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Ethnopharmacological relevance: An aqueous decoction of root bark of Alstonia congensis Engl. (Apocynaceae) is used in several African countries to treat various ailments including malaria. Materials and methods: Extracts of different polarity and isolated constituents were tested in vitro for their antiplasmodial activity against the chloroquine-resistant strain Plasmodium falciparum K1 and the chloroquine-sensitive strain P. falciparum NF54A19A, as well as for their cytotoxic effects againt MRC-5 cells (human lung fibroblasts). Extracts and fractions were evaluated in vivo against the chloroquine-resistant strain P. yoelii N67 and the chloroquine-sensitive strain P. berghei berghei ANKA. Results: The aqueous extract, the 80% methanol extract and the alkaloid-enriched extract exhibited strong antiplasmodial activity against P. falciparum K1 with IC50 values < 10 mu g/ml and against P. falciparum NF54 A19A with IC50 values < 0.02 mu g/ml. In vivo against P. yoelii N67, at the highest oral dose of 400 mg/kg body weight, all extracts produced 70-73% chemosuppression, while against P. berghei berghei, more than 75% chemosuppression was observed. With regard to the isolated constituents, boonein was inactive in vitro against P. falci-parwn K-1 (IC50 > 64 mu M), while echitamine, 6,7-seco-angustilobine B and beta-amyrin exhibited moderate activity (IC50 < 30 mu M). Against P. falciparum NF54 A19A, boonein was inactive (IC50 > 64 mu M), while echitamine, 6,7-secoangustilobine and beta-amyrin showed moderate IC50 values of 11.07, 21.26 and 40.70 mu M, respectively. Conclusion: These results demonstrated that all extracts from A. congensis root bark possessed antiplasmodial activity in vitro and in vivo. They can be used as raw materials for the preparation of ameliorated remedies for the treatment of uncomplicated malaria. The observed antiplasmodial activity may be due in part to the presence of indole alkaloids.

A series of eleven double prodrug derivatives of a fosmidomycin surrogate were synthesized and investigated for their ability to inhibit in vitro growth of P. falciparum and M. tuberculosis. A pivaloyloxymethyl (POM) phosphonate prodrug modification was combined with various prodrug derivatisations of the hydroxamate moiety. The majority of compounds showed activity comparable with or inferior to fosmidomycin against P. falciparum. N-benzyl substituted carbamate prodrug 6f was the most active antimalarial analog with an IC50 value of 0.64 mu M. Contrary to fosmidomycin and parent POM-prodrug 5, 2-nitrofuran and 2-nitrothiophene prodrugs 6i and 6j displayed promising antitubercular activities.

Human African trypanosomiasis is a neglected parasitic disease for which the current treatment options are quite limited. Trypanosomes are not able to synthesize purines de novo and thus solely depend on purine salvage from the host environment. This characteristic makes players of the purine salvage pathway putative drug targets. The activity of known nucleoside analogues such as tubercidin and cordycepin led to the development of a series of C7-substituted nucleoside analogues. Here, we use RNA interference (RNAi) libraries to gain insight into the mode-of-action of these novel nucleoside analogues. Whole-genome RNAi screening revealed the involvement of adenosine kinase and 4E interacting protein into the mode-of-action of certain antitrypanosomal nucleoside analogues. Using RNAi lines and gene-deficient parasites, 4E interacting protein was found to be essential for parasite growth and infectivity in the vertebrate host. The essential nature of this gene product and involvement in the activity of certain nucleoside analogues indicates that it represents a potential novel drug target.

The discovery of novel antiparasitic drugs for neglected tropical diseases (NTDs) constitutes a global urgency and requires a range of innovative strategies to ensure a sustainable pipeline of lead compounds. Thus far, primaquine (PQ) is the only transmission-blocking antimalarial that is clinically available, displaying marked activity against gametocytes of all causative species of human malaria (Plasmodium spp.). Chagas disease, caused by Trypanosoma cruzi, is another PQ-sensitive illness besides malaria. One of the major drawbacks of PQ is its metabolism into carboxyprimaquine (CPQ), which is less active than the parent drug. In this study, we developed different synthetic pathways to confer N-protection to PQ through introduction of thioxo-imidazolidin-4-one. The introduction of this group prevents the formation of CPQ, counteracting one major drawback of the parent drug. After that, we evaluated the potential biological activity of the novel 2-thioxo-imidazolidin-4-one derivative of PQ, which showed relevant in vitro activity against Trypanosoma cruzi (IC50 1.4 μM) compared to PQ (IC50 1.7 μM) and the reference drug benznidazole (IC50 1.6 μM). Noting its acceptable pharmacokinetic profile, this PQ conjugate may be a potential scaffold for novel drug exploration against Chagas disease.

