Do natural catastrophic events and exceptional climatic conditions also affect parasites?

Parasites and parasitologists have always lived together in good and bad luck in a sort of forced marriage. In recent times bad luck certainly prevailed, because of increasing man-made emergencies such as wars, chemical disasters, but also because of natural disasters, amplified by climate change, that condition more and more parasite­host equilibrium. The symposium at the National Congress of the Italian Society for Parasitology, was a first occasion for Italian parasitologists to reason about 'disaster parasitology' and researchers' responsibilities. Extreme weather events and their impacts on parasites' epidemiology are illustrated, comparing disasters that recently occurred in Italy with literature data. In particular, the Sardinian Island was hit subsequently by fires and floods exacerbating the effects on ecosystems and parasite­host-relationships. Examples of Cryptosporidium outbreaks in man and Fasciola hepatica infections in various hosts after heavy rains are reviewed and effects of droughts on pasture borne parasites, such as gastro-intestinal nematodes of ruminants are discussed. Heavy rains may also cause dissemination of toxic substances released accidentally from chemical plants as happened e.g. in Milan province (IT) after the dioxin hazard. The overlapping effects of strictly man-made disasters with climate change dependent extreme weather events is further challenging the understanding of what are the consequences of disasters on ecosystems and parasite epidemiology.GIS applications combined with AI programs may help to face the complex challenges, allowing the collection and analysis of spatial/temporal data at whatever level desired. Examples illustrated in the article suggest their employment also in a more systematic, prevention-oriented manner.

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.

In vitro Multistage Malaria Transmission Blocking Activity of Selected Malaria Box Compounds.

PURPOSE: Continuous efforts into the discovery and development of new antimalarials are required to face the emerging resistance of the parasite to available treatments. Thus, new effective drugs, ideally able to inhibit the Plasmodium life-cycle stages that cause the disease as well as those responsible for its transmission, are needed. Eight compounds from the Medicines for Malaria Venture (MMV) Malaria Box, potentially interfering with the parasite polyamine biosynthesis were selected and assessed in vitro for activity against malaria transmissible stages, namely mature gametocytes and early sporogonic stages. METHODS: Compound activity against asexual blood stages of chloroquine-sensitive 3D7 and chloroquine-resistant W2 strains of Plasmodium falciparum was tested measuring the parasite lactate dehydrogenase activity. The gametocytocidal effect was determined against the P. falciparum 3D7elo1-pfs16-CBG99 strain with a luminescent method. The murine P. berghei CTRP.GFP strain was employed to assess compounds activities against early sporogonic stage development in an in vitro assay simulating mosquito midgut conditions. RESULTS: Among the eight tested molecules, MMV000642, MMV000662 and MMV006429, containing a 1,2,3,4-tetrahydroisoquinoline-4-carboxamide chemical skeleton substituted at N-2, C-3 and C-4, displayed multi-stage activity. Activity against asexual blood stages of both strains was confirmed with values of IC50 (50% inhibitory concentration) in the range of 0.07-0.13 µM. They were also active against mature stage V gametocytes with IC50 values below 5 µM (range: 3.43-4.42 µM). These molecules exhibited moderate effects on early sporogonic stage development, displaying IC50 values between 20 and 40 µM. CONCLUSION: Given the multi-stage, transmission-blocking profiles of MMV000642, MMV000662, MMV006429, and their chemical characteristics, these compounds can be considered worthy for further optimisation toward a TCP5 or TCP6 target product profile proposed by MMV for transmission-blocking antimalarials.

Plasmodium stage-selective antimalarials from Lophira lanceolata stem bark.

Targeting the transmissible stages of the Plasmodium parasite that develop in the human and mosquito host is a crucial strategy for malaria control and elimination. Medicinal plants offer a prolific source for the discovery of new antimalarial compounds. The recent identification of the gametocytocidal activity of lophirone E, obtained from the African plant Lophira lanceolata (Ochnaceae), inspired the evaluation of the plant also against early sporogonic stages of the parasite development. The bioassay-guided phytochemical study led to the isolation of two known lanceolins and of a new glycosylated bichalcone, named glucolophirone C. Its stereostructure, including absolute configuration of the bichalcone moiety, was elucidated by means of NMR, HRMS, ECD and computational calculations. Lanceolin B proved to be a potent inhibitor of the development of Plasmodium early sporogonic stages indicating that the plant produces two different stage-specific antimalarial agents acting on transmissible stages in the human and mosquito host.

