific lactate dehydrogenase (PfLDH) enzyme inhibition. MethodsTo carry out enzyme inhibition studies, lactate dehydrogenase was cloned from Plasmodium falciparum 3D7 strain using expression vector pET28a and expressed in Escherichia coli BL21 (DE3). Protein purification was carried out by Ni-affinity chromatography. This protein was used for the inhibition study. Methanol extract of E. alsinoids was standardized by high performance thin layer chromatography described by previous literature and screened for PfLDH enzyme inhibitory activity and compared with gossypol (a known PfLDH enzyme inhibitor). ResultsIt was found that E. alsinoids possesses a compond scopoletin and potentially inhibits PfLDH [(25.04依0.51)%]. ConclusionsMethanol extract of E. alsinoids showed significant PfLDH inhibition as evidenced from the experiments performed. The activity may be attributed to the presence of various polyphenolic and flavanoids compounds.

Antiplasmodial activity of synthetic ellipticine derivatives and an isolated analog

Ellipticine has been shown previously to exhibit excellent in vitro antiplasmodial activity and in vivo antimalarial properties that are comparable to those of the control drug chloroquine in a mouse malaria model. Ellipticine derivatives and analogs exhibit antimalarial potential however only a few have been studied to date. Herein, ellipticine and a structural analog were isolated from Aspidosperma vargasii bark. A-ring brominated and nitrated ellipticine derivatives exhibit good in vitro inhibition of Plasmodium falciparum K1 and 3D7 strains. Several of the compounds were found not to be toxic to human fetal lung fibroblasts. 9-Nitroellipticine (IC50=0.55μM) exhibits greater antiplasmodial activity than ellipticine. These results are further evidence of the antimalarial potential of ellipticine derivatives.

Gellan sulfate inhibits Plasmodium falciparum growth and invasion of red blood cells in vitro.

Here, we assessed the sulfated derivative of the microbial polysaccharide gellan gum and derivatives of λ and κ-carrageenans for their ability to inhibit Plasmodium falciparum 3D7 and Dd2 growth and invasion of red blood cells in vitro. Growth inhibition was assessed by means of flow cytometry after a 96-h exposure to the inhibitors and invasion inhibition was assessed by counting ring parasites after a 20-h exposure to them. Gellan sulfate strongly inhibited invasion and modestly inhibited growth for both P. falciparum 3D7 and Dd2; both inhibitory effects exceeded those achieved with native gellan gum. The hydrolyzed λ-carrageenan and oversulfated κ-carrageenan were less inhibitory than their native forms. In vitro cytotoxicity and anticoagulation assays performed to determine the suitability of the modified polysaccharides for in vivo studies showed that our synthesized gellan sulfate had low cytotoxicity and anticoagulant activity.

Bioassay-guided isolation and characterization of active antiplasmodial compounds from Murraya koenigii extracts against Plasmodium falciparum and Plasmodium berghei