Nine cyclopeptide alkaloids (1−9), of which five (compounds 2, 3, 5, 8, and 9) are described herein for the first time, were isolated from roots of Ziziphus oxyphylla by means of conventional separation methods as well as semipreparative HPLC with DAD and ESIMS detection and LC-DAD-SPE-NMR. Structure elucidation was done by spectroscopic means. Nummularine-R (1), a previously known constituent from this species, was isolated along with its new derivatives O-desmethylnummularine-R (2) and Odesmethylnummularine- R N-oxide (3). In addition, the known compounds hemsine-A (4) and ramosine-A (6), as well as hemsine-A N-oxide (5), were isolated. Moreover, oxyphylline-C (7), a known constituent of Z. oxyphylla stems, was obtained, and two new compounds were identified, oxyphyllines-E (8) and -F (9). Just like oxyphylline-C, oxyphyllines-E and -F belong to the relatively rare class of neutral cyclopeptide alkaloids. The antiplasmodial activity and cytotoxicity of compounds 1, 2, 4, 6, and 9 were evaluated, and the most promising activity was found for O-desmethylnummularine-R (2), which exhibited an IC50 value of 3.2 ± 2.6 μM against Plasmodium falciparum K1, whereas an IC50 value of >64.0 μM was evident for its cytotoxicity against MRC-5 cells.

Background: Nepal is very rich in biodiversity, and no extensive effort has yet been carried out to screen plants that are used by traditional healers against parasitic diseases. The aim of this study was to evaluate the in vitro antileishmanial and antimalarial activity of crude methanolic or ethanolic extracts of 29 plant species that are currently used by local people of Nepal for treating different ailments. Methods: Crude extracts of leaves, twigs, aerial parts, and/or roots of the selected plants were evaluated for in vitro inhibitory activity against intracellular amastigotes of Leishmania infantum and against erythrocytic stages of Plasmodium falciparum. To determine the selectivity index (SI), cytotoxicity was assessed on MRC-5 cells in parallel. Results: Three plant species, namely Phragmites vallatoria and Ampelocissus tomentosa, for which no antiprotozoal activity has previously been reported, and Terminalia chebula revealed antiprotozoal activity. The extract of A. tomentosa exhibited moderate activity against L. infantum with an inhibitory concentration 50% (IC50) of 13.2 +/- 4.3 mu g/ml and SI >3, while T. chebula exhibited fairly good antiplasmodial activity with IC50 values of 4.5 +/- 2.4 mu g/ml and SI values >5. Conclusion: In countries like Nepal, where the current health system is unable to combat the burden of endemic parasitic diseases, evaluation of local plants as a potential source of the drug can help in expanding the treatment options. The extent of untapped resources available in these countries provides an opportunity for future bioprospecting.

Biofilm-associated infections are hard to treat because of their high antibiotic resistance and the presence of a very persistent subpopulation of bacteria. The second messenger molecule cyclic di-guanosine monophosphate (c-di-GMP) plays a very important role in this biofilm physiology. Here, we evaluated the role of YddV, an enzyme with a c-di-GMP synthesis function, in the formation and maturation of Escherichia coli biofilms. Our results suggest that YddV stimulates biofilm growth via its role in the production of c-di-GMP and this likely by influencing the production of matrix (e.g. poly-N-acetylglucosamine (PGA)). However, lowering the YddV expression did not alter the biofilm formation since there was no significant difference between the biofilm phenotypes of WT E. coli and YddV-knockout bacteria. Additionally, YddV expression had no significant influence on the amount of persister cells within the biofilm population, questioning the use of YddV as therapeutic target. (C) 2016 Published by Elsevier B.V.

Four cyclopeptide alkaloids (1-4) were isolated from the root bark of Hymenocardia acida by means of semipreparative HPLC with DAD and ESIMS detection and conventional separation methods. Structure elucidation was performed by spectroscopic means. In addition to the known compound hymenocardine (1), three other alkaloids were isolated for the first time from a natural source. These included a hymenocardine derivative with a hydroxy group instead of a carbonyl group that was named hymenocardinol (2), as well as hymenocardine N-oxide (3) and a new cyclopeptide alkaloid containing an unusual histidine moiety named hymenocardine-H (4). The isolated cyclopeptide alkaloids were tested for their antiplasmodial activity and cytotoxicity. All four compounds showed moderate antiplasmodial activity, with IC50 values ranging from 12.2 to 27.9 mu M, the most active one being hymenocardine N-oxide (3), with an IC50 value of 12.2 +/- 6.6 mu M. Compounds 2-4 were found not to be cytotoxic against MRC-5 cells (IC50 > 64.0 mu M), but hymenocardine (1) showed some cytotoxicity, with an IC50 value of 51.1 +/- 17.2 mu M.