A murine malaria protocol for characterizing transmission blocking benefits of antimalarial drug combinations.

BACKGROUND: Current efforts towards malaria elimination include the discovery of new transmission blocking (TB) drugs and identification of compounds suitable to replace primaquine, recommended as transmission blocking post treatment after artemisinin combination therapy (ACT). High through put screening of compound libraries has allowed to identify numerous compounds active in vitro against gametocytes and insect early sporogonic stages, but few studies have been performed to characterize TB compounds in vivo. Here we propose a double TB drug Direct Feeding Assay (2TB-DFA), suitable to assess the combined effects of TB compounds. MATERIALS AND METHODS: Plasmodium berghei GFPcon (PbGFPcon), BALB/c mice and Anopheles stephensi mosquitoes were used. Artemisinin (ART) and artesunate (AS) served as examples of artemisinins, NeemAzal® (NA), as a known TB-product with sporontocidal activity. DFA experiments were performed to assess the appropriate time point of administration before mosquito feeding and estimate suitable sub-optimal doses of the three compounds that allow combination effects to be appreciated. RESULTS: Suboptimal dosages, that reduce about 50% of oocyst development, were recorded with ART in the range of 16-30 mg/ kg, AS 14-28 mg/kg and NA 31-38mg/kg. Ten hours before mosquito feeding (corresponding to 3.5 days after mouse infection) was determined as a suitable time point for mouse treatment with ART and AS and 1 hour for post-treatment with NA. ART given at 35 mg/kg in combination with NA at 40 mg/kg reduced oocyst density by 94% and prevalence of infection by 59%. Similarly, the combination of ART at 25 mg/kg plus NA at 35 mg/kg decreased oocyst density by 95% and prevalence of infection by 34%. In the 2TB-DFA, conducted with AS (20 mg/kg) and NA (35 mg/kg) the combination treatment reduced oocyst density by 71% and did not affect prevalence of infection. Applying 'Highest Single Agent' analysis and considering as readout oocyst density and prevalence of infection, cooperative effects of the combination treatments, compared with the single compound treatments emerged. CONCLUSION: This study suggests the 2TB-DFA to be suitable for the profiling of new TB candidates that could substitute primaquine as a post-treatment to ACT courses.

Identification of a potent and selective gametocytocidal antimalarial agent from the stem barks of Lophira lanceolata.

Bioassay-guided fractionation of the organic extract obtained from stem barks of the African plant Lophira lanceolata has led to the isolation of seven biflavonoids, including the new α'-chlorolophirone E (5) and 5'-chlorolophirone D (6). Among the isolated compounds, the bichalcone lophirone E was identified as a potent gametocytocidal agent with an IC50 value in the nanomolar range and negligible cytotoxicity (selectivity index = 570). Lophirone E proved to be about 100 times more active against P. falciparum stage V gametocytes than on asexual blood stages, thus exhibiting a unique stage-specific activity profile. The isolation of structural analogues allowed to draw preliminary structure-activity relationships, identifying the critical positions on the chemical scaffold of lophirone E.

A formative study of disposal and re-use of old mosquito nets by communities in Malindi, Kenya.

BACKGROUND: About 30 million insecticide treated mosquito nets have been distributed in Kenya since 2001 and ownership is approaching full coverage. As a consequence of this achievement, Kenya is faced with the challenge of disposing old mosquito nets that are no longer in use. The study aimed at investigating ways of disposal and re-use of old and torn nets by end users. MATERIALS AND METHODS: A formative study was conducted in the former Malindi District, which is comprised of Malindi and Magarini sub-counties of Kilifi County in Coastal Kenya. A total of 6 Focus Group Discussions, 10 Key Informant Interviews and 9 transect walks/drives were undertaken. Data from the different sources were analysed separately and triangulated for similarities and differences. RESULTS: There were variations in disposal and re-use of old nets between urban and rural or peri-urban residents. In all settings, people adopted innovative and beneficial ways of re-using old, expired nets, and those that were damaged beyond repair. Common causes of damage were fire, children, domestic animals sharing the sleeping room and friction from the bed poles while hanging or tacking it in under a sleeping mat. Re-use was most prominent in farming activities (78%) and less to for use in mosquito control, like window screening (15%). The remaining 8% was related to making ropes, swings, footballs, goal posts and fishing nets. Advantageous texture and nature of the netting material, perceived economic benefit and lack of guidelines for disposal were the main reasons cited by residents for re-using old nets. CONCLUSIONS: It is important that re-use and disposal of old mosquito nets is distinguished from misuse of newly distributed mosquito nets. Alternative uses of old nets as opposed to misuse of new nets was found to be common in our study.

