In silico study and in vitro antileishmanial and antitrypanosomal evaluation of azopyrazoles and azopyrimidines.

Hydrazones 1-6, azo-pyrazoles 7-9 and azo-pyrimidines 10-15 are compounds that exhibit antibacterial activity. The mode of action and structures of these derivatives have been previously confirmed as antibacterial. In this investigation, biological screening and molecular docking studies were performed for derivatives 1-15, with compounds 2, 7, 8, 14 and 15 yielding the best energy scores (from -20.7986 to -10.5302 kcal/mol). Drug-likeness and in silico ADME prediction for the most potent derivatives, 2, 7, 8, 14 and 15, were predicted (from 84.46 to 96.85%). The latter compounds showed good recorded physicochemical properties and pharmacokinetics. Compound 8 demonstrated the strongest inhibition, which was similar to the positive control (eflornithine) against Trypanosoma brucei brucei (WT), with an EC50 of 25.12 and 22.52µM, respectively. Moreover, compound 14 exhibited the best activity against Leishmania mexicana promastigotes and Leishmania major promastigotes (EC50 =46.85; 40.78µM, respectively).

Immunotherapy Combining Mimotopes Selected by Phage Display Plus Amphotericin B Is Effective for Treatment Against Visceral Leishmaniasis.

The treatment for visceral leishmaniasis (VL) causes toxicity in patients, entails high cost and/or leads to the emergence of resistant strains. No human vaccine exists, and diagnosis presents problems related to the sensitivity or specificity of the tests. Here, we tested two phage clones, B1 and D11, which were shown to be protective against Leishmania infantum infection in a murine model as immunotherapeutics to treat mice infected with this parasite species. The phages were used alone or with amphotericin B (AmpB), while other mice received saline, AmpB, a wild-type phage (WTP) or WTP/AmpB. Results showed that the B1/AmpB and D11/AmpB combinations induced polarised Th1-type cellular and humoral responses, which were primed by high levels of parasite-specific IFN-γ, IL-12, TNF-α, nitrite and IgG2a antibodies, which reflected in significant reductions in the parasite load in distinct organs of the animals when analyses were performed 1 and 30 days after the treatments. Reduced organic toxicity was also found in these animals, as compared with the controls. In conclusion, preliminary data suggest the potential of the B1/AmpB and D11/AmpB combinations as immunotherapeutics against L. infantum infection.

Imported leishmaniasis in Denmark.

Leishmaniasis is transmitted by sandflies and involves cutaneous, mucocutaneous, or visceral disease. Sporadic, imported cases in Denmark emphasize the need for greater awareness. The incidence is stable with at least ten verified cases per year. Diagnostic methods include PCR- and antibody tests with a high positivity rate for PCR (17%) and a low positivity rate for antibody (1.4%). The latter should be used only when visceral disease is suspected. Immunosuppressed patients are at particular risk. Treatment strategies are chosen according to the severity of the condition, as argued in this review.

Preparation and characterization of artemether-loaded niosomes in Leishmania major-induced cutaneous leishmaniasis.

Cutaneous leishmaniasis is the most prevalent form of leishmaniasis worldwide. Although various anti-leishmanial regimens have been considered, due to the lack of efficacy or occurrence of adverse reactions, design and development of novel topical delivery systems would be essential. This study aimed to prepare artemether (ART)-loaded niosomes and evaluate their anti-leishmanial effects against Leishmania major. ART-loaded niosomes were prepared through the thin-film hydration technique and characterized in terms of particle size, zeta potential, morphology, differential scanning calorimetry, drug loading, and drug release. Furthermore, anti-leishmanial effect of the preparation was assessed in vitro and in vivo. The prepared ART-loaded niosomes were spherical with an average diameter of about 100 and 300 nm with high encapsulation efficiencies of > 99%. The results of in vitro cytotoxicity revealed that ART-loaded niosomes had significantly higher anti-leishmanial activity, lower general toxicity, and higher selectivity index (SI). Half-maximal inhibitory concentration (IC50) values of ART, ART-loaded niosomes, and liposomal amphotericin B were 39.09, 15.12, and 20 µg/mL, respectively. Also, according to the in vivo study results, ART-loaded niosomes with an average size of 300 nm showed the highest anti-leishmanial effects in animal studies. ART-loaded niosomes would be promising topical drug delivery system for the management of cutaneous leishmaniasis.