Nymphoides indica (L.) Kuntze (Menyanthaceae) is traditionally used in the Indian subcontinent. However, scientific data reporting its constituents are poor. This study aimed at evaluating its phytochemical constituents and various biological activities. Phytochemical investigations of the extracts and fractions resulted in the isolation of 5 lipophilic compounds, i.e. azelaic (nonanedioic) acid (1) and 4-methyl-heptanedioic acid (3), hexadecanoic (2) and stearic acid (5) and the fatty alcohol hexadecanol (4); 3 seco-iridoids, i.e. 7-epiexaltoside (6), 6″,7″-dihydro-7-epiexaltoside (7) and menthiafolin (8); 3 flavonoids, i.e. 3,7-di-O-methylquercetin-4′-O-β-glucoside (9), 3-O-methylquercetin-7-O-β-glucoside (10) and 3,7-di-O-methylquercetin (11); scopoletin (12) and ferulic acid (13); and the monoterpenoids foliamenthoic acid (14) and 6,7-dihydrofoliamenthoic acid methyl ester (15). Compounds 1–5 showed moderate antimicrobial activities, whereas compound 9 presented mild antiprotozoal activities against Trypanosoma brucei (IC50 8 μM), Leishmania infantum (IC50 32 μM) and Trypanosoma cruzi (IC50 30 μM). Antiglycation activity was shown by compounds 7 (IC50 0.36 mM), 10 (IC50 0.42 mM) and 15 (IC50 0.61 mM). Finally α-glucosidase inhibition was shown by compounds 7, 9, 11 and 13–15. It could be concluded that N. indica leaf extracts possess mild to moderate antimicrobial, antiprotozoal, antioxidant and antidiabetic activities. Copyright © 2016 John Wiley & Sons, Ltd.

The bioactive petroleum ether fraction of Verbesina encelioides, previously studied by the authors, was chosen for the isolation of antiprotozoal metabolites. Pseudotaraxasterol-3β-acetate (1), benzyl 2,6-dimethoxy benzoate (2), 16β-hydroxy-pseudotaraxasterol-3β-palmitate (3) and pseudotaraxasterol (4), in addition to β-sitosterol glucoside (5) and β-sitosterol galactoside (6) were isolated and identified based on one-dimensional and two-dimensional spectral analysis. This is the first report describing (3) and (6) in genus Verbesina. The isolated compounds were tested in vitro against Plasmodium falciparum, Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum. Cytotoxicity was evaluated on MRC-5 cells. Compound 1 showed moderate to weak activity against L. infantum T. brucei and P. falciparum and was inactive against T. cruzi. Compound 3 showed moderate activity against L. infantum, compound 4 revealed weak activity against T. cruzi, while 5 and 6 were inactive against all tested protozoa. All compounds were non-cytotoxic. The isolated constituents showed less antiprotozoal activity than the crude fraction.

Many topical commercial products are currently available for the treatment of onychomycosis. However, limited data are available concerning their antifungal activity. Using an in vitro onychomycosis model, the daily application of seven nail formulations was compared to the antifungal reference drug amorolfine (Loceryl(®) ) and evaluated for inhibitory activity against Trichophyton mentagrophytes using an agar diffusion test. Of all commercial nail formulations, only Excilor(®) and Nailner(®) demonstrated inhibitory activity, which was much lower compared to the daily application of Loceryl(®) . However, Excilor(®) showed similar efficacy compared to the conventional weekly application of Loceryl(®) . These results suggest a role for organic acids in the antifungal effect of Excilor(®) (acetic acid, ethyl lactate) and Nailner(®) (lactic acid, citric acid, ethyl lactate) as all tested formulations without organic acids were inactive.

Streptococcus pneumoniae is the leading cause of bacterial pneumonia. Infection is linked to high morbidity and mortality rates and antibiotic resistance within this pathogen is on the rise. Therefore, there is a need for novel antimicrobial therapies. To lower the time and costs of the drug discovery process, alternative in vivo models should be considered. As such, Galleria mellonella larvae can be of great value. The larval immunity consisting of several types of haemocytes is remarkably similar to the human innate immune system. Furthermore, these larvae don’t require specific housing, are cheap and are easy to handle. In this study, the use of a G. mellonella infection model to study early pneumococcal infections and treatment is proposed. Firstly, the fitness of this model to study pneumococcal virulence factors is confirmed using streptococcal strains TIGR4, ATCC®49619, D39 and its capsule-deficient counterpart R6 at different inoculum sizes. The streptococcal polysaccharide capsule is considered the most important virulence factor without which streptococci are unable to sustain an in vivo infection. Kaplan–Meier survival curves showed indeed a higher larval survival after infection with streptococcal strain R6 compared to strain D39. Then, the infection was characterized by determining the number of haemocytes, production of oxygen free radicals and bacterial burden at several time points during the course of infection. Lastly, treatment of infected larvae with the standard antibiotics amoxicillin and moxifloxacin was evaluated. Treatment has proven to have a positive outcome on the course of infection, depending on the administered dosage. These data imply that G. mellonella larvae can be used to evaluate antimicrobial therapies against S. pneumoniae, apart from using the larval model to study streptococcal properties. The in-depth knowledge acquired regarding this model, makes it more suitable for use in future research.