Malaria is an overwhelming impact in the poorest countries in the world due to their prevalence, virulence and drug resistance ability. Currently, there is inadequate armoury of drugs for the treatment of malaria. This underscores the continuing need for the discovery and development of new effective and safe antimalarial drugs. To evaluate the in vitro and in vivo antimalarial activity of the leaf ethyl acetate extract of Murraya koenigii, bioassay-guided chromatographic fractionation was employed for the isolation and purification of antimalarial compounds. The in vitro antimalarial activity was assayed by the erythrocytic stages of chloroquine-sensitive strain of Plasmodium falciparum (3D7) in culture using the fluorescence-based SYBR Green I assay. The in vivo assay was done by administering mice infected with Plasmodium berghei (NK65) four consecutive daily doses of the extracts through oral route following Peter's 4-day curative standard test. The percentage suppression of parasitaemia was calculated for each dose level by comparing the parasitaemia in untreated control with those of treated mice. Cytotoxicity was determined against HeLa cells using MTT assay. Histopathology was studied in kidney, liver and spleen of isolated compound-treated Swiss albino mice. The leaf crude ethyl acetate extract of M. koenigii showed good in vitro antiplasmodial activity against P. falciparum. The in vivo test of the leaf crude ethyl acetate extract (600 mg/kg) showed reduced malaria parasitaemia by 86.6% against P. berghei in mice. Bioassay-guided fractionation of the leaf ethyl acetate extract of M. koenigii led to the isolation of two purified fractions C3B2 (2.84 g) and C3B4 (1.97 g). The purified fractions C3B2 and C3B4 were found to be active with IC50 values of 10.5 ± 0.8 and 8.25 ± 0.2 μg/mL against P. falciparum, and in vivo activity significantly reduced parasitaemia by 82.6 and 88.2% at 100 mg/kg/body weight on day 4 against P. berghei, respectively. The isolated fractions C3B2 and C3B4 were monitored by thin-layer chromatography until a single spot was obtained with R f values of 0.36 and 0.52, respectively. The pure compounds obtained in the present investigation were subjected to UV-visible spectroscopy, Fourier transformer infrared spectroscopy, 1D and 2D (1)H-Nuclear magnetic resonance (NMR), (13)C NMR, DEPT, COSY and Mass spectral analysis. Based on the spectral analysis, it is concluded that the isolated compounds were myristic acid (C3B2) and β-caryophyllene (C3B4). The cytotoxic effect of myristic acid and β-caryophyllene showed the TC50 values of >100 and 80.5 μg/mL, respectively against HeLa cell line. The histopathology study showed that protection against nephrotoxicity of kidney, hepatic damage of liver and splenocytes protection in spleen was achieved with the highest dose tested at 100 mg/kg/body weight. The present study provides evidence of antiplasmodial compounds from M. koenigii and is reported for the first time.

Diverted total synthesis of falcitidin acyl tetrapeptides as new antimalarial leads

We report not only the convergent total synthesis of falcitidin, a natural inhibitor of falcipain-2 from myxobacterium Chitinophaga, but also its diversification into a new antimalarial class of N-acyl tetrapeptides (Acyl-His-Ile-Val-Pro-NH2). Despite the lack of whole-cell activity of falcitidin itself, our study led to the identification of a trifluoromethyl (CF3) analogue displaying sub-micromolar IC50 activity against Plasmodium falciparum 3D7 in a standard blood-cell assay, but only when N-tritylated on its histidine (imidazole) residue.

Anti-plasmodial potential of crude alkaloidal extract of three plants used in traditional medicine in India

Malaria is one of the most important tropical diseases and the greatest cause of morbidity and mortality in India. The search for new antimalarial compounds has been necessitated by Plasmodium falciparum resistance to standard antimalarial drugs. Plants are important source of biologically active compounds and have potential for the development of novel antimalarial drugs. Since a number of alkaloids have been successfully used for the treatment of malaria since ancient time In this study the crude alkaloid extract of three young plants Carica papaya Linn. (Family: Caricaceae), Datura innoxia Mill. (Family: Solonaceae) and Ricinus communis Linn (Euphorbiaceae) were evaluated against Plasmodium falciparum 3D7. The mean inhibitory concentration (IC50), the mean cytotoxic concentration and the selectivity index were estimated. The cytotoxicity was estimated on Madin-Darby Canine Kidney (MDCK) cell line in maximum dose tested. The alkaloidal extract of C. papaya showed stronger antiplasmodial activity as compared to D. innoxia and R. communis, the IC50 values in the range of 28.35 to 93.17μg/mL. Results demonstrated alkaloids as the putative active compound showing promising antimalarial effect.