Susceptibility of Leishmania to novel pentavalent organometallics: Investigating impact on DNA and membrane integrity in antimony(III)-sensitive and -resistant strains.

The aim the present study was to investigate the impact of novel pentavalent organobismuth and organoantimony complexes on membrane integrity and their interaction with DNA, activity against Sb(III)-sensitive and -resistant Leishmania strains and toxicity in mammalian peritoneal macrophages. Ph3M(L)2 type complexes were synthesized, where M = Sb(V) or Bi(V) and L = deprotonated 3-(dimethylamino)benzoic acid or 2-acetylbenzoic acid. Both organobismuth(V) and organoantimony(V) complexes exhibited efficacy at micromolar concentrations against Leishmania amazonensis and L. infantum but only the later ones demonstrated biocompatibility. Ph3Sb(L1)2 and Ph3Bi(L1)2 demonstrated distinct susceptibility profiles compared to inorganic Sb(III)-resistant strains of MRPA-overexpressing L. amazonensis and AQP1-mutated L. guyanensis. These complexes were able to permeate the cell membrane and interact with the Leishmania DNA, suggesting that this effect may contribute to the parasite growth inhibition via apoptosis. Taken altogether, our data substantiate the notion of a distinct mechanism of uptake pathway and action in Leishmania for these organometallic complexes, distinguishing them from the conventional inorganic antimonial drugs.

In vitro antileishmanial activities of hydro-methanolic crude extracts and solvent fractions of Clematis simensis fresen leaf, and Euphorbia abyssinica latex.

As a result of increasing drug resistance, crossover resistance development, prolonged therapy, and the absence of different agents with innovative methods for implementation, the efficacy of recent antileishmanial medications is severely declining. So, it is vital to look for other medications from botanical remedies that have antileishmanial activity. The latex of Euphorbia abyssinica (E abyssinica) and the leaves of Clematis simensis fresen (C simensis) were macerated in methanol (80%). In vitro antileishmanial activity of the preparation was tried on promastigotes of Leishmania aethiopica (L aethiopica) and Leishmania donovani (L donovani) using resazurin assay, and fluorescence intensity was measured. One percent of dimethyl sulfoxide (DMSO) and media as negative control and amphotericin B as positive control were used. Additionally, hemolytic & phytochemical tests of the preparation were done. The mean and standard errors of each extract were evaluated and interpreted for statistical significance using one-way analysis of variance. From sigmoidal dose-response curves of % inhibition, half maximal inhibitory concentration (IC50) values were determined by GraphPad Prism and Microsoft Excel; outcomes were presented as mean ±â€…standard error of mean of triplicate trials. P < .05 was statistical significance. The phytochemical screening of C simensis and E abyssinica confirmed the existence of steroids, phenols, tannins, saponins, alkaloids, terpenoids, flavonoids and glycosides. C simensis possesses antileishmanial activity with IC50 outcomes of 46.12 ±â€…0.03 and 8.18 ±â€…0.10 µg/mL on the promastigotes of L aethiopica and L donovani, respectively. However, E abyssinica showed stronger activity with IC50 outcomes of 16.07 ±â€…0.05 µg/mL and 4.82 ±â€…0.07 µg/mL on L aethiopica and L donovani, respectively. C simensis and E abyssinica have a less hemolytic effect on human red blood cells at low concentrations. The outcomes from this investigation demonstrated that the preparation of C simensis and E abyssinica indicated significant antileishmanial activity. Therefore, further in vivo assessment of antileishmanial, cytotoxicity activity and quantitative identification of secondary metabolites are highly recommended.

Discovery and Optimization of Tambjamines as a Novel Class of Antileishmanial Agents.