Although miltefosine (MIL) has only been approved for the treatment of visceral leishmaniasis (VL) in 2002, its application in monotherapy already led to the development of two confirmed MIL-resistant isolates by 2009. Although liposomal amphotericin B is recommended as first-line treatment in Europe, MIL is still occasionally used in HIV co-infected patients. Since their immune system is incapable of controlling the infection, high parasite burdens and post-treatment relapses are common. Linked to the particular pharmacokinetic profile of MIL, successive treatment of recurrent relapses could in principle facilitate the emergence of drug resistance. This study evaluated the effect of immunosuppression (cyclophosphamide 150 mg/kg once weekly) on the development of MIL-resistance in Syrian golden hamsters infected with Leishmania infantum. The hamsters were treated with MIL (20 mg/kg orally for 5 days) whenever clinical signs of infection or relapse were observed. The immunosuppression resulted in a significant depletion of CD4+ lymphocytes and MHCII-expressing cells in peripheral blood, and a concomitant increase in tissue parasite burdens and shorter time to relapse, but the strain's susceptibility upon repeated MIL exposure remained unaltered. This study demonstrates that immunosuppression accelerates the occurrence of relapse without expediting MIL resistance development., Graphical abstract Image 1, Highlights • Successive treatment cycles of L. infantum in the Syrian golden hamster model do not result in MIL resistance. • Immunosuppression of infected hamsters with cyclophosphamide (CPA) does not expedite the emergence of MIL resistance. • Weekly CPA administration decreases the time-to-relapse and increases parasite burdens in liver, spleen and bone marrow. • Immunosuppression with CPA results in a depletion of CD4+ lymphocytes and MHCII-expressing cells in peripheral blood.

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The available treatments for leishmaniasis are less than optimal due to inadequate efficacy, toxic side-effects and the emergence of resistant strains, clearly endorsing the urgent need for discovery and development of novel drug candidates. Ideally, these should act via an alternative mechanism-of-action to avoid cross-resistance with the current drugs. As cyclic nucleotide specific phosphodiesterases (PDEs) of L. major have been postulated as putative drug targets, a series of potential inhibitors of Leishmania PDEs was explored. Several displayed potent and selective in vitro activity against L. infantum intracellular amastigotes. One imidazole derivative, compound 35, was shown to reduce the parasite loads in vivo and dose-dependently increase the cellular cAMP level at just 2* and 5* the IC50, indicating a correlation between antileishmanial activity and increased cellular cAMP. Docking studies and molecular dynamics simulations pointed to imidazole 35 exerting its activity through PDE inhibition. This study establishes for the first time that inhibition of cAMP PDEs can potentially be exploited for new antileishmanial chemotherapy., Funding from the EC 7th Framework Programme (FP7-HEALTH-2013 INNOVATION-1, PDE4NPD no. 602666), MINECO (Grant SAF2015-65740-R), RICET (RD16/0027/0010), FEDER funds and MECD (Grant FPU15/1465 to V. S.-P.) is acknowledged.

Tuberculosis (TB) still has a major impact on public health. In order to efficiently eradicate this life-threatening disease, the exploration of novel anti-TB drugs is of paramount importance. As part of our program to design new 2-azaanthraquinones with anti-mycobacterial activity, various “out-of-plane” tetrahydro- and octahydrobenzo[j]phenanthridinediones were synthesized. In this study, the scaffold of the most promising hits was further optimized in an attempt to improve the bioactivity and to decrease enzymatic degradation. The rudiment bio-evaluation of a small library of fluorinated tetrahydrobenzo[j]phenanthridine-7,12-dione derivatives indicated no significant improvement of the bio-activity against intracellular and extracellular Mycobacterium tuberculosis (Mtb). Though, the derivatives showed an acceptable toxicity against J774A.1 macrophages and early signs of genotoxicity were absent. All derivatives showed to be metabolic stabile in the presence of both phase I and phase II murine or human microsomes. Finally, the onset of reactive oxygen species within Mtb after exposure to the derivatives was measured by electron paramagnetic resonance (EPR). Results showed that the most promising fluorinated derivative is still a possible candidate for the subversive inhibition of mycothione reductase.

