Genome-Wide Transcriptional Analysis and Functional Validation Linked a Cluster of Epsilon Glutathione S-Transferases with Insecticide Resistance in the Major Malaria Vector Anopheles funestus across Africa.

Resistance is threatening the effectiveness of insecticide-based interventions in use for malaria control. Pinpointing genes associated with resistance is crucial for evidence-based resistance management targeting the major malaria vectors. Here, a combination of RNA-seq based genome-wide transcriptional analysis and RNA-silencing in vivo functional validation were used to identify key insecticide resistance genes associated with DDT and DDT/permethrin cross-resistance across Africa. A cluster of glutathione-S-transferase from epsilon group were found to be overexpressed in resistant populations of Anopheles funestus across Africa including GSTe1 [Cameroon (fold change, FC: 2.54), Ghana (4.20), Malawi (2.51)], GSTe2 [Cameroon (4.47), Ghana (7.52), Malawi (2.13)], GSTe3 [Cameroon (2.49), Uganda (2.60)], GSTe4 in Ghana (3.47), GSTe5 [Ghana (2.94), Malawi (2.26)], GSTe6 [Cameroun (3.0), Ghana (3.11), Malawi (3.07), Uganda (3.78)] and GSTe7 (2.39) in Ghana. Validation of GSTe genes expression profiles by qPCR confirmed that the genes are differentially expressed across Africa with a greater overexpression in DDT-resistant mosquitoes. RNAi-based knock-down analyses supported that five GSTe genes are playing a major role in resistance to pyrethroids (permethrin and deltamethrin) and DDT in An. funestus, with a significant recovery of susceptibility observed when GSTe2, 3, 4, 5 and GSTe6 were silenced. These findings established that GSTe3, 4, 5 and 6 contribute to DDT resistance and should be further characterized to identify their specific genetic variants, to help design DNA-based diagnostic assays, as previously done for the 119F-GSTe2 mutation. This study highlights the role of GSTes in the development of resistance to insecticides in malaria vectors and calls for actions to mitigate this resistance.

Synthesis of Biogenic Gold Nanoparticles from Terminalia mantaly Extracts and the Evaluation of Their In Vitro Cytotoxic Effects in Cancer Cells.

Scientists have demonstrated the potential of plant materials as 'green' reducing and stabilizing agents for the synthesis of gold nanoparticles (AuNPs) and opened new ecofriendly horizons to develop effective and less harmful treatment strategies. The current study demonstrated the use of Terminalia mantaly (TM) extracts to synthesize AuNPs with enhanced cytotoxic effects. The TM-AuNPs were synthesized at 25 and 70 °C using water (WTM) and methanolic (MTM) extracts of the leaf, root and stem/bark parts of the plant. The TM-AuNPs were characterized using UV-visible spectrophotometry, dynamic light scattering (DLS), transmission electron microscopy, energy dispersive X-ray (EDX), selection area electron diffraction (SAED) and Fourier transform infrared (FTIR) spectroscopy. Majority of the TM-AuNPs were spherical with a mean diameter between 22.5 and 43 nm and were also crystalline in nature. The cytotoxic effects of TM-AuNPs were investigated in cancer (Caco-2, MCF-7 and HepG2) and non-cancer (KMST-6) cell lines using the MTT assay. While the plant extracts showed some cytotoxicity towards the cancer cells, some of the TM-AuNPs were even more toxic to the cells. The IC50 values (concentrations of the AuNPs that inhibited 50% cell growth) as low as 0.18 µg/mL were found for TM-AuNPs synthesized using the root extract of the plant. Moreover, some of the TM-AuNPs demonstrated selective toxicity towards specific cancer cell types. The study demonstrates the potential of TM extracts to produce AuNPs and describe the optimal conditions for AuNPs using TM extracts. The toxicity of some the TM-AuNPs can possibly be explored in the future as an antitumor treatment.

Antiplasmodial Properties and Cytotoxicity of Endophytic Fungi from Symphonia globulifera (Clusiaceae).

There is continuing need for new and improved drugs to tackle malaria, which remains a major public health problem, especially in tropical and subtropical regions of the world. Natural products represent credible sources of new antiplasmodial agents for antimalarial drug development. Endophytes that widely colonize healthy tissues of plants have been shown to synthesize a great variety of secondary metabolites that might possess antiplasmodial benefits. The present study was carried out to evaluate the antiplasmodial potential of extracts from endophytic fungi isolated from Symphonia globulifera against a chloroquine-resistant strain of Plasmodium falciparum (PfINDO). Sixty-one fungal isolates with infection frequency of 67.77% were obtained from the bark of S. globulifera. Twelve selected isolates were classified into six different genera including Fusarium, Paecilomyces, Penicillium, Aspergillus, Mucor, and Bipolaris. Extracts from the 12 isolates were tested against PfINDO, and nine showed good activity (IC50 < 10 μg·mL−1) with three fungi including Paecilomyces lilacinus (IC50 = 0.44 μg·mL−1), Penicillium janthinellum (IC50 = 0.2 μg·mL−1), and Paecilomyces sp. (IC50 = 0.55 μg·mL−1) showing the highest promise. These three isolates were found to be less cytotoxic against the HEK293T cell line with selectivity indices ranging from 24.52 to 70.56. Results from this study indicate that endophytic fungi from Symphonia globulifera are promising sources of hit compounds that might be further investigated as novel drugs against malaria. The chemical investigation of active extracts is ongoing.