Acridone alkaloids with cytotoxic and antimalarial activities from Zanthoxylum simullans Hance

Background:Zanthoxylum simullans Hance is a popular natural spice belonging to the Rutaceae family and it is one of the common prescribed herbs in traditional Chinese medicine.Materials and Methods:The chemical constituents were mainly isolated and purified by silica gel column chromatography and semi-preparative High Performance Liquid Chromatography. Their structures were identified by comparing the spectral data with those reported in the literature. Cytotoxic activities for the isolated acridone alkaloids were evaluated against two prostate cancer cell lines PC-3M and Lymph Node Carcinoma of Prostate (LNCaP), and their antimalarial activities were tested against two different strains of the parasite Plasmodium falciparum 3D7, and Dd2.Results:The root bark MeOH extract of Z. simullans Hance afforded β-sitosterol, 4-methoxy benzoic acid, daucosterol, and five acridone alkaloids, normelicopidine, normelicopine, melicopine, melicopidine, and melicopicine. All five acridone alkaloids were isolated from this plant for the first time and exhibited certain cytotoxic and antimalarial activities in vitro.Conclusion:Normelicopidine was the most active against PC-3M, LNCaP and Dd2 with IC50 values of 12.5, 21.1, and 18.9 ug/mL respectively.

Brine shrimp toxicity and invitro antimalarial activity of Citrus aurantifolia (Christm.) Swingle against Plasmodium falciparum 3D7

Malaria is one of the most prevalent, devastating parasitic infectious diseases in the world. In an attempt to search for new antimalarial compounds efforts are being made to do preliminary screening of aerial parts of the plant Citrus aurantifolia (Christm.) Swingle (family Rutaceae) at early flowering and fruiting stages against chloroquine sensitive Plasmodium falciparum 3D7 strain. The plant extract were prepared in three extract forms: Ethyl acetate, Methanol and aqueous extract using standard procedure. The highest antimalarial activity was observed with ethyl acetate extract (IC50 42.73 μg/mL) as compared to methanolic extract (IC50 94.18 μg/mL) followed by aqueous extract (IC50 >100μg/mL).The toxicity of the extract was monitored with the brine shrimp lethality assay and showed no toxicity in all solvent extract tested. The preliminary phytochemical screening of aerial parts of the plant showed the presence of alkaloids, saponins, flavonoids and glycosides in all the three solvent extracts may contain antiplasmodial molecule that could be isolated by bio-guided phytochemical studies. Keywords: Plasmodium falciparum, Citrus aurantifolia, antimalarial plants, Brine shrimp

Adamantyl derivative as a potent inhibitor of Plasmodium FK506 binding protein 35.

FKBP35, FK506 binding protein family member, in Plasmodium species displays a canonical peptidyl-prolyl isomerase (PPIase) activity and is intricately involved in the protein folding process. Inhibition of PfFKBP35 by FK506 or its analogues were shown to interfere with the in vitro growth of Plasmodium falciparum. In this study, we have synthesized adamantyl derivatives, Supradamal (SRA/4a) and its analogues SRA1/4b and SRA2/4c, which demonstrate submicromolar inhibition of Plasmodium falciparum FK506 binding domain 35 (FKBD35) PPIase activity. SRA and its analogues not only inhibit the in vitro growth of Plasmodium falciparum 3D7 strain but also show stage specific activity by inhibiting the trophozoite stage of the parasite. SRA/4a also inhibits the Plasmodium vivax FKBD35 PPIase activity and our crystal structure of PvFKBD35 in complex with the SRA provides structural insights in achieving selective inhibition against Plasmodium FKBPs.

Chemical investigation of an antimalarial Chinese medicinal herb Picrorhiza scrophulariiflora

An antimalarial medicinal plant Picrorhiza scrophulariiflora was chemically investigated as part of our ongoing research in traditional chinese medicines (TCM). Mass directed fractionation of the active part of the crude extract led to the isolation of ten main components, three new compounds (1–3) and seven known compounds (4–10). Compound 10 inhibited the growth of the Plasmodium falciparum 3D7 malarial parasite line, with an IC50 value of 8.3μM. This compound accounted for ∼95% of P. falciparum growth inhibitory activity in the crude extract confirming, for this TCM, that a single compound was responsible for the antimalarial activity.