Primary mouse macrophages are frequently used to provide an in vitro intracellular model to evaluate antileishmanial drug efficacy. The present study compared the phenotypic characteristics of Swiss, BALB/c, and C57BL/6 mouse bone marrow-derived macrophages and peritoneal exudate cells using different stimulation and adherence protocols upon infection with a Leishmania infantum laboratory strain and two clinical isolates. Evaluation parameters were susceptibility to infection, permissiveness to amastigote multiplication, and impact on drug efficacy. Observed variations in infection of peritoneal exudate cells can mostly be linked to changes in the inflammatory cytokine profiles (IL-6, TNF-α, KC/GRO) rather than to differences in initial production of nitric oxide and reactive oxygen species. Optimization of the cell stimulation and adherence conditions resulted in comparable infection indices among peritoneal exudate cells and the various types of bone marrow-derived macrophages. BALB/c-derived bone marrow-derived macrophages were slightly more permissive to intracellular amastigote replication. Evaluation of antileishmanial drug potency in the various cell systems revealed minimal variation for antimonials and paromomycin, and no differences for miltefosine and amphotericin B. The study results allow to conclude that drug evaluation can be performed in all tested primary macrophages as only marginal differences are observed in terms of susceptibility to infection and impact of drug exposure. Combined with some practical considerations, the use of 24-h starch-stimulated, 48-h adhered, Swiss-derived peritoneal exudate cells can be advocated as an efficient, reliable, relatively quick, and cost-effective tool for routine drug susceptibility testing in vitro whenever the use of primary cells is feasible.

Several trypanosomatid cyclic nucleotide phosphodiesterases (PDEs) possess a unique, parasite-specific cavity near the ligand-binding region that is referred to as the P-pocket. One of these enzymes, Trypanosoma brucei PDE B1 (TbrPDEB1), is considered a drug target for the treatment of African sleeping sickness. Here, we elucidate the molecular determinants of inhibitor binding and reveal that the P-pocket is amenable to directed design. By iterative cycles of design, synthesis, and pharmacological evaluation and by elucidating the structures of inhibitor-bound TbrPDEB1, hPDE4B, and hPDE4D complexes, we have developed 4a,5,8,8a-tetrahydrophthalazinones as the first selective TbrPDEB1 inhibitor series. Two of these, 8 (NPD-008) and 9 (NPD-039), were potent (Ki = 100 nM) TbrPDEB1 inhibitors with antitrypanosomal effects (IC50 = 5.5 and 6.7 μM, respectively). Treatment of parasites with 8 caused an increase in intracellular cyclic adenosine monophosphate (cAMP) levels and severe disruption of T. brucei cellular organization, chemically validating trypanosomal PDEs as therapeutic targets in trypanosomiasis.

Here is reported the anti

Prevention of severe lower respiratory tract infections in infants caused by the human respiratory syncytial virus (hRSV) remains a major public health priority. Currently, the major focus of vaccine development relies on the RSV fusion (F) protein since it is the main target protein for neutralizing antibodies induced by natural infection. The protein conserves 5 N-glycosylation sites, two of which are located in the F2 subunit (N27 and N70), one in the F1 subunit (N500) and two in the p27 peptide (N116 and N126). To study the influence of the loss of one or more N-glycosylation sites on RSV F immunogenicity, BALB/c mice were immunized with plasmids encoding RSV F glycomutants. In comparison with F WT DNA immunized mice, higher neutralizing titres were observed following immunization with F N116Q. Moreover, RSV A2-K-line19F challenge of mice that had been immunized with mutant F N116Q DNA was associated with lower RSV RNA levels compared with those in challenged WT F DNA immunized animals. Since p27 is assumed to be post-translationally released after cleavage and thus not present on the mature RSV F protein, it remains to be elucidated how deletion of this glycan can contribute to enhanced antibody responses and protection upon challenge. These findings provide new insights to improve the immunogenicity of RSV F in potential vaccine candidates.

An aqueous decoction of stem bark of Triclisia gilletii (De Wild.) Staner (Menispermaceae) is used in several African countries to treat various diseases including malaria. The aqueous extract and the total alkaloid extract were evaluated in vitro for their antiplasmodial activity against the chloroquine-resistant Plasmodium falciparum strain K-1, the chloroquine-sensitive strain NF54 A19A, and in vivo in mice infected with P. berghei berghei and P. yoelii N67. Both extracts were active in vitro with IC50 values of 0.75 and 0.25 mu g/ml against P. falciparum Kl, respectively; and 1.15 and < 0.02 mu g/ml against P. falciparum NF54 A19A, respectively. With regard to the in vivo activity, at the highest oral dose of 400 mg/kg body weight, the aqueous and the total alkaloid extracts produced 73.0% and 80.7% chemosuppression against P. berghei berghei, respectively, while against P. yoelii N67, a chemosuppression of 70.1%, and 78.4%, respectively, was observed. Two bisbenzylisoquinoline alkaloids were isolated from the total alkaloid extract, i.e. (-)-pycmanilline and (-)-phaeanthine in a yield of 0.20% and 0.40%, respectively. They were active in vitro against P. falciparum K-1 (IC50 1.6 +/- 0.3 and 0.8 +/- 0.3 mu M, respectively), and P. falciparum NF54 A19 A (IC50 0.07 +/- 0.01 and 0.03 +/- 0.01 mu M, respectively). Also against other protozoa IC50 values in the micromolar range were observed. (-)-Pycmanilline is reported for the first time.