Potent antiplasmodial extracts and fractions from Terminalia mantaly and Terminalia superba.

BACKGROUND: The emergence and spread of malaria parasites resistant to artemisinin-based combination therapy stresses the need for novel drugs against malaria. Investigating plants used in traditional medicine to treat malaria remains a credible option for new anti-malarial drug development. This study was aimed at investigating the antiplasmodial activity and selectivity of extracts and fractions from Terminalia mantaly and Terminalia superba (Combretaceae) that are used in Cameroon to treat malaria. METHODS: Twelve methanolic (m) and water (w) extracts obtained by maceration of powdered dried leaves (l), stem bark (sb) and root (r) of Terminalia mantaly (Tm) and Terminalia superba (Ts) and 12 derived fractions of hexane, chloroform, ethyl acetate and 4 final residues of selected extracts were assessed for antiplasmodial potential in vitro against the chloroquine-resistant PfINDO and the chloroquine-sensitive Pf3D7 strains of Plasmodium falciparum using the SYBR green I-based fluorescence assay. The cytotoxicity of potent extracts and fractions was evaluated in vitro using the MTT assay on HEK239T cell line. RESULTS: The antiplasmodial IC50 of extracts from both plants ranged from 0.26 to > 25 µg/mL. Apart from the extracts Tmrm and Tsrw that exerted moderate antiplasmodial activities (IC50: 5-20 µg/mL) and Tmrw that was found to be non-active at the tested concentrations (IC50 > 25 µg/mL), all other tested crude extracts exhibited potent activities with IC50 < 5 µg/mL. The aqueous extracts from the stem bark of Terminalia mantaly (Tmsbw) and the leaf of Terminalia superba (Tslw) displayed the highest antiplasmodial activities (IC50: 0.26-1.26 µg/mL) and selectivity (SI > 158) on both resistant PfINDO and sensitive Pf3D7 strains. Four fractions upon further extraction with chloroform and ethyl acetate (TmlwChl, TmsbwChl, TmsbwEA, TsrmEA) afforded from three selected crude extracts (Tmlw, Tmsbw, Tsrm) exhibited highly potent activities against both P. falciparum strains (IC50 < 2 µg/mL) and high selectivity (SI > 109). CONCLUSIONS: The results achieved in this work validate the reported traditional use of Terminalia mantaly and Terminalia superba to treat malaria. Moreover, the highly potent and selective fractions warrant further investigation to characterize the active antiplasmodial principles and progress them to rodent malaria models studies if activity and selectivity are evidenced.

Open Source Drug Discovery with the Malaria Box Compound Collection for Neglected Diseases and Beyond.

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts.

Investigation of minor species Candida africana, Candida stellatoidea and Candida dubliniensis in the Candida albicans complex among Yaoundé (Cameroon) HIV-infected patients.

Minor species of the Candida albicans complex may cause overestimation of the epidemiology of C. albicans, and misidentifications could mask their implication in human pathology. Authors determined the occurrence of minor species of the C. albicans complex (C. africana, C. dubliniensis and C. stellatoidea) among Yaoundé HIV-infected patients, Cameroon. Stool, vaginal discharge, urine and oropharyngeal samples were analysed by mycological diagnosis. Isolates were identified by conventional methods and mass spectrometry (MS; carried out by the matrix-assisted laser desorption-ionisation time-of-flight MS protocol). Candida albicans isolates were thereafter submitted to the PCR amplification of the Hwp1 gene. The susceptibility of isolates to antifungal drugs was tested using the Clinical and Laboratory Standards Institute M27-A3 protocol. From 115 C. albicans obtained isolates, neither C. dubliniensis nor C. stellatoidea was observed; two strains of C. africana (422PV and 448PV) were identified by PCR electrophoretic profiles at 700 bp. These two C. africana strains were vaginal isolates. The isolate 448PV was resistant to ketoconazole at the minimal inhibitory concentration of 2 µg ml(-1), and showed reduced susceptibility to amphotericin B at 1 µg ml(-1). This first report on C. africana occurrence in Cameroon brings clues for the understanding of the global epidemiology of this yeast as well as that of minor species of the C. albicans complex.

Repurposing the open access malaria box to discover potent inhibitors of Toxoplasma gondii and Entamoeba histolytica.

