Novel quinolinepiperazinyl-aryltetrazoles targeting the blood stage of Plasmodium falciparum.

The emergence of drug resistance against the frontline antimalarials is a major challenge in the treatment of malaria. In view of emerging reports on drug-resistant strains of Plasmodium against artemisinin combination therapy, a dire need is felt for the discovery of novel compounds acting against novel targets in the parasite. In this study, we identified a novel series of quinolinepiperazinyl-aryltetrazoles (QPTs) targeting the blood stage of Plasmodium. In vitro anti-plasmodial activity screening revealed that most of the compounds showed IC50 < 10 µM against chloroquine-resistant PfINDO strain, with the most promising lead compounds 66 and 75 showing IC50 values of 2.25 and 1.79 µM, respectively. Further, compounds 64-66, 68, 75-77 and 84 were found to be selective (selectivity index >50) in their action against Pf over a mammalian cell line, with compounds 66 and 75 offering the highest selectivity indexes of 178 and 223, respectively. Explorations into the action of lead compounds 66 and 75 revealed their selective cidal activity towards trophozoites and schizonts. In a ring-stage survival assay, 75 showed cidal activity against the early rings of artemisinin-resistant PfCam3.1R539T. Further, 66 and 75 in combination with artemisinin and pyrimethamine showed additive to weak synergistic interactions. Of these two in vitro lead molecules, only 66 restricted rise in the percentage of parasitemia to about 10% in P. berghei-infected mice with a median survival time of 28 days as compared to the untreated control, which showed the percentage of parasitemia >30%, and a median survival of 20 days. Promising antimalarial activity, high selectivity, and additive interaction with artemisinin and pyrimethamine indicate the potential of these compounds to be further optimized chemically as future drug candidates against malaria.

Identification of Novel, Potent, and Selective Compounds against Malaria Using Glideosomal-Associated Protein 50 as a Drug Target.

Phylum apicomplexan consists of parasites, such as Plasmodium and Toxoplasma. These obligate intracellular parasites enter host cells via an energy-dependent process using specialized machinery, called the glideosome. In the present study, we used Plasmodium falciparum GAP50, a glideosome-associated protein, as a target to screen 951 different compounds from diverse chemical libraries. Using different screening methods, eight compounds (Hayatinine, Curine, MMV689758 (Bedaquiline), MMV1634402 (Brilacidin), and MMV688271, MMV782353, MMV642550, and USINB4-124-8) were identified, which showed promising binding affinity (KD < 75 µM), along with submicromolar range antiparasitic efficacy and selectivity index > 100 fold for malaria parasite. These eight compounds were effective against Chloroquine-resistant PfINDO and Artemisinin-resistant PfCam3.1R359T strains. Studies on the effect of these compounds at asexual blood stages showed that these eight compounds act differently at different developmental stages, indicating the binding of these compounds to other Plasmodium proteins, in addition to PfGAP50. We further studied the effects of compounds (Bedaquiline and USINB4-124-8) in an in vivoPlasmodium berghei mouse model of malaria. Importantly, the oral delivery of Bedaquiline (50 mg/kg b. wt.) showed substantial suppression of parasitemia, and three out of seven mice were cured of the infection. Thus, our study provides new scaffolds for the development of antimalarials that can act at multiple Plasmodium lifecycle stages.

Marmesin isolated from Celtis durandii Engl. root bioactive fraction inhibits ß-hematin formation and contributes to antiplasmodial activity.

ETHNOPHARMACOLOGICAL RELEVANCE: Malaria is a leading cause of death in many developing countries, especially in sub-Saharan Africa. Nigeria is endowed with an abundance of medicinal plants, many of which are used to treat malaria. Celtis durandii Engl. is one such plant used as a traditional antimalarial remedy in southeast Nigeria. However, its antiplasmodial potential is poorly explored. AIM OF THE STUDY: The study aimed at identifying the antiplasmodial components of C. durandii root extract through antiplasmodial activity-guided fractionation. MATERIALS AND METHODS: Dichloromethane/methanol mixture extract (1:1 v/v) of C. durandii root was prepared and partitioned against water to obtain the organic phase, which was further separated by column chromatography into nine (C1 - C9) fractions. The antiplasmodial activity was evaluated by in vitro screening of the different fractions against drug-sensitive and drug-resistant Plasmodium falciparum strains. Further purification of the active column fractions resulted in a potent anti-Plasmodial compound that was subsequently investigated for its effect on ß-hematin formation. Additionally, the isolated compound was characterized and identified as marmesin using mass spectrometry and nuclear magnetic resonance spectroscopy. RESULTS: Celtis durandii root extract exhibited promising antiplasmodial activity {IC50 (µg/ml) 5.92, 6.04, and 6.92} against PfW2mef, PfINDO, and Pf3D7 respectively. Pooled fractions with good antiplasmodial activity {IC50 (µg/ml) Pf3D7: 3.99; PfINDO: 2.24} and selectivity for the parasites (SI: 21) yielded a compound that was fourteen-fold potent in antiplasmodial activity against Pf3D7(IC50: 0.28 µg/ml). It also inhibited ß-hematin formation with an IC50 = 150 µM. Further studies using spectral data, literature, and chemical databases identified the purified compound as marmesin. CONCLUSION: This work has demonstrated that Celtis durandii root extract has good antiplasmodial activity against drug-sensitive and drug-resistant P. falciparum. The inhibition of ß-hematin formation by marmesin accounts in part for this activity.

Antiplasmodial activity of the bulbs of Fritillaria cirrhosa D.Don (Syn: Fritillaria roylei Hook.): UPLC-IM-Q-TOF-MS/MS-based biochemometric approach for the identification of marker compounds.

ETHNOPHARMACOLOGICAL RELEVANCE: Fritillaria cirrhosa D.Don (Syn: Fritillaria roylei Hook.) (Hindi name: Kshirakakoli) is a critically endangered Himalayan medicinal plant, well documented in Ayurveda for its therapeutic uses against various disorders such as jvara (fever), kasa (respiratory tract disease) etc. Its bulbs are also used as Szechuan-Pei-Mu for their antipyretic properties in the traditional Chinese medicine. However, despite its ethnomedicinal usage, the therapeutic use of F. cirrhosa bulbs for jvara (fever) related conditions such as malaria has remained unexplored. Hence in the context of increasing global concerns about drug-resistant malaria, it is important to investigate the antiplasmodial activity of F. cirrhosa bulbs for novel antimalarial agents. AIM OF THE STUDY: To investigate the antiplasmodial effects of the extracts/fractions of F. cirrhosa bulbs by the biochemometric approach and to rationalize its ethnopharmacological usage for jvara (fever) related conditions such as malaria. MATERIAL AND METHODS: This study involves the UHPLC-MS-based plant material selection, preparation, quantification, and assessment of F. cirrhosa bulb extracts against CQ-sensitive Pf 3D7 & CQ-resistant Pf INDO strains. Further, UPLC-IM-Q-TOF-MS-based biochemometric approach has been applied for the identification of marker compounds responsible for the observed antiplasmodial effects. The identified marker compounds were also assessed for their in silico ADMET properties and binding efficacy with the drug transporter Pf CRT. RESULTS: Different F. cirrhosa bulb extracts/fractions showed promising antiplasmodial activity with IC50 values 2.71-19.77 µg/mL for CQ-resistant Pf INDO strain and 1.76-21.52 µg/mL for CQ-sensitive Pf 3D7 strain. UPLC-IM-Q-TOF-MS/MS-based biochemometric analysis revealed four marker compounds i.e., peimine (m/z 432.3448), peimisine (m/z 428.3504), puqiedinone (m/z 414.3379), and puqiedine (m/z 416.3509) responsible for the observed antiplasmodial activity. The identified marker compounds showed excellent binding efficacy with Pf CRT and suitable drug-like properties in silico. CONCLUSIONS: The study demonstrated promising antiplasmodial activity of the chloroform and alkaloid enriched fractions of F. cirrhosa bulbs and further identified the four marker compounds responsible for the promising antiplasmodial activity. These marker compounds i.e., peimine, peimisine, puqiedinone and puqiedine were identified by the biochemometric analysis as the putative antiplasmodial constituents of the F. cirrhosa bulbs. Further, in silico studies indicated the good binding affinity of the marker compounds with Pf CRT along with suitable ADMET properties. Overall, the study elucidates the antiplasmodial activity of F. cirrhosa bulbs from the western Himalayan region and provides nascent scientific evidence for their ethnopharmacological usage in jvara (fever) related conditions such as malaria.