Several methods have been developed for the detection of Leishmania, mostly targeting the minicircle kinetoplast DNA (kDNA). A new RNA real-time quantitative PCR (qPCR) assay was developed targeting the conserved and highly expressed spliced-leader (SL) mini-exon sequence. This study compared the limits of detection of various real-time PCR assays in hamsters infected with Leishmania infantum, in spiked human blood, and in clinical blood samples from visceral leishmaniasis patients. The SL-RNA assay showed an excellent analytical sensitivity in tissues (0.005 and 0.002 parasites per mg liver and spleen, respectively) and was not prone to false-positive reactions. Evaluation of the SL-RNA assay on clinical samples demonstrated lower threshold cycle values than the kDNA qPCR, an excellent interrun stability of 97%, a 93% agreement with the kDNA assay, and an estimated sensitivity, specificity, and accuracy of 93.2%, 94.3%, and 93.8%, respectively. The SL-RNA qPCR assay was equally efficient for detecting Leishmania major, Leishmania tropica, Leishmania mexicana, Leishmania guayensis, Leishmania panamensis, Leishmania braziliensis, L. infantum, and Leishmania donovani and revealed similar SL-RNA levels in the different species and the occurrence of polycistronic SL-containing transcripts in Viannia species. Collectively, this single SL-RNA qPCR assay enables universal Leishmania detection and represents a particularly useful addition to the widely used kDNA assay in clinical studies in which the detection of viable parasites is pivotal to assess parasitological cure.

Here is reported the anti Leishmania infantum activity of 48 hexane, CH2Cl2 and MeOH extracts from 16 macroalgae collected on the Iberian Coast. Seven hexane and CH2Cl2 Cystoseira baccata, Cystoseira barbata, Cystoseira tamariscifolia, Cystoseira usneoides, Dictyota spiralis and Plocamium cartilagineum extracts were active towards promastigotes (IC50 29.8-101.8 mu g/mL) inducing strong morphological alterations in the parasites. Hexane extracts of C. baccata and C. barbata were also active against intracellular amastigotes (IC50 5.1 and 6.8 mu g/mL, respectively). Fatty acids, triacylglycerols, carotenoids, steroids and meroterpenoids were detected by nuclear magnetic resonance (NMR), and gas chromatography in the Cystoseira extracts. These results suggest that Cystoseira macroalgae contain compounds with antileishmanial activity, which could be explored as scaffolds to the development of novel sources of antiparasitic derivatives. [GRAPHICS] . Foundation for Science and Technology (FCT)Portuguese Foundation for Science and Technology Portuguese National Budget [PTDC/MAR/103957/2008, CCMAR/Multi/04326, GHTM-UID/Multi/04413/2013, SFRH/BD/78062/2011, SFRH/BD/81425/2011, IF/00049/2012, IF/00743/2015] Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)CAPES Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)National Council for Scientific and Technological Development (CNPq) info:eu-repo/semantics/publishedVersion

During the past decades, visceral leishmaniasis therapy has been faced with the rapid emergence of drug resistance against the pentavalent antimonials which had been used as mainstay of treatment for over 70 years. Even though cutaneous leishmaniasis cannot be linked to development of drug resistance, the huge species- and strain-specific variations in drug susceptibilities severely complicate effective treatment as well. A new challenge in leishmaniasis control has arisen with increasing numbers of treatment failures against all of the currently used anti-leishmanial standard drugs. The exact causes of these treatment failures are still not fully comprehended, but they are most likely a consequence of the complex interplay between parasite, host and drug. In this chapter, the generally accepted underlying factors of treatment failure are discussed along with their consequences for therapy, drug design and other related challenges.

Chemotherapy is currently the only effective approach to treat all forms of leishmaniasis. However, its effectiveness is severely limited due to high toxicity, long treatment length, drug resistance, or inadequate mode of administration. As a consequence, there is a need to identify new molecular scaffolds and targets as potential therapeutics for the treatment of this disease. We report a small series of 1,2‐substituted‐1H‐benzo[d]imidazole derivatives (9ad) showing affinity in the submicromolar range (Ki = 0.150.69 μM) toward Leishmania mexicanaCPB2.8ΔCTE, one of the more promising targets for antileishmanial drug design. The compounds confirmed activity in vitro against intracellular amastigotes of Leishmania infantum with the best result being obtained with derivative 9d (IC50 = 6.8 μM), although with some degree of cytotoxicity (CC50 = 8.0 μM on PMM and CC50 = 32.0 μM on MCR‐5). In silico molecular docking studies and ADME‐Tox properties prediction were performed to validate the hypothesis of the interaction with the intended target and to assess the drug‐likeness of these derivatives.

In an effort to identify novel molecular warheads able to inhibit Leishmania mexicana cysteine protease CPB2.8CTE, fused benzo[b]thiophenes and ,'-triketones emerged as covalent inhibitors binding the active site cysteine residue. Enzymatic screening showed a moderate-to-excellent activity (12%-90% inhibition of the target enzyme at 20m). The most promising compounds were selected for further profiling including in vitro cell-based assays and docking studies. Computational data suggest that benzo[b]thiophenes act immediately as non-covalent inhibitors and then as irreversible covalent inhibitors, whereas a reversible covalent mechanism emerged for the 1,3,3'-triketones with a Y-topology. Based on the predicted physicochemical and ADME-Tox properties, compound 2b has been identified as a new drug-like, non-mutagen, non-carcinogen, and non-neurotoxic lead candidate.

The diverse pharmacological properties of the diaryltriazenes have sparked the interest to investigate their potential to be repurposed as antitubercular drug candidates. In an attempt to improve the antitubercular activity of a previously constructed diaryltriazene library, eight new halogenated nitroaromatic triazenides were synthesized and underwent biological evaluation. The potency of the series was confirmed against the Mycobacterium tuberculosis lab strain H37Ra, and for the most potent derivative, we observed a minimal inhibitory concentration of 0.85 μm. The potency of the triazenide derivatives against M. tuberculosis H37Ra was found to be highly dependent on the nature of the halogenated phenyl substituent and less dependent on cationic species used for the preparation of the salts. Although the inhibitory concentration against J774A.1 macrophages was observed at 3.08 μm, the cellular toxicity was not mediated by the generation of nitroxide intermediate as confirmed by electron paramagnetic resonance spectroscopy, whereas no in vitro mutagenicity could be observed for the new halogenated nitroaromatic triazenides when a trifluoromethyl substituent was present on both the aryl moieties.

Objectives Three new chemical series (bicyclic nitroimidazoles, aminopyrazoles and oxaboroles) were selected by Drugs for Neglected Diseases initiative as potential new drug leads for leishmaniasis. Pharmacodynamics studies included both in vitro and in vivo efficacy, cross-resistance profiling against the current antileishmanial reference drugs and evaluation of their cidal activity potential. Methods Efficacy against the reference laboratory strains of Leishmania infantum (MHOM/MA(BE)/67/ITMAP263) and L. donovani (MHOM/ET/67/L82) was evaluated in vitro on intracellular amastigotes and in vivo in the early curative hamster model. Cidal activity was assessed over a period of 15 days in an in vitro ‘time-to-kill’ assay. Cross-resistance was assessed in vitro on a panel of L. infantum strains with different degrees of resistance to either antimony, miltefosine or paromomycin. Results All lead compounds showed potent and selective in vitro activity against the Leishmania strains tested and no cross-resistance could be demonstrated against any of the current antileishmanial drugs. Cidal activity was obtained in vitro for all series within 15 days of exposure with some differences noted between L. donovani and L. infantum. When evaluated in vivo, all lead compounds showed high efficacy and no adverse effects were observed. Conclusions The new lead series were shown to have cidal pharmacodynamic activity. The absence of cross-resistance with any of the current antileishmanial drugs opens possibilities for combination treatment to reduce the likelihood of treatment failures and drug resistance., Graphical abstract Image 1, Highlights • Good efficacy was evaluated for all series in vitro and in vivo. • No cross-resistance towards current anti-leishmanial drugs was observed. • Cidal activity was obtained in vitro for all series within 15 days of exposure. • Some differences were observed between L. infantum and L. donovani.

The Calophyllum genus is well-known for its antimicrobial and cytotoxic activities, and therefore, we analyzed these biological activities for resins of Calophyllum antillanum and Calophyllum inophyllum growing in Cuba. C. antillanum resins sbowed a potent activity against Plasmodium falciparum (IC50 0.3 0.1 μg/mL), while its cytotoxicity against MRC-5 cells was much lower (IC50 2L6:t1.1 μglmL). In contrary, the resin of C. inophyllum sbowed an unspecific activity. The presence of apetalic acid, isoapetalic acid, calolongic acid, pinetoric acid pinetoric acid u, isocalolongic acid, pinetoric acid m, and isopinetoric acid m in c. antillanum resins was also confinned. These resuJts demonstrated for tbe first time tbe potential activity of C. antillanum resins against P.falciparum. Copyright© 2015 Jobo Wiley Sons, Ltd.

Objectives Widespread antimony resistance in the Indian subcontinent has enforced a therapy shift in visceral leishmaniasis treatment primarily towards miltefosine and secondarily also towards paromomycin. In vitro selection of miltefosine resistance in Leishmania donovani turned out to be quite challenging. Although no increase in IC50 was detected in the standard intracellular amastigote susceptibility assay, promastigote back-transformation remained positive at high miltefosine concentrations, suggesting a more resistant phenotype. This observation was explored in a large set of Nepalese clinical isolates from miltefosine cure and relapse patients to assess its predictive value for patient treatment outcome. Methods The predictive value of the promastigote back-transformation for treatment outcome of a set of Nepalese L. donovani field isolates (n = 17) derived from miltefosine cure and relapse patients was compared with the standard susceptibility assays on promastigotes and intracellular amastigotes. Results In-depth phenotypic analysis of the clinical isolates revealed no correlation between the different susceptibility assays, nor any clear link to the actual treatment outcome. In addition, the clinical isolates proved to be phenotypically heterogeneous, as reflected by the large variation in drug susceptibility among the established clones. Conclusions This in vitro laboratory study shows that miltefosine treatment outcome is not necessarily exclusively linked with the susceptibility profile of pre-treatment isolates, as determined in standard susceptibility assays. The true nature of miltefosine treatment failures still remains ill defined.

Since biofilms are important in many clinical, industrial, and environmental settings, reliable methods to quantify these sessile microbial populations are crucial. Most of the currently available techniques do not allow the enumeration of the viable cell fraction within the biofilm and are often time consuming. This paper proposes flow cytometry (FCM) using the single-stain viability dye TO-PRO®-3 iodide as a fast and precise alternative. Mature biofilms of Candida albicans and Escherichia coli were used to optimize biofilm removal and dissociation, as a single-cell suspension is needed for accurate FCM enumeration. To assess the feasibility of FCM quantification of biofilms, E. coli and C. albicans biofilms were analyzed using FCM and crystal violet staining at different time points. A combination of scraping and rinsing proved to be the most efficient technique for biofilm removal. Sonicating for 10 min eliminated the remaining aggregates, resulting in a single-cell suspension. Repeated FCM measurements of biofilm samples revealed a good intraday precision of approximately 5 %. FCM quantification and the crystal violet assay yielded similar biofilm growth curves for both microorganisms, confirming the applicability of our technique. These results show that FCM using TO-PRO®-3 iodide as a single-stain viability dye is a valid fast alternative for the quantification of viable cells in a biofilm.

Blocking the 2-C-methyl-D-erythrithol-4-phosphate (MEP) pathway for isoprenoid biosynthesis offers interesting prospects for inhibiting Plasmodium or Mycobacterium spp. growth. Fosmidomycin (1) and its homologue FR900098 (2) potently inhibit 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr), a key enzyme in this pathway. Here we introduced aryl or aralkyl substituents at the β-position of the hydroxamate analogue of 2. While direct addition of a β-aryl moiety resulted in poor inhibition, longer linkers between the carbon backbone and the phenyl ring were generally associated with better binding to the enzymes. X-ray structures of the parasite Dxr-inhibitor complexes show that the "longer" compounds generate a substantially different flap structure, in which a key tryptophan residue is displaced, and the aromatic group of the ligand lies between the tryptophan and the hydroxamate's methyl group. Although the most promising new Dxr inhibitors lack activity against Escherichia coli and Mycobacterium smegmatis, they proved to be highly potent inhibitors of Plasmodium falciparum in vitro growth. (Figure Presented).

Fosmidomycin inhibits IspC (Dxr, 1-deoxy-D-xylulose 5-phosphate reductoisomerase), a key enzyme in nonmevalonate isoprenoid biosynthesis that is essential in Plasmodium falciparum. The drug has been used successfully to treat malaria patients in clinical studies, thus validating IspC as an antimalarial target. However, improvement of the drugs pharmacodynamics and pharmacokinetics is desirable. Here, we show that the conversion of the phosphonate moiety into acyloxymethyl and alkoxycarbonyloxymethyl groups can increase the in vitro activity against asexual blood stages of P. falciparum by more than 1 order of magnitude. We also synthesized double prodrugs by additional esterification of the hydroxamate moiety. Prodrugs with modified hydroxamate moieties are subject to bioactivation in vitro. All prodrugs demonstrated improved antiplasmodial in vitro activity. Selected prodrugs and parent compounds were also tested for their cytotoxicity toward HeLa cells and in vivo in a Plasmodium berghei malaria model as well as in the SCID mouse P. falciparum model.

Summary Invasive aspergillosis (IA) has become increasingly common and is characterised by high morbidity and mortality. Upcoming resistance threatens treatment with azoles and highlights the continuous need for novel therapeutics. This laboratory study investigated the in vitro and in vivo potential of the alkylphospholipid oleylphosphocholine (OlPC) against Aspergillus. In vitro activities of OlPC, miltefosine, posaconazole and voriconazole were determined for Aspergillus fumigatus, A. niger, A. terreus and A. flavus. In vivo efficacy of OlPC was evaluated in a systemic A. fumigatus mouse model, adopting a short-term and long-term oral or intraperitoneal dosing regimen. OlPC showed good in vitro activity against A. fumigatus (IC50 = 1.04 μmol l−1). Intraperitoneal administration of 50 mg kg−1 day−1 OlPC significantly reduced the fungal organ burdens at 4 days post-infection (dpi). Although 5- and 10-day OlPC treatment improved survival, organ burdens were not affected at 10 and 15 dpi. While this study showed excellent in vitro activity of OlPC against Aspergