Effects of Azadirachta indica seed kernel extracts on early erythrocytic schizogony of Plasmodium berghei and pro-inflammatory response in inbred mice

Background: Medicinal plant research may contribute to develop new pharmacological control tools for vector borne diseases, such as malaria. Methods: The effects of methanol extracts (ME) obtained from seed kernel of ripe and unripe Azadirachta indica fruits were studied on erythrocytic proliferation of the rodent malaria parasite Plasmodium berghei strain ANKA and on mice pro-inflammatory response, as evaluated by measuring the matrix-metalloproteinase-9 (MMP-9) and tumour necrosis factor (TNF) plasma levels, in two mouse strains (C57BL/6 and BALB/c) which are considered as prototypical of Th1 and Th2 immune response, respectively. Results: ME obtained from seed kernel of unripe Azadirachta indica fruits decreased by about 30% the proportion of erythrocytes infected with the malaria parasite in C57BL/6 mice in the 4 days suppressive test. In this treatment group, MMP-9 and TNF levels were notably higher than those measured in the same mouse strain treated with the anti-malarial drug artesunate, Azadirachta indica kernel extracts from ripe fruits or solvent. In BALB/c mice, treatment with kernel extracts did not influence parasitaemia. MMP-9 and TNF levels measured in this mouse strain were notably lower than those recorded in C57BL/6 mice and did not vary among treatment groups. Conclusions: The effects of the ME on the parasite-host interactions appeared to be mouse strain-dependent, but also related to the ripening stage of the neem fruits, as only the unripe fruit seed kernel extracts displayed appreciable bioactivity

Streamlined SMFA and mosquito dark-feeding regime significantly improve malaria transmission-blocking assay robustness and sensitivity

Background The development of malaria transmission-blocking strategies including the generation of malaria refractory mosquitoes to replace the wild populations through means of gene drives hold great promise. The standard membrane feeding assay (SMFA) that involves mosquito feeding on parasitized blood through an artificial membrane system is a vital tool for evaluating the efficacy of transmission-blocking interventions. However, despite the availability of several published protocols, the SMFA remains highly variable and broadly insensitive. Methods The SMFA protocol was optimized through coordinated culturing of Anopheles coluzzii mosquitoes and Plasmodium falciparum parasite coupled with placing mosquitoes under a strict dark regime before, during, and after the gametocyte feed. Results A detailed description of essential steps is provided toward synchronized generation of highly fit An. coluzzii mosquitoes and P. falciparum gametocytes in preparation for an SMFA. A dark-infection regime that emulates the natural vector-parasite interaction system is described, which results in a significant increase in the infection intensity and prevalence. Using this optimal SMFA pipeline, a series of putative transmission-blocking antimicrobial peptides (AMPs) were screened, confirming that melittin and magainin can interfere with P. falciparum development in the vector. Conclusion A robust SMFA protocol that enhances the evaluation of interventions targeting human malaria transmission in laboratory setting is reported. Melittin and magainin are identified as highly potent antiparasitic AMPs that can be used for the generation of refractory Anopheles gambiae mosquitoes

Converting endogenous genes of the malaria mosquito into simple non-autonomous gene drives for pope ion replacement

Gene drives for mosquito population replacement are promising tools for malaria control. However, there is currently no clear pathway for safely testing such tools in endemic countries. The lack of well-characterized promoters for infection-relevant tissues and regulatory hurdles are further obstacles for their design and use. Here we explore how minimal genetic modifications of endogenous mosquito genes can convert them directly into non-autonomous gene drives without disrupting their expression. We co-opted the native regulatory sequences of three midgut-specific loci of the malaria vector Anopheles gambiae to host a prototypical antimalarial molecule and guide-RNAs encoded within artificial introns that support efficient gene drive. We assess the propensity of these modifications to interfere with the development of Plasmodium falciparum and their effect on fitness. Because of their inherent simplicity and passive mode of drive such traits could form part of an acceptable testing pathway of gene drives for malaria eradication

Synthetic spirocyclic endoperoxides: New antimalarial scaffolds

Here we report the development of a straightforward synthetic procedure for the preparation of spirocyclic endoperoxides as synthetic analogues of the natural product dihydroplakortin. The peroxides presented here are more potent antiplasmodials than dihydroplakortin itself and we proved for the first time their antimalarial activity in vivo. This journal i

Synthesis and biological evaluation of benzhydryl-based antiplasmodial agents possessing Plasmodium falciparum chloroquine resistance transporter (PfCRT) inhibitory activity

Due to the surge in resistance to common therapies, malaria remains a significant concern to human health worldwide. In chloroquine (CQ)-resistant (CQ-R) strains of Plasmodium falciparum, CQ and related drugs are effluxed from the parasite's digestive vacuole (DV). This process is mediated by mutant isoforms of a protein called CQ resistance transporter (PfCRT). CQ-R strains can be partially re-sensitized to CQ by verapamil (VP), primaquine (PQ) and other compounds, and this has been shown to be due to the ability of these molecules to inhibit drug transport via PfCRT. We have previously developed a series of clotrimazole (CLT)-based antimalarial agents that possess inhibitory activity against PfCRT (4a,b). In our endeavor to develop novel PfCRT inhibitors, and to perform a structure-activity relationship analysis, we synthesized a new library of analogues. When the benzhydryl system was linked to a 4-aminoquinoline group (5a-f) the resulting compounds exhibited good cytotoxicity against both CQ-R and CQ-S strains of P. falciparum. The most potent inhibitory activity against the PfCRT-mediated transport of CQ was obtained with compound 5k. When compared to the reference compound, benzhydryl analogues of PQ (5i,j) showed a similar activity against blood-stage parasites, and a stronger in vitro potency against liver-stage parasites. Unfortunately, in the in vivo transmission blocking assays, 5i,j were inactive against gametocytes

Transmission blocking effects of Azadirachta indica limonoids on early sporogonic development of Plasmodium: activity and bioavailability of seed fractions and isolated compounds

Azadirachta indica (Meliaceae) possesses a wide spectrum of biological properties, conferred to the plant by secondary metabolites. A. indica seeds contain abundantly limonoid molecules such as azadirone, nimbin, salanin and azadirachtins (A to L), azadirachtin A (AzaA) being one of the most bio-active molecules. AzaA has been shown to inhibit Plasmodium berghei microgamete formation and an AzaA rich commercial kernel extract (NeemAzal®) was found to completely block the transmission of P. berghei to Anopheles stephensi females when administered to gametocytemic mice at an AzaA dose of 50 mg/kg before exposure to mosquitoes. The present study was aimed at i) elucidating early sporogonic stage specific effects of A. indica seed fractions and their main constituents; ii) assessing the bioavailability of a fraction rich in AzA and the isolated AzaA molecule through a biological response-based assay. Ex vivo and in vitro assays were performed with the murine malaria parasites P. berghei ANKA strain and P. berghei CTRPp.GFP. Fractions were obtained from A. indica seeds collected in Burkina Faso and from NeemAzal® (NA, provided by Trifolio-M GmbH, Lahnau, Germany) by column chromatography. Constituents were identified by NMR spectroscopy. NA, AzaA, nimbin and salannin rich fractions from unripe seeds tested at 50 µg/ml, revealed inhibitory activity on early sporogonic stages in vitro. Nimbin and salannin were found to interfere with ookinete maturation while NA and AzaA showed multiple effects on early sporogonic development. The IC50 value determined for NA was 6.8 µg/ml (CI95: 5.95- 7.86), about half of that of AzaA IC50 (12.4 µg/ml; CI95: 11.0- 14.04). The stronger activity of NA, when compared to AzaA, appeared not to be due to an additive or synergistic effect of other azadirachtins (B, D and I) present in NA, since the addition of these compounds at 50 µM to AzaA did not evidence any decrease of the IC50. Also, bioavailability of AzaA, administered as constituent of NA, compared to pure AzaA appeared to be increased. Ex vivo exflagellation tests using blood sampled from mice treated with NA at an AzaA dosage of 150 mg/kg, revealed a half life of NA anti-plasmodial compounds of up to 7 hours. Accumulated evidence on bioavailability and anti-plasmodial activity of limonoids against Plasmodium stages developing in the human and mosquito host, suggests A. indica as a valid resource for the design of limonoid dosed, transmission blocking phytomedicines