Exploring communities' and health workers' perceptions of indicators and drivers of malaria decline in Malindi, Kenya.

BACKGROUND: Since 2000, a decrease in malaria burden has been observed in most endemic countries. Declining infection rates and disease burden and reduction in asymptomatic carriers are the outcome of improved quality of care and related health system factors. These include improved case management through better diagnosis, implementation of highly effective antimalarial drugs and increased use of bednets. We studied communities' and health workers' perceptions of indicators and drivers in the context of decreasing malaria transmission in Malindi, Kenya. MATERIALS AND METHODS: A variety of qualitative methods that included participatory rural appraisal (PRA) tools such as community river of life and trend lines, focus group discussions (FGDs) and key informant interviews were used. Studies took place between November 2013 and April 2014. RESULTS: Providing residents with bednets contributed to malaria reduction, and increasing community awareness on the causes and symptoms of malaria and improved malaria treatment were also perceived to contribute to the decline of malaria. The study identified three perceived drivers to the reported decline in malaria: a) community health workers' enhanced awareness creation towards household owners regarding malaria-related activities through visitations and awareness sessions, b) Women involvement in Savings Internal Lending Community was perceived to have increased their financial base, thereby improving their decision-making power towards the care of their sick child(ren), c) Non Governmental Organizations (NGOs) and partners played a promoter part in health and general economic development initiatives. CONCLUSIONS: To achieve the goal of malaria elimination, collaboration between governmental and NGOs will be crucial when improving the financial base of women and enhancing participation of community health workers.

Daucovirgolides I-L, four congeners of the antimalarial daucovirgolide G from Daucus virgatus.

Repeated chromatographic purifications of aerial parts of the Tunisian plant Daucus virgatus led to the isolation of four new germacranolides, named daucovirgolides I-L (2-5), along with the Plasmodium transmission-blocking agent daucovirgolide G. The chemical structures of the new compounds were defined as mono- or di-angeloylated germacrane-type sesquiterpenoids by spectroscopic (mainly 1D and 2D NMR) and spectrometric methods (ESIMS). The low potency exhibited by daucovirgolides I-L further supports the observation that strict structural requirements do exist for the Plasmodium transmission blocking activity in the daucovirgolide series. In particular, the endocyclic double bond system seems to be crucial for bioactivity.

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.

Adenosine Triphosphate-Binding Cassette Transporters Are Not Involved In the Detoxification of Azadirachta indica Extracts In Anopheles stephensi Larvae.

Detoxifying pathways of mosquitoes against the neem (Azadirachta indica) extracts are still unclear. The aim of the present study was to investigate the role of adenosine triphosphate-binding cassette (ABC) transporters in this process in Anopheles stephensi, one of the main malaria vectors in southern Asia. Third-stage larvae of An. stephensi were fed with fish food alone or in combination with neem extract at 0.5%, 1%, 5%, and 10%. Six ABC-transporter genes from 3 different subfamilies (B, C, and G) were analyzed to assess their relative expression compared with controls. A bioassay was also performed to assess larval mortality rate at different concentrations and in combination with verapamil, an ABC-transporter inhibitor. No significant variation in the expression levels of any transporter belonging to the B, C, and G subfamilies was detected. Furthermore, the use of verapamil did not induce an increase in mortality at any of the tested neem extract concentrations, indicating that ABC transporters are not involved in the detoxification of neem extracts in An. stephensi larvae.

In Vivo and In Vitro Activities and ADME-Tox Profile of a Quinolizidine-Modified 4-Aminoquinoline: A Potent Anti-P. falciparum and Anti-P. vivax Blood-Stage Antimalarial.

Natural products are a prolific source for the identification of new biologically active compounds. In the present work, we studied the in vitro and in vivo antimalarial efficacy and ADME-Tox profile of a molecular hybrid (AM1) between 4-aminoquinoline and a quinolizidine moiety derived from lupinine (Lupinus luteus). The aim was to find a compound endowed with the target product profile-1 (TCP-1: molecules that clear asexual blood-stage parasitaemia), proposed by the Medicine for Malaria Venture to accomplish the goal of malaria elimination/eradication. AM1 displayed a very attractive profile in terms of both in vitro and in vivo activity. By using standard in vitro antimalarial assays, AM1 showed low nanomolar inhibitory activity against chloroquine-sensitive and resistant P. falciparum strains (range IC50 16-53 nM), matched with a high potency against P. vivax field isolates (Mean IC50 29 nM). Low toxicity and additivity with artemisinin derivatives were also demonstrated in vitro. High in vivo oral efficacy was observed in both P.berghei and P. yoelii mouse models with IC50 values comparable or better than those of chloroquine. The metabolic stability in different species and the pharmacokinetic profile in the mouse model makes AM1 a compound worth further investigation as a potential novel schizonticidal agent.

Angeloylated Germacranolides from Daucus virgatus and Their Plasmodium Transmission Blocking Activity.

Phytochemical investigation of the aerial parts of the Tunisian plant Daucus virgatus led to the isolation of eight new germacranolides named daucovirgolides A-H (1-8). The stereostructures of these sesquiterpene lactones, decorated by either one or two angeloyl groups, have been determined by a combination of MS, NMR spectroscopy, chemical derivatization, and comparison of experimental electronic circular dichroism curves with TDDFT-predicted data. Daucovirgolide G (7) proved to be the single member of this family to possess a marked inhibitory activity (92% at 50 µg/mL) on the development of Plasmodium early sporogonic stages, the nonpathogenic transmissible stages of malaria parasites, devoid of general cytotoxicity. The selective activity of daucovirgolide G points to the existence of strict structural requirements for this transmission-blocking activity and therefore of a well-defined, although yet unidentified, biological target.

Novel metalloantimalarials: Transmission blocking effects of water soluble Cu(I), Ag(I) and Au(I) phosphane complexes on the murine malaria parasite Plasmodium berghei.

The water soluble phosphane complexes [M(L)4]PF6 (M=Cu(I), Ag(I)) and [Au(L)4]Cl (L=thp (tris(hydroxymethyl)phosphane) or PTA (1,3,5-triaza-7-phosphaadamantane)) showed notable in vitro activity against Plasmodium early sporogonic stages, the sexual forms of the malaria parasite that are responsible for infection of the mosquito vector. Effects varied according to both, the type of metal and phosphane ligands. [Ag(thp)4]PF6 was the best performing complex exhibiting a half inhibitory concentration (IC50) value in the low micromolar range (0.3-15.6µM). The silver complex [Ag(thp)4]PF6 was characterized by X-ray crystallography revealing that the structure comprises the cationic complex [Ag(thp)4]+, the PF6- anion, and a water molecule of crystallization. Our results revealed that Cu(I), Ag(I) and Au(I) phosphanes complexes elicited similar activity profiles showing potential for the development of antimalarial, transmission blocking compounds. Molecules targeting the sexual parasite stages in the human and/or mosquito host are urgently needed to complement current artemisinin based treatments and next generation antimalarials in a vision not only to cure the disease but to interrupt its transmission.

In vitro and ex vivo activity of an Azadirachta indica A.Juss. seed kernel extract on early sporogonic development of Plasmodium in comparison with azadirachtin A, its most abundant constituent.

BACKGROUND: NeemAzal® (NA) is a quantified extract from seed kernels of neem, Azadirachta indica A.Juss. (Meliaceae), with a wide spectrum of biological properties, classically ascribed to its limonoid content. NA contains several azadirachtins (A to L), azadirachtin A (AzaA) being its main constituent. AzaA has been shown to inhibit microgamete formation of the rodent malaria parasite Plasmodium berghei, and NA was found to completely inhibit the transmission of Plasmodium berghei to Anopheles stephensi mosquitoes when administered to gametocytemic mice at a corresponding AzaA dose of 50mg/kg before exposure to mosquitoes. PURPOSE: The present study was aimed at i) assessing the pharmacodynamics and duration of action of NA and AzaA against P. berghei exflagellation in systemic circulation in mice and ii) elucidating the transmission blocking activity (TBA) of the main NA constituents. STUDY DESIGN: The NA and AzaA pharmacodynamics on exflagellation were assessed through ex vivo exflagellation assays, while TBA of NA constituents was evaluated through in vitro ookinete development assay. METHODS: Pharmacodynamics experiments: Peripheral blood from P. berghei infected BALB/c mice with circulating mature gametocytes, were treated i.p. with 50mg/kg and 100mg/kg pure AzaA and with NeemAzal® (Trifolio-M GmbH) at the corresponding AzaA concentrations. The effect magnitude and duration of action of compounds was estimated by counting exflagellation centers, formed by microgametocytes in process of releasing flagellated gametes, at various time points after treatment in ex vivo exflagellation tests. Ookinete Development Assay: The direct effects of NeemAzal® and AzaA on ookinete development were measured by fluorescence microscopy after incubation of gametocytemic blood with various concentrations of test substances in microplates for 24h. RESULTS: The exflagellation tests revealed an half-life of NA anti-plasmodial compounds of up to 7h at a NA dose corresponding to 100mg/kg equivalent dose of AzaA. The ookinete development assay showed an increased activity of NA against early sporogonic stages compared to that of AzaA. The IC50 value determined for NA was 6.8µg/ml (CI95: 5.95-7.86), about half of the AzaA IC50 (12.4µg/ml; CI95: 11.0-14.04). CONCLUSION: The stronger activity of NA, when compared to AzaA, could not be explained by an additive or synergistic effect by other azadirachtins (B, D and I) present in NA. In fact, the addition of these compounds at 50µM concentration to AzaA did not evidence any decrease of the IC50 against early sporogonic stages to that obtained with AzaA alone. It is likely that other non-limonoid compounds present in NA may contribute to AzaA activity and enhanced pharmacodynamics against exflagellation both in vitro and in vivo.