Identification of new oxospiro chromane quinoline-carboxylate antimalarials that arrest parasite growth at ring stage.

Malaria still threatens half the globe population despite successful Artemisinin-based combination therapy. One of the reasons for our inability to eradicate malaria is the emergence of resistance to current antimalarials. Thus, there is a need to develop new antimalarials targeting Plasmodium proteins. The present study reported the design and synthesis of 4, 6 and 7-substituted quinoline-3-carboxylates 9(a-o) and carboxylic acids 10(a-b) for the inhibition of Plasmodium N-Myristoyltransferases (NMTs) using computational biology tools followed by chemical synthesis and functional analysis. The designed compounds exhibited a glide score of -9.241 to -6.960 kcal/mol for PvNMT and -7.538 kcal/mol for PfNMT model proteins. Development of the synthesized compounds was established via NMR, HRMS and single crystal X-ray diffraction study. The synthesized compounds were evaluated for their in vitro antimalarial efficacy against CQ-sensitive Pf3D7 and CQ-resistant PfINDO lines followed by cell toxicity evaluation. In silico results highlighted the compound ethyl 6-methyl-4-(naphthalen-2-yloxy)quinoline-3-carboxylate (9a) as a promising inhibitor with a glide score of -9.084 kcal/mol for PvNMT and -6.975 kcal/mol for PfNMT with IC50 values of 6.58 µM for Pf3D7 line. Furthermore, compounds 9n and 9o exhibited excellent anti-plasmodial activity (Pf3D7 IC50 = 3.96, 6.71 µM, and PfINDO IC50 = 6.38, 2.8 µM, respectively). The conformational stability of 9a with the active site of the target protein was analyzed through MD simulation and was found concordance with in vitro results. Thus, our study provides scaffolds for the development of potent antimalarials targeting both Plasmodium vivax and Plasmodium falciparum.Communicated by Ramaswamy H. Sarma.

Antiplasmodial diterpenoid alkaloid from Aconitum heterophyllum Wall. ex Royle: Isolation, characterization, and UHPLC-DAD based quantification.