Leishmaniasis is a neglected tropical disease that is estimated to afflict over 12 million people. Current drugs for leishmaniasis suffer from serious deficiencies, including toxicity, high cost, modest efficacy, primarily parenteral delivery, and emergence of widespread resistance. We have discovered and developed a natural product-inspired tambjamine chemotype, known to be effective against Plasmodium spp, as a novel class of antileishmanial agents. Herein, we report in vitro and in vivo antileishmanial activities, detailed structure-activity relationships, and metabolic/pharmacokinetic profiles of a large library of tambjamines. A number of tambjamines exhibited excellent potency against both Leishmania mexicana and Leishmania donovani parasites with good safety and metabolic profiles. Notably, tambjamine 110 offered excellent potency and provided partial protection to leishmania-infected mice at 40 and/or 60 mg/kg/10 days of oral treatment. This study presents the first account of antileishmanial activity in the tambjamine family and paves the way for the generation of new oral antileishmanial drugs.

Designing Antitrypanosomal and Antileishmanial BODIPY Derivatives: A Computational and In Vitro Assessment.

Leishmaniasis and Human African trypanosomiasis pose significant public health threats in resource-limited regions, accentuated by the drawbacks of the current antiprotozoal treatments and the lack of approved vaccines. Considering the demand for novel therapeutic drugs, a series of BODIPY derivatives with several functionalizations at the meso, 2 and/or 6 positions of the core were synthesized and characterized. The in vitro activity against Trypanosoma brucei and Leishmania major parasites was carried out alongside a human healthy cell line (MRC-5) to establish selectivity indices (SIs). Notably, the meso-substituted BODIPY, with 1-dimethylaminonaphthalene (1b) and anthracene moiety (1c), were the most active against L. major, displaying IC50 = 4.84 and 5.41 µM, with a 16 and 18-fold selectivity over MRC-5 cells, respectively. In contrast, the mono-formylated analogues 2b and 2c exhibited the highest toxicity (IC50 = 2.84 and 6.17 µM, respectively) and selectivity (SI = 24 and 11, respectively) against T. brucei. Further insights on the activity of these compounds were gathered from molecular docking studies. The results suggest that these BODIPYs act as competitive inhibitors targeting the NADPH/NADP+ linkage site of the pteridine reductase (PR) enzyme. Additionally, these findings unveil a range of quasi-degenerate binding complexes formed between the PRs and the investigated BODIPY derivatives. These results suggest a potential correlation between the anti-parasitic activity and the presence of multiple configurations that block the same site of the enzyme.

4-Quinolinylhydrazone analogues kill Leishmania (Leishmania) amazonensis by inducing apoptosis and mitochondria-dependent pathway cell death.

Despite efforts, available alternatives for the treatment of leishmaniasis are still scarce. In this work we tested a class of 15 quinolinylhydrazone analogues and presented data that support the use of the most active compound in cutaneous leishmaniasis caused by Leishmania amazonensis. In general, the compounds showed activity at low concentrations for both parasitic forms (5.33-37.04 µM to promastigotes, and 14.31-61.98 µM to amastigotes). In addition, the best compound (MHZ15) is highly selective for the parasite. Biochemical studies indicate that the treatment of promastigotes with MHZ15 leads the loss of mitochondrial potential and increase in ROS levels as the primary effects, which triggers accumulation of lipid droplets, loss of plasma membrane integrity and apoptosis hallmarks, including DNA fragmentation and phosphatidylserine exposure. These effects were similar in the intracellular form of the parasite. However, in this parasitic form there is no change in plasma membrane integrity in the observed treatment time, which can be attributed to metabolic differences and the resilience of the amastigote. Also, ultrastructural changes such as vacuolization suggesting autophagy were observed. The in vivo effectiveness of MHZ15 in the experimental model of cutaneous leishmaniasis was carried out in mice of the BALB/c strain infected with L. amazonensis. The treatment by intralesional route showed that MHZ15 acted with great efficiency with significantly reduction in the parasite load in the injured paws and draining lymph nodes, without clinical signs of distress or compromise of animal welfare. In vivo toxicity was also evaluated and null alterations in the levels of hepatic enzymes aspartate aminotransferase, and alanine aminotransferase was observed. The data presented herein demonstrates that MHZ15 exhibits a range of favorable characteristics conducive to the development of an antileishmanial agent.