Transmission blocking effects of neem (Azadirachta indica) seed kernel limonoids on Plasmodium berghei early sporogonic development.

Azadirachta indica, known as neem tree and traditionally called "nature's drug store" makes part of several African pharmacopeias and is widely used for the preparation of homemade remedies and commercial preparations against various illnesses, including malaria. Employing a bio-guided fractionation approach, molecules obtained from A. indica ripe and green fruit kernels were tested for activity against early sporogonic stages of Plasmodium berghei, the parasite stages that develop in the mosquito mid gut after an infective blood meal. The limonoid deacetylnimbin (3) was identified as one the most active compounds of the extract, with a considerably higher activity compared to that of the close analogue nimbin (2). Pure deacetylnimbin (3) appeared to interfere with transmissible Plasmodium stages at a similar potency as azadirachtin A. Considering its higher thermal and chemical stability, deacetylnimbin could represent a suitable alternative to azadirachtin A for the preparation of transmission blocking antimalarials.

Neemazal ® as a possible alternative control tool for malaria and African trypanosomiasis?

BACKGROUND: Research efforts to identify possible alternative control tools for malaria and African trypanosomiasis are needed. One promising approach relies on the use of traditional plant remedies with insecticidal activities. METHODS: In this study, we assessed the effect of blood treated with different doses of NeemAzal ® (NA, neem seed extract) on mosquitoes (Anopheles coluzzii) and tsetse flies (Glossina palpalis gambiensis) (i) avidity to feed on the treated blood, (ii) longevity, and (iii) behavioural responses to human and calf odours in dual-choice tests. We also gauged NeemAzal ® toxicity in mice. RESULTS: In An. coluzzii, the ingestion of NA in bloodmeals offered by membrane feeding resulted in (i) primary antifeedancy; (ii) decreased longevity; and (iii) reduced response to host odours. In G. palpalis gambiensis, NA caused (i) a knock-down effect; (ii) decreased or increased longevity depending on the dose; and (iii) reduced response to host stimuli. In both cases, NA did not affect the anthropophilic rate of activated insects. Overall, the most significant effects were observed with NA treated bloodmeals at a dose of 2000 µg/ml for mosquitoes and 50 µg/ml for tsetse flies. Although no mortality in mice was observed after 14 days of follow-up at oral doses of 3.8, 5.6, 8.4 and 12.7 g/kg, behavioural alterations were noticed at doses above 8 g/kg. CONCLUSION: This study revealed promising activity of NA on A. coluzzii and G. palpalis gambiensis but additional research is needed to assess field efficacy of neem products to be possibly integrated in vector control programmes.

Plasmodium transmission blocking activities of Vernonia amygdalina extracts and isolated compounds.