ETHNOPHARMACOLOGICAL RELEVANCE: Aconitum heterophyllum Wall. ex Royle is a traditionally important medicinal plant having numerous therapeutic actions as documented in Ayurveda. This plant is traditionally known for combating worm infestation, fever, respiratory tract disease, vomiting, diarrhoea, diabetes, skin disorders, anaemia, and joint disorders. Further, it has been used alone and in combination with other plants to prepare various anti-malarial formulations. However, there is no report on the assessment of its anti-plasmodial activity, and the metabolite(s) responsible for this activity. AIM OF THE STUDY: The main aim of this study was to conduct phytochemical investigation of A. heterophyllum roots for the preparation of extract, fractions, and isolation of pure molecules to identify active fractions/molecules responsible for the anti-plasmodial activity, and development of UHPLC-DAD based analytical method which can be used for the quantification of marker compounds in the extracts and fractions. MATERIALS AND METHODS: Hydroalcoholic extract (1:1 v/v) and fractions (n-hexane, chloroform, ethyl acetate, n-butanol, and water) were prepared from the dried powdered roots of A. heterophyllum. Fractions were further subjected to silica gel column chromatography to isolate pure specialized secondary metabolites from this plant. All extracts, fractions, and pure molecules were evaluated against the chloroquine resistant Pf INDO and chloroquine sensitive Pf3D7 strains in culture for calculating their IC50 values. UHPLC-DAD based analytical method was also developed for the first time for the quantification of marker compounds and quality assessment of this commercially important Himalayan medicinal plant. RESULTS: Phytochemical investigation of A. heterophyllum root led to the isolation of six specialized metabolites viz. 2-O-cinnamoyl hetisine (1), atisinium (2), 4-oxabicyclo [3.2.2] nona-1(7),5,8-triene (3), atisinium cinnamate (4), aconitic acid (5), and atisinium formate (6). Compound 1 is a new hetisine type diterpenoid alkaloid, compounds 4 and 6 are new counter ionic forms observed with atisinium ion, and compound 3 is being reported for the first time from this genus. Chloroform fraction was found to be the most active with IC50 (µg/mL) 1.01 (Pf INDO) and 1.32 (Pf3D7). The molecule 2-O-cinnamoyl hetisine (1), a new diterpenoid alkaloid isolated from chloroform fraction, showed promising antiplasmodial activities with IC50 (µM) 1.92 (Pf INDO) and 10.8 (Pf 3D7). The activity of chloroform fraction was further validated by the developed UHPLC-DAD based method as the quantity of 2-O-cinnamoyl hetisine (1) was higher in the chloroform fraction (≅200 mg/g) than in all other fractions (<7 mg/g). Atisinium (2) and 2-O-cinnamoyl hetisine (1) were found to be the main marker compounds of this plant based on quantity and antiplasmodial activity, respectively. CONCLUSION: This study provides the scientific rationale for the traditional use of this plant in treating malaria. Further, this study revealed that the anti-malarial potential of this plant might be due to the presence of diterpenoid alkaloids.

Antifungal effects of tulsi, garlic, cinnamon and lemongrass in powder and oil form on Candida albicans: An in vitro study.

INTRODUCTION: The use of plants for treating diseases is as old as the human species. Medicinal plants have been a major source of therapeutic agents for alleviation and cure of diseases. OBJECTIVES: The objective of the study was to evaluate and compare the antifungal activity of garlic, cinnamon, lemongrass and tulsi in powder and oil form at different concentrations on Candida albicans. MATERIALS AND METHODS: Powder and oil of garlic, cinnamon, lemongrass and Tulsi dissolved in inert solvent dimethyl formamide to obtain different concentration. Stock solution of different concentration was inoculated on Petri plates containing C. albicans and incubated at 30°C for 48 h. The inhibition zones were measured in millimeters using Vernier caliper. The collected data were analyzed using statistical test like mean value and one-way analysis of variance. RESULTS: Maximum zone of inhibition for the C. albicans was 42 mm at concentrations of 50% for the oil of lemongrass; followed by cinnamon 40 mm, garlic 24 mm and tulsi 20 mm. The P value obtained 0.050, 0.040, 0.036 and 0.031 were found to be statically significant for C. albicans at 20%, 30%, 40% and 50% concentrations of the various oil preparations, respectively. The P value obtained 0.043, 0.033, 0.032 and 0.027 were found to be statically significant for C. albicans at 20%, 30%, 40% and 50% concentrations of various plant powder, respectively. CONCLUSIONS: Lemongrass and cinnamon oil shows best antifungal effect against C. albicans as compared to garlic and tulsi. Compared to powder preparations, the oil preparations are better to inhibit the growth and higher the concentrations, greater the zone of inhibition seen in all the plant extracts and in oil.

Plasmodium falciparum PhIL1-associated complex plays an essential role in merozoite reorientation and invasion of host erythrocytes.

The human malaria parasite, Plasmodium falciparum possesses unique gliding machinery referred to as the glideosome that powers its entry into the insect and vertebrate hosts. Several parasite proteins including Photosensitized INA-labelled protein 1 (PhIL1) have been shown to associate with glideosome machinery. Here we describe a novel PhIL1 associated protein complex that co-exists with the glideosome motor complex in the inner membrane complex of the merozoite. Using an experimental genetics approach, we characterized the role(s) of three proteins associated with PhIL1: a glideosome associated protein- PfGAPM2, an IMC structural protein- PfALV5, and an uncharacterized protein-referred here as PfPhIP (PhIL1 Interacting Protein). Parasites lacking PfPhIP or PfGAPM2 were unable to invade host RBCs. Additionally, the downregulation of PfPhIP resulted in significant defects in merozoite segmentation. Furthermore, the PfPhIP and PfGAPM2 depleted parasites showed abrogation of reorientation/gliding. However, initial attachment with host RBCs was not affected in these parasites. Together, the data presented here show that proteins of the PhIL1-associated complex play an important role in the orientation of P. falciparum merozoites following initial attachment, which is crucial for the formation of a tight junction and hence invasion of host erythrocytes.

Novel halogenated arylvinyl-1,2,4 trioxanes as potent antiplasmodial as well as anticancer agents: Synthesis, bioevaluation, structure-activity relationship and in-silico studies.

Herein, we have synthesized a series of lipophilic, halogenated-arylvinyl-1,2,4-trioxanes 8a-g (28 compounds) and assessed for their in vitro anti-plasmodial activity in Plasmodium falciparum culture using SYBRgreen-I fluorescence assay against chloroquine-resistant Pf INDO and artemisinin-resistant Pf Cam 3.1R539T (MRA-1240) strains. Alongside, the cell cytotoxic potential of 8a-g has also been determined against the HEK293 cell line in vitro. Out of twenty-eight halogenated-arylvinyl-1,2,4-trioxanes; ten analogues (8a2, 8a4, 8b2, 8b4, 8d4, 8e1, 8e2, 8e4,8f2, and 8g4) have shown potent in vitro antiplasmodial activity with IC50 < 27 nM (IC50 range = 4.48-26.58 nM). Also, the selectivity index (SI) for these ten analogues were found in the range of 72.00-3972.50 which indicates their selective potential towards Plasmodium cells. Results of the cell cycle stage specificity with two of the most potent compounds 8a4 {(IC50 = 4.48 nM; SI = 3972.50) more potent than chloroquine (IC50 = 546 nM; SI = 36.64) and artesunate (IC50 = 6.6 nM; SI = 4333.33)} and 8e2 (IC50 = 9.69 nM; SI = 1348) against Pf INDO indicated all three stages to be the target of the action of 8e2 while only rings and trophozoites appeared to be targeted by 8a4. Ring stage survival assay against artemisinin-resistant Pf Cam 3.1R539T indicated that 8a4 may be well suited to replace artemisinin from current ACTs which are experiencing in vivo delayed parasite clearance. With intraperitoneal (i.p.) and oral (p.o.) route at the dose of 50 mg/kg/day × 4 days; 8a4 has also shown 100% suppression of parasitemia in P. berghei ANKA infected Balb C mice. Further, the in vitro anticancer activity of 8a-g performed against human lung (A549) and liver (HepG2) cancer cell lines as also against immortalized normal lung (BEAS-2B) and liver (LO2) cell lines has revealed that most of the derivatives are endowed also with promising anticancer activity (IC50 = 0.69-15 µM; SI = 1.02-20.61) in comparison with standard drugs such as chloroquine (IC50 = 100 µM; SI = 0.03), artemisinin (IC50 = 100 µM), and artesunic acid (IC50 = 9.85 µM; SI = 0.76), respectively. All the derivatives have shown moderate anticancer activity against liver (HepG2) cancer cell lines. Arylvinyl-1,2,4-trioxanes 8f2 (IC50 = 0.69 µM; SI = 16.66), the most active compound of the series, has shown ∼145 fold more cytotoxic potential with higher selectivity in comparison to reference drugs chloroquine (IC50 = 100 µM; SI = 0.03) and artemisinin (IC50 = 100 µM), respectively against the lung (A549) cancer cell line. Finally, the in-silico docking studies of the potent halogenated 1,2,4-trioxanes along with reference drug molecules against epidermal growth factor receptor (EGFR; PDB ID: 1M17) have demonstrated the strong virtual interaction.