Essential oil of oregano (Origanum vulgare L.) reduces infection and proliferation of Toxoplasma gondii in BeWo cells with induction of autophagy and death of tachyzoites through a mechanism similar to necrosis.

Toxoplasmosis poses a global health threat, ranging from asymptomatic cases to severe, potentially fatal manifestations, especially in immunocompromised individuals and congenital transmission. Prior research suggests that oregano essential oil (OEO) exhibits diverse biological effects, including antiparasitic activity against Toxoplasma gondii. Given concerns about current treatments, exploring new compounds is important. This study was to assess the toxicity of OEO on BeWo cells and T. gondii tachyzoites, as well as to evaluate its effectiveness in in vitro infection models and determine its direct action on free tachyzoites. OEO toxicity on BeWo cells and T. gondii tachyzoites was assessed by MTT and trypan blue methods, determining cytotoxic concentration (CC50), inhibitory concentration (IC50), and selectivity index (SI). Infection and proliferation indices were analyzed. Direct assessments of the parasite included reactive oxygen species (ROS) levels, mitochondrial membrane potential, necrosis, and apoptosis, as well as electron microscopy. Oregano oil exhibited low cytotoxicity on BeWo cells (CC50: 114.8 µg/mL ± 0.01) and reduced parasite viability (IC50 12.5 ± 0.06 µg/mL), demonstrating 9.18 times greater selectivity for parasites than BeWo cells. OEO treatment significantly decreased intracellular proliferation in infected cells by 84% after 24 h with 50 µg/mL. Mechanistic investigations revealed increased ROS levels, mitochondrial depolarization, and lipid droplet formation, linked to autophagy induction and plasma membrane permeabilization. These alterations, observed through electron microscopy, suggested a necrotic process confirmed by propidium iodide labeling. OEO treatment demonstrated anti-T. gondii action through cellular and metabolic change while maintaining low toxicity to trophoblastic cells.

Novel Effective Medical Therapy for Acanthamoeba Keratitis.

PURPOSE: To report first clinical use of novel medical treatment for Acanthamoeba keratitis. METHODS: Interventional observational case series. Two patients with Acanthamoeba keratitis were unsuccessfully treated with polihexanide (PHMB) 0.02% and propamidine 0.1% for 6 weeks, then all were shifted in a compassionate use of PHMB 0.08% with novel standardized protocol. The postinterventional follow-up of patients was at least 7 months. RESULTS: PHMB 0.08% eyedrops in a novel standardized protocol improved infection resolution and led to complete healing of the lesion after 4 weeks in the two cases. Corneal opacities and neovascularization decreased slowly, best-corrected visual acuity slightly improved and progressively increased in the further 7 months, and no infection recurrence occurred. CONCLUSIONS: This preliminary report of two cases shows promising response to polihexanide 0.08% lowering drastically the illness duration, with reduced chance of recurrence, and mostly improving patients' quality of life.

Natural resistance to meglumine antimoniate is associated with treatment failure in cutaneous leishmaniasis caused by Leishmania (Viannia) panamensis.

The multifactorial basis of therapeutic response can obscure the relation between antimicrobial drug susceptibility and clinical outcome. To discern the relationship between parasite susceptibility to meglumine antimoniate (SbV) and therapeutic outcome of cutaneous leishmaniasis, risk factors for treatment failure were considered in evaluating this relationship in ninety-one cutaneous leishmaniasis patients and corresponding clinical strains of Leishmania (Viannia) panamensis. Parasite susceptibility to 32 µg SbV/mL (plasma Cmax) was evaluated in primary human macrophages, PBMCs, and U937 macrophages. Early parasitological response to treatment was determined in lesions of a subgroup of patients, and pathogenicity of Sb-resistant and sensitive clinical strains was compared in BALB/c mice. Parasite survival in cell models and patient lesions was determined by qRT-PCR of Leishmania 7SLRNA transcript. Parasite loads in BALB/c mice were quantified by limiting dilution analysis. The disparate Sb-susceptibility of parasite subpopulations distinguished by isoenzyme profiles (zymodemes) was manifest in all cell models. Notably, Sb-resistance defined by parasite survival, was most effectively discerned in U937 macrophages compared with primary human host cells, significantly higher among strains from patients who failed treatment than cured and, significantly associated with treatment failure. Each unit increase in transformed survival rate corresponded to a 10.6-fold rise in the odds of treatment failure. Furthermore, treatment failure was significantly associated with naturally Sb-resistant zymodeme 2.3 strains, which also produced larger lesions and parasite burdens in BALB/c mice than Sb-sensitive zymodeme 2.2 strains. The confounding effect of host risk factors for treatment failure in discerning this association was evidenced in comparing strains from patients with and without the defined risk factors for treatment failure. These results establish the association of natural resistance to meglumine antimoniate with treatment failure, the importance of host risk factors in evaluating drug susceptibility and treatment outcome, and the clinical and epidemiological relevance of natural Sb-resistance in L. (V.) panamensis subpopulations.

Design and Synthesis of 1,3-Diarylpyrazoles and Investigation of Their Cytotoxicity and Antiparasitic Profile.

Herein, we report a series of 1,3-diarylpyrazoles that are analogues of compound 26/HIT 8. We previously identified this molecule as a 'hit' during a high-throughput screening campaign for autophagy inducers. A variety of synthetic strategies were utilized to modify the 1,3-diarylpyrazole core at its 1-, 3-, and 4-position. Compounds were assessed in vitro to identify their cytotoxicity properties. Of note, several compounds in the series displayed relevant cytotoxicity, which warrants scrutiny while interpreting biological activities that have been reported for structurally related molecules. In addition, antiparasitic activities were recorded against a range of human-infective protozoa, including Trypanosoma cruzi, T. brucei rhodesiense, and Leishmania infantum. The most interesting compounds displayed low micromolar whole-cell potencies against individual or several parasitic species, while lacking cytotoxicity against human cells.

Design, synthesis and identification of novel molecular hybrids based on naphthoquinone aromatic hydrazides as potential trypanocide and leishmanicidal agents.

In pursuit of potential agents to treat Chagas disease and leishmaniasis, we report the design, synthesis, and identification novel naphthoquinone hydrazide-based molecular hybrids. The compounds were subjected to in vitro trypanocide and leishmanicidal activities. N'-(1,4-Dioxo-1,4-dihydronaphthalen-2-yl)-3,5-dimethoxybenzohydrazide (13) showed the best performance against Trypanosoma cruzi (IC50 1.83 µM) and Leishmania amazonensis (IC50 9.65 µM). 4-Bromo-N'-(1,4-dioxo-1,4-dihydronaphthalen-2-yl)benzohydrazide (16) exhibited leishmanicidal activity (IC50 12.16 µM). Regarding trypanocide activity, compound 13 was low cytotoxic to LLC-MK2 cells (SI = 95.28). Furthermore, through molecular modeling studies, the cysteine proteases cruzain, rhodesain and CPB2.8 were identified as the potential biological targets.

Py-CoMFA, docking, and molecular dynamics simulations of Leishmania (L.) amazonensis arginase inhibitors.

Leishmaniasis is a disease caused by a protozoan of the genus Leishmania, affecting millions of people, mainly in tropical countries, due to poor social conditions and low economic development. First-line chemotherapeutic agents involve highly toxic pentavalent antimonials, while treatment failure is mainly due to the emergence of drug-resistant strains. Leishmania arginase (ARG) enzyme is vital in pathogenicity and contributes to a higher infection rate, thus representing a potential drug target. This study helps in designing ARG inhibitors for the treatment of leishmaniasis. Py-CoMFA (3D-QSAR) models were constructed using 34 inhibitors from different chemical classes against ARG from L. (L.) amazonensis (LaARG). The 3D-QSAR predictions showed an excellent correlation between experimental and calculated pIC50 values. The molecular docking study identified the favorable hydrophobicity contribution of phenyl and cyclohexyl groups as substituents in the enzyme allosteric site. Molecular dynamics simulations of selected protein-ligand complexes were conducted to understand derivatives' interaction modes and affinity in both active and allosteric sites. Two cinnamide compounds, 7g and 7k, were identified, with similar structures to the reference 4h allosteric site inhibitor. These compounds can guide the development of more effective arginase inhibitors as potential antileishmanial drugs.

Development of a topical treatment for tegumentary leishmaniasis using 8-hydroxyquinoline.

In the context of neglected diseases, tegumentary leishmaniasis (TL) presents an emerging and re-emerging character in the national territory and in the world. The treatment of TL has limitations, such as intravenous administration route, high toxicity, and high treatment costs. Thus, several researchers work on new therapeutic strategies to improve the effectiveness of the treatment of leishmaniasis. In this light, the present study used a topical formulation, containing 8-hydroquinoline (8-HQN), for the treatment of Balb/c mice infected with L. amazonensis. After the treatment, the mean diameter of the lesion was measured, as well as the parasite load in organs and immunological parameters associated with the treatment. The results showed that the animals treated with 8-HQN 5%, when compared to controls, showed a reduction in the mean diameter of the lesion and in the parasite load. The animals treated with the ointment showed a type 1 cellular immune response profile associated with the production of cytokines such as INF-γ and TNF-α. In addition, the treatment did not demonstrate toxicity to mice. Therefore, the topical formulation containing 8-HQN 5% is a promising candidate in the topical treatment and could be considered, in the future, as an alternative for the treatment of TL.

The antiprotozoal drug nitazoxanide improves experimental liver fibrosis in mice.

Nitazoxanide is an FDA-approved antiprotozoal drug. Our previous studies find that nitazoxanide and its metabolite tizoxanide affect AMPK, STAT3, and Smad2/3 signals which are involved in the pathogenesis of liver fibrosis, therefore, in the present study, we examined the effect of nitazoxanide on experimental liver fibrosis and elucidated the potential mechanisms. The in vivo experiment results showed that oral nitazoxanide (75, 100 mg·kg-1) significantly improved CCl4- and bile duct ligation-induced liver fibrosis in mice. Oral nitazoxanide activated the inhibited AMPK and inhibited the activated STAT3 in liver tissues from liver fibrosis mice. The in vitro experiment results showed that nitazoxanide and its metabolite tizoxanide activated AMPK and inhibited STAT3 signals in LX-2 cells (human hepatic stellate cells). Nitazoxanide and tizoxanide inhibited cell proliferation and collagen I expression and secretion of LX-2 cells. Nitazoxanide and tizoxanide inhibited transforming growth factor-ß1 (TGF-ß1)- and IL-6-induced increases of cell proliferation, collagen I expression and secretion, inhibited TGF-ß1- and IL-6-induced STAT3 and Smad2/3 activation in LX-2 cells. In mouse primary hepatic stellate cells, nitazoxanide and tizoxanide also activated AMPK, inhibited STAT3 and Smad2/3 activation, inhibited cell proliferation, collagen I expression and secretion. In conclusion, nitazoxanide inhibits liver fibrosis and the underlying mechanisms involve AMPK activation, and STAT3 and Smad2/3 inhibition.

Evaluation and discovery of novel benzothiazole derivatives as promising hits against Leishmania infantum.

An early exploration of the benzothiazole class against two kinetoplastid parasites, Leishmania infantum and Trypanosoma cruzi, has been performed after the identification of a benzothiazole derivative as a suitable antileishmanial initial hit. The first series of derivatives focused on the acyl fragment of its class, evaluating diverse linear and cyclic, alkyl and aromatic substituents, and identified two other potent compounds, the phenyl and cyclohexyl derivatives. Subsequently, new compounds were designed to assess the impact of the presence of diverse substituents on the benzothiazole ring or the replacement of the endocyclic sulfur by other heteroatoms. All compounds showed relatively low cytotoxicity, resulting in decent selectivity indexes for the most active compounds. Ultimately, the in vitro ADME properties of these compounds were assessed, revealing a satisfying water solubility, gastrointestinal permeability, despite their low metabolic stability and high lipophilicity. Consequently, compounds 5 and 6 were identified as promising hits for further hit-to-lead exploration within this benzothiazole class against L. infantum, thus providing promising starting points for the development of antileishmanial candidates.

A Theileria annulata parasite with a single mutation, methionine 128 to isoleucine (M128I), in cytochrome B is resistant to buparvaquone.

Tropical theileriosis is a fatal leukemic-like disease of cattle caused by the tick-transmitted protozoan parasite Theileria annulata. The economics of cattle meat and milk production is severely affected by theileriosis in endemic areas. The hydroxynaphtoquinone buparvaquone (BPQ) is the only available drug currently used to treat clinical theileriosis, whilst BPQ resistance is emerging and spreading in endemic areas. Here, we chronically exposed T. annulata-transformed macrophages in vitro to BPQ and monitored the emergence of drug-resistant parasites. Surviving parasites revealed a significant increase in BPQ IC50 compared to the wild type parasites. Drug resistant parasites from two independent cloned lines had an identical single mutation, M128I, in the gene coding for T. annulata cytochrome B (Tacytb). This in vitro generated mutation has not been reported in resistant field isolates previously, but is reminiscent of the methionine to isoleucine mutation in atovaquone-resistant Plasmodium and Babesia. The M128I mutation did not appear to exert any deleterious effect on parasite fitness (proliferation and differentiation to merozoites). To gain insight into whether drug-resistance could have resulted from altered drug binding to TaCytB we generated in silico a 3D-model of wild type TaCytB and docked BPQ to the predicted 3D-structure. Potential binding sites cluster in four areas of the protein structure including the Q01 site. The bound drug in the Q01 site is expected to pack against an alpha helix, which included M128, suggesting that the change in amino acid in this position may alter drug-binding. The in vitro generated BPQ resistant T. annulata is a useful tool to determine the contribution of the various predicted docking sites to BPQ resistance and will also allow testing novel drugs against theileriosis for their potential to overcome BPQ resistance.

Antiprotozoal Activity of Plants Used in the Management of Sleeping Sickness in Angola and Bioactivity-Guided Fractionation of Brasenia schreberi J.F.Gmel and Nymphaea lotus L. Active against T. b. rhodesiense.

Folk medicine is widely used in Angola, even for human African trypanosomiasis (sleeping sickness) in spite of the fact that the reference treatment is available for free. Aiming to validate herbal remedies in use, we selected nine medicinal plants and assessed their antitrypanosomal activity. A total of 122 extracts were prepared using different plant parts and solvents. A total of 15 extracts from seven different plants exhibited in vitro activity (>70% at 20 µg/mL) against Trypanosoma brucei rhodesiense bloodstream forms. The dichloromethane extract of Nymphaea lotus (leaves and leaflets) and the ethanolic extract of Brasenia schreberi (leaves) had IC50 values ≤ 10 µg/mL. These two aquatic plants are of particular interest. They are being co-applied in the form of a decoction of leaves because they are considered by local healers as male and female of the same species, the ethnotaxon "longa dia simbi". Bioassay-guided fractionation led to the identification of eight active molecules: gallic acid (IC50 0.5 µg/mL), methyl gallate (IC50 1.1 µg/mL), 2,3,4,6-tetragalloyl-glucopyranoside, ethyl gallate (IC50 0.5 µg/mL), 1,2,3,4,6-pentagalloyl-ß-glucopyranoside (IC50 20 µg/mL), gossypetin-7-O-ß-glucopyranoside (IC50 5.5 µg/mL), and hypolaetin-7-O-glucoside (IC50 5.7 µg/mL) in B. schreberi, and 5-[(8Z,11Z,14Z)-heptadeca-8,11,14-trienyl] resorcinol (IC50 5.3 µg/mL) not described to date in N. lotus. Five of these active constituents were detected in the traditional preparation. This work provides the first evidence for the ethnomedicinal use of these plants in the management of sleeping sickness in Angola.