BACKGROUND: Medicinal plants are a validated source for discovery of new leads and standardized herbal medicines. The aim of this study was to assess the activity of Vernonia amygdalina leaf extracts and isolated compounds against gametocytes and sporogonic stages of Plasmodium berghei and to validate the findings on field isolates of Plasmodium falciparum. METHODS: Aqueous (Ver-H2O) and ethanolic (Ver-EtOH) leaf extracts were tested in vivo for activity against sexual and asexual blood stage P. berghei parasites. In vivo transmission blocking effects of Ver-EtOH and Ver-H2O were estimated by assessing P. berghei oocyst prevalence and density in Anopheles stephensi mosquitoes. Activity targeting early sporogonic stages (ESS), namely gametes, zygotes and ookinetes was assessed in vitro using P. berghei CTRPp.GFP strain. Bioassay guided fractionation was performed to characterize V. amygdalina fractions and molecules for anti-ESS activity. Fractions active against ESS of the murine parasite were tested for ex vivo transmission blocking activity on P. falciparum field isolates. Cytotoxic effects of extracts and isolated compounds vernolide and vernodalol were evaluated on the human cell lines HCT116 and EA.hy926. RESULTS: Ver-H2O reduced the P. berghei macrogametocyte density in mice by about 50% and Ver-EtOH reduced P. berghei oocyst prevalence and density by 27 and 90%, respectively, in An. stephensi mosquitoes. Ver-EtOH inhibited almost completely (>90%) ESS development in vitro at 50 µg/mL. At this concentration, four fractions obtained from the ethylacetate phase of the methanol extract displayed inhibitory activity >90% against ESS. Three tested fractions were also found active against field isolates of the human parasite P. falciparum, reducing oocyst prevalence in Anopheles coluzzii mosquitoes to one-half and oocyst density to one-fourth of controls. The molecules and fractions displayed considerable cytotoxicity on the two tested cell-lines. CONCLUSIONS: Vernonia amygdalina leaves contain molecules affecting multiple stages of Plasmodium, evidencing its potential for drug discovery. Chemical modification of the identified hit molecules, in particular vernodalol, could generate a library of druggable sesquiterpene lactones. The development of a multistage phytomedicine designed as preventive treatment to complement existing malaria control tools appears a challenging but feasible goal.

Poly-Electrophilic Sesquiterpene Lactones from Vernonia amygdalina: New Members and Differences in Their Mechanism of Thiol Trapping and in Bioactivity.

In addition to known compounds, the leaves of Vernonia amygdalina afforded the new sesquiterpene lactones 14-O-methylvernolide (2), 3'-deoxyvernodalol (6), and vernomygdalin (8). These and related compounds were evaluated for modulation of a series of thiol trapping-sensitive transcription factors (NF-κB, STAT3, and Nrf2), involved in the maintenance of the chronic inflammatory condition typical of human degenerative diseases. Vernolide (1) emerged as a potent inhibitor of STAT3 and NF-κB and showed cytostatic activity toward the prostate cancer cell line DU45, arresting the cell cycle at the S phase. The exomethylene lactones are characterized by multiple Michael acceptor sites, as exemplified by vernolide (1) and vernodalol (5). By using the nuclear magnetic resonance-based cysteamine assay, the most reactive thiophilic site could be identified in both compounds, and competitive experiments qualified vernolide (1) as being more thiophilic than vernodalol (5), in agreement with the results of the pharmacological assays.

Salinomycin and other ionophores as a new class of antimalarial drugs with transmission-blocking activity.

The drug target profile proposed by the Medicines for Malaria Venture for a malaria elimination/eradication policy focuses on molecules active on both asexual and sexual stages of Plasmodium, thus with both curative and transmission-blocking activities. The aim of the present work was to investigate whether the class of monovalent ionophores, which includes drugs used in veterinary medicine and that were recently proposed as human anticancer agents, meets these requirements. The activity of salinomycin, monensin, and nigericin on Plasmodium falciparum asexual and sexual erythrocytic stages and on the development of the Plasmodium berghei and P. falciparum mosquito stages is reported here. Gametocytogenesis of the P. falciparum strain 3D7 was induced in vitro, and gametocytes at stage II and III or stage IV and V of development were treated for different lengths of time with the ionophores and their viability measured with the parasite lactate dehydrogenase (pLDH) assay. The monovalent ionophores efficiently killed both asexual parasites and gametocytes with a nanomolar 50% inhibitory concentration (IC50). Salinomycin showed a fast speed of kill compared to that of standard drugs, and the potency was higher on stage IV and V than on stage II and III gametocytes. The ionophores inhibited ookinete development and subsequent oocyst formation in the mosquito midgut, confirming their transmission-blocking activity. Potential toxicity due to hemolysis was excluded, since only infected and not normal erythrocytes were damaged by ionophores. Our data strongly support the downstream exploration of monovalent ionophores for repositioning as new antimalarial and transmission-blocking leads.