Chemical profiling and quantification of potential active constituents responsible for the antiplasmodial activity of Cissampelos pareira.

ETHNOPHARMACOLOGICAL RELEVANCE: Cissampelos pareira is used traditionally in India as a remedy for the treatment of various diseases including malaria but the active ingredients responsible for antiplasmodial activity have not yet been investigated. AIM OF THE STUDY: The identification and quantification of compounds responsible for antiplasmodial activity in different parts (leaf, stem and root) of C. pareira is the target of current study. MATERIAL AND METHODS: The hydro ethanolic parent extracts of different parts of C. pareira and fractions prepared from these extracts were evaluated against Pf3D7 (chloroquine sensitive) and PfINDO (chloroquine resistance) strains in culture to quantify the IC50 for extracts and fractions. Promising fractions of root part of plant were subjected to silica gel column chromatography to obtain pure compounds and their structures were elucidated by detailed spectroscopic analysis. Pure compounds were also tested against Pf3D7 and PfINDO strains. A rapid and simple UPLC-DAD method was developed for the identification and quantification of pharmaceutically important metabolites of C. pareira. RESULTS: Among different extracts, the hydro ethanolic extract of root part of C. pareira was found most active with IC50 values (µg/ml) of 1.42 and 1.15 against Pf 3D7 and Pf INDO, respectively. Tested against Pf 3D7 the most potent fractions were root ethyl acetate fraction (IC50 4.0 µg/ml), stem water fraction (IC50 4.4 µg/ml), and root water fraction (IC50 8.5 µg/ml). Further, phytochemical investigation of active fractions of root part led to the isolation and characterization of a new isoquinoline alkaloid, namely pareirarine (8), along with five known compounds magnoflorine (5), magnocurarine (10), salutaridine (11), cissamine (13) and hayatinine (15). Hayatinine (15), a bisbenzylisoquinoline alkaloid, isolated from root ethyl acetate fraction was most promising compound with IC50 of 0.41 µM (Pf INDO) and 0.509 µM (Pf 3D7). Magnocurarine (10) and cissamine (13) were also found active with IC50 values of 12.51 and 47.34 µM against Pf INDO and 12.54 and 8.76 µM against Pf 3D7, respectively. A total of thirty compounds were detected in studied extracts and fractions, structures were assigned to 15 of these and five of these biologically important compounds were quantified. Isolation of saluteridine (11) from C. pareira and the evaluation of antiplasmodial activity of pure compound from C. pariera is disclosed for the first time. CONCLUSION: This study concludes that the antimalarial potential of C. pareira may be attributed to isoquinoline type alkaloids present in this plant and also provides the scientific evidence for the traditional use of this plant in treatment of malaria.

Design, Synthesis, Structure-Activity Relationship and Docking Studies of Novel Functionalized Arylvinyl-1,2,4-Trioxanes as Potent Antiplasmodial as well as Anticancer Agents.