Toxoplasmosis and amebiasis are important public health concerns worldwide. The drugs currently available to control these diseases have proven limitations. Therefore, innovative approaches should be adopted to identify and develop new leads from novel scaffolds exhibiting novel modes of action. In this paper, we describe results from the screening of compounds in the Medicines for Malaria Venture (MMV) open access Malaria Box in a search for new anti-Toxoplasma and anti-Entamoeba agents. Standard in vitro phenotypic screening procedures were adopted to assess their biological activities. Seven anti-Toxoplasma compounds with a 50% inhibitory concentration (IC50) of <5 µM and selectivity indexes (SI) of >6 were identified. The most interesting compound was MMV007791, a piperazine acetamide, which has an IC50 of 0.19 µM and a selectivity index of >157. Also, we identified two compounds, MMV666600 and MMV006861, with modest activities against Entamoeba histolytica, with IC50s of 10.66 µM and 15.58 µM, respectively. The anti-Toxoplasma compounds identified in this study belong to scaffold types different from those of currently used drugs, underscoring their novelty and potential as starting points for the development of new antitoxoplasmosis drugs with novel modes of action.

Antiplasmodial sesquiterpenes from the seeds of Salacia longipes var. camerunensis.

Phytochemical investigation of the seeds of Salacia longipes var. camerunensis led to the isolation of four sesquiterpenoid derivatives, salaterpene A (1) (1α,2ß,8ß-triacetoxy-6ß,9ß-dibenzoyloxy-4ß-hydroxy-dihydro-ß-agarofuran), salaterpene B (2) (1α,2ß,8ß-triacetoxy-9ß-benzoyloxy-6ß-cinnamoyloxy-4ß-hydroxy-dihydro-ß-agarofuran), salaterpene C (3) (1α,2ß-diacetoxy-6ß,9ß-dibenzoyloxy-4ß-hydroxy-dihydro-ß-agarofuran) and salaterpene D (4) (2ß-acetoxy-1α,6ß-dibenzoyloxy-4ß-hydroxy-9ß-nicotinoyloxy-dihydro-ß-agarofuran) together with two known compounds (5 and 6). The structures of the compounds were established by means of NMR spectroscopy. Compounds 1-4 and 6 were tested in vitro for their antiplasmodial activity against Plasmodium falciparum chloroquine-resistant strain W2. All the tested compounds exhibited a moderate potency with IC50 below 2.7 µM.

Compounds from Sorindeia juglandifolia (Anacardiaceae) exhibit potent anti-plasmodial activities in vitro and in vivo.

BACKGROUND: Discovering new lead compounds against malaria parasites is a crucial step to ensuring a sustainable global pipeline for effective anti-malarial drugs. As far as we know, no previous phytochemical or pharmacological investigations have been carried out on Sorindeia juglandifolia. This paper describes the results of an anti-malarial activity-driven investigation of the fruits of this Cameroonian plant. METHODS: Air-dried fruits were extracted by maceration using methanol. The extract was fractionated by flash chromatography followed by column chromatography over silica gel, eluting with gradients of hexane-ethyl acetate mixtures. Resulting fractions and compounds were tested in vitro against the Plasmodium falciparum chloroquine-resistant strain W2, against field isolates of P. falciparum, and against the P. falciparum recombinant cysteine protease falcipain-2. Promising fractions were assessed for acute toxicity after oral administration in mice. One of the promising isolated compounds was assessed in vivo against the rodent malaria parasite Plasmodium berghei. RESULTS: The main end-products of the activity-guided fractionation were 2,3,6-trihydroxy benzoic acid (1) and 2,3,6-trihydroxy methyl benzoate (2). Overall, nine fractions tested against P. falciparum W2 and falcipain-2 were active, with IC50 values of 2.3-11.6 µg/ml for W2, and 1.1-21.9 µg/ml for falcipain-2. Purified compounds (1) and (2) also showed inhibitory effects against P. falciparum W2 (IC50s 16.5 µM and 13.0 µM) and falcipain-2 (IC50s 35.4 and 6.1 µM). In studies of P. falciparum isolates from Cameroon, the plant fractions demonstrated IC50 values of 0.14-19.4 µg/ml and compounds (1) and (2) values of 6.3 and 36.1 µM. In vivo assessment of compound (1) showed activity against P. berghei strain B, with mean parasitaemia suppressive dose and curative dose of 44.9 mg/kg and 42.2 mg/kg, respectively. Active fractions were found to be safe in mice after oral administration of 7 g/kg body weight. CONCLUSIONS: Fractions of Sorindeia juglandifolia and two compounds isolated from these fractions were active against cultured malaria parasites, the P. falciparum protease falcipain-2, and in a rodent malaria model. These results suggest that further investigation of the anti-malarial activities of natural products from S. juglandifolia will be appropriate.

Antimalarial compounds from the stem bark of Vismia laurentii.

A phytochemical study of the stem bark of Vismia laurentii resulted in the isolation of a tetracyclic triterpene, tirucalla-7,24-dien-3-one (1), and seven other known compounds: 3-geranyloxyemodin (2), vismiaquinone A (3), vismiaquinone B (4), bivismiaquinone (5), epifriedelinol (6), betulinic acid (7) and stigmasta-7,22-dien-3-ol (8). The structure of all these compounds was elucidated by spectroscopic means. The stem bark extract and compounds 1 and 3 showed good antimalarial activity against the W2 strain of Plasmodium falciparum.