The discovery, synthesis and antimalarial evaluation of natural product-based polyamine alkaloids

Bioassay-​guided fractionation of an antimalarial ext. derived from the fungus Ramaria subaurantiaca afforded the known polyamine alkaloid, pistillarin. Nine pistillarin analogs were synthesized via EDC-​mediated chem. and these compds. along with the previously reported natural product polyamines, ianthelliformisamines A-​C and spermatinamine, were evaluated against Plasmodium falciparum (3D7) parasites and a normal human cell line to det. parasite-​specific activity. Spermatinamine (IC50 0.23 μM) and pistillarin (IC50 1.9 μM) were the two most potent antimalarials identified during these studies.

Discovery, Synthesis and Antimalarial Evaluation of Natural Product-based Polyamines

Bioassay-guided fractionation of an antimalarial organic extract derived from the Australian mushroom Ramaria subaurantica resulted in the isolation of the known spermidine-based alkaloid, pistillarin (1). This molecule was shown to inhibit a chloroquine-sensitive Plasmodium falciparum (3D7) line with an IC50 value of 1.9µM, and displayed low cytotoxicity against neonatal foreskin fibroblast (NFF) cells with an IC50 > 100µM. Substructure searching of an in-house compound library using the spermidine fragment found in 1 identified four other related and previously isolated polyamines, ianthelliformisamines A-C, and spermatinamine that warranted antimalarial investigation. Concomitantly, a small library of pistillarin analogues was synthesized and all compounds were tested in vitro against P. falciparum (3D7) parasites. The synthesis and structure activity relationships of this polyamine alkaloid library will be presented.

In vivo antimalarial activity of Keetia leucantha twigs extracts and in vitro antiplasmodial effect of their constituents

Ethnopharmacological relevanceThe West African tree Keetia leucantha (Rubiaceae) is used in traditional medicine in Benin to treat malaria. The twigs dichloromethane extract was previously shown to inhibit in vitro Plasmodium falciparum growth with no cytotoxicity (>100µg/ml on human normal fibroblasts). Materials and methodsThe dichloromethane and aqueous extracts of twigs of K. leucantha were evaluated in vivo against Plasmodium berghei NK 173 by the 4-day suppressive test and in vitro against a chloroquine-sensitive strain of Plasmodium falciparum (3D7) using the measurement of the plasmodial lactate dehydrogenase activity. Bioguided fractionations were realized and compounds were structurally elucidated using extensive spectroscopic analysis. ResultsThe in vivo antimalarial activity of K. leucantha dichloromethane and aqueous twigs extracts were assessed in mice at the dose of 200mg/kg/day. Both extracts exhibited significant effect in inhibiting parasite growth by 56.8% and 53.0% (p<0.0001) on day 7-postinfection. An LC–MS analysis and bioguided fractionations on the twigs dichloromethane extract led to the isolation and structural determination of scopoletin (1), stigmasterol (2), three phenolic compounds: vanillin (3), hydroxybenzaldehyde (4) and ferulaldehyde (5), eight triterpenic esters (6–13), oleanolic acid and ursolic acid. The antiplasmodial activity of the mixture of the eight triterpenic esters showed an antiplasmodial activity of 1.66±0.54µg/ml on the 3D7 strain, and the same range of activity was observed for isolated isomers mixtures. ConclusionsThis is the first report on the in vivo activity of K. leucantha extracts, the isolation of thirteen compounds and analysis of their antiplasmodial activity. The results obtained may partially justify the traditional use of K. leucantha to treat malaria in Benin.

Two series of new semisynthetic triterpene derivatives: differences in anti-malarial activity, cytotoxicity and mechanism of action

BackgroundThe discovery and development of anti-malarial compounds of plant origin and semisynthetic derivatives thereof, such as quinine (QN) and chloroquine (CQ), has highlighted the importance of these compounds in the treatment of malaria. Ursolic acid analogues bearing an acetyl group at C-3 have demonstrated significant anti-malarial activity. With this in mind, two new series of betulinic acid (BA) and ursolic acid (UA) derivatives with ester groups at C-3 were synthesized in an attempt to improve anti-malarial activity, reduce cytotoxicity, and search for new targets. In vitro activity against CQ-sensitive Plasmodium falciparum 3D7 and an evaluation of cytotoxicity in a mammalian cell line (HEK293T) are reported. Furthermore, two possible mechanisms of action of anti-malarial compounds have been evaluated: effects on mitochondrial membrane potential (ΔΨm) and inhibition of β-haematin formation.ResultsAmong the 18 derivatives synthesized, those having shorter side chains were most effective against CQ-sensitive P. falciparum 3D7, and were non-cytotoxic. These derivatives were three to five times more active than BA and UA. A DiOC6(3) ΔΨm assay showed that mitochondria are not involved in their mechanism of action. Inhibition of β-haematin formation by the active derivatives was weaker than with CQ. Compounds of the BA series were generally more active against P. falciparum 3D7 than those of the UA series.ConclusionsThree new anti-malarial prototypes were obtained from natural sources through an easy and relatively inexpensive synthesis. They represent an alternative for new lead compounds for anti-malarial chemotherapy.

Prelimanary Quantitative Structure-Activity Relationship Study of 9,10- Anthraquinone Analogues Based on their Antiplasmodial Activity

Anthraquinones isolated from the roots of Rennellia elliptica Korth. demonstrated interesting antiplasmodial activity. In addition, a series of 9,10-anthraquinones resembling those isolated from R. elliptica were synthesized and preliminary two dimensional quantitative SAR (2D-QSAR) study was performed to examine physico-chemical properties essential for antiplasmodial activity of 9,10-anthraquinones. The analogues of bioactive natural anthraquinones were synthesized through Friedel-Craft reaction between phthalic anhydride and various benzene derivatives in the presence of eutectic mixture of aluminium chloride and sodium chloride. The antiplasmodial activity was determined based on inhibition of the compounds against Plasmodium falciparum (3D7) growth in vitro. The 2D-QSAR models were developed using 24 anthraquinones as data set using Vlife MDS 3.5 software. Multiple linear regression (MLR) and principal component regression (PCR) methods were used coupled with various feature selection methods i.e. stepwise forward, stepwise forward-backward, genetic algorithm and simulated annealing. The models were accepted considering the term selection criterion such as r, q, rse and pred_r. The training and test set were selected using random selection method by selecting randomly 30 % of molecules from data set for test set. Dipole moment and SaaCHE-index descriptors gave negative contribution towards the antiplasmodial activity of 9,10anthraquinones. The coefficient of regression is moderate with weak internal and external predictive power (40 60 %). The descriptors selected did not show clear physical meaning for mechanism of action of anthraquinones. The descriptors selected are mainly structural rather than providing an insight into physico-chemical properties of anthraquinones. The standard errors for all models were relatively high (> 0.9) probably due to human error. This error is probably due the nature of the antiplasmodial assay which requires manual estimation of parasitemia using light microscope. The biological data obtained were not normally distributed which could be due to lack of structural diversity of 9,10-anthraquinones. The presence of outliers in the correlation plots could be due to several factors; the experimental error or the molecules may act as antiplasmodial on a different mechanism of action.

An improved approach to mate-paired library preparation for Illumina sequencing

High quality data from mate-pair libraries provides long range sequence linkage across the genome, which is crucial for de novo assembly and structural-variant detection. Current commercial methods available for the construction of such libraries have differing limitations and are often linked to a single sequencing platform in a kit format, which may not be cost effective. We present an alternative mate-paired protocol, demonstrated using Illumina sequencing platforms, combining the specificity of hybridisation and ligation, to circularise fragments with high yield. An adapter sequence is incorporated between the junction site of the mate pairs, the length of which is evenly controlled by nick translation. We present a comparison of results from 3 Kb E. coli and Plasmodium falciparum 3D7 mate-pairs made with our protocol, alongside commercial mate-pair methods. Furthermore, we present the results of a set of 3 and 6 Kb mate-pair libraries from seven different mouse strains made with our mate-pair protocol to demonstrate its reliability and robustness.