A novel series of synthetic functionalized arylvinyl-1,2,4-trioxanes (8 a-p) has been prepared and assessed for their in vitro antiplasmodial activity against the chloroquine-resistant Pf INDO strain of Plasmodium falciparum by using a SYBR green-I fluorescence assay. Compounds 8 g (IC50 =0.051 µM; SI=589.41) and 8 m (IC50 =0.059 µM; SI=55.93) showed 11-fold and >9-fold more potent antiplasmodial activity, respectively, as compared to chloroquine (IC50 =0.546 µM; SI=36.63). Different in silico docking studies performed on many target proteins revealed that the most active arylvinyl-1,2,4-trioxanes (8 g and 8 m) showed dihydrofolate reductase (DHFR) binding affinities on a par with those of chloroquine and artesunate. The in vitro cytotoxic potentials of 8 a-p were also evaluated against human lung (A549) and liver (HepG2) cancer cell lines along with immortalized normal lung (BEAS-2B) and liver (LO2) cell lines. Following screening, five derivatives viz. 8 a, 8 h, 8 l, 8 m and 8 o (IC50 =1.65-31.7 µM; SI=1.08-10.96) were found to show potent cytotoxic activity against (A549) lung cancer cell lines, with selectivity superior to that of the reference compounds artemisinin (IC50 =100 µM), chloroquine (IC50 =100 µM) and artesunic acid (IC50 =9.85 µM; SI=0.76). In fact, the most active 4-naphthyl-substituted analogue 8 l (IC50 =1.65 µM; SI >10) exhibited >60 times more cytotoxicity than the standard reference, artemisinin, against A549 lung cancer cell lines. In silico docking studies of the most active anticancer compounds, 8 l and 8 m, against EGFR were found to validate the wet lab results. In summary, a new series of functionalized aryl-vinyl-1,2,4-trioxanes (8 a-p) has been shown to display dual potency as promising antiplasmodial and anticancer agents.

Strain-transcending neutralization of malaria parasite by antibodies against Plasmodium falciparum enolase.

BACKGROUND: Plasmodium enolase is a target for the growth neutralizing antibodies. Interestingly, the three invasive stages i.e. sporozoites, merozoites, and ookinetes express this protein on their cell surface. Polyclonal anti-Plasmodium falciparum enolase (Pfeno) antibodies disrupt traversal of ookinete through mosquito mid-gut wall as well as have inhibitory effect on parasite growth at erythrocytic stage. In a recent study, it was observed that immunization with a unique epitope of parasite enolase (EWGWS) could confer partial protection against mouse malaria. Further validation is needed for the protective potential of this unique epitope in otherwise highly conserved enolase. METHODS: In order to investigate the efficacy of growth inhibitory potential of the epitope of P falciparum enolase, a monoclonal antibody specific to EWGWS is generated. In vitro parasite growth inhibition assays and passive immunization of Plasmodium yoelii (or Plasmodium berghei) infected mice were used to assess the parasite growth neutralizing activity of the antibody. RESULTS: Screening a panel of monoclonal antibodies raised against recombinant Pfeno that were specific to EWGWS resulted in isolation of H12E1. This antibody recognized only EWGWS epitope containing enolases. H12E1 strongly inhibited parasite growth in culture. This inhibition was strain transcending. Passive infusion of this antibody in P. yoelii or P. berghei infected mice showed significant reduction in parasitemia as compared to controls (p < 0.001). Surface Plasmon Resonance measurements indicated high affinity binding of H12E1 to P. falciparum enolase (KD ~ 7.6 × 10-9M). CONCLUSIONS: A monoclonal antibody directed against EWGWS epitope of Pfeno was shown to inhibit the growth of blood stage malarial parasites. This inhibition was species/strain transcending and is likely to arise due to blockade of enolase on the surface of merozoites, functionally implicating Pfeno in invasion related events. Presence of enolase on the cell surface of merozoites and ookinetes could potentially result in inhibition of host cell invasions at erythrocytic and transmission stages in the parasite life cycle. It is suggested that antibodies against EWGWS epitope have the potential to confer dual stage, species and strain transcending protection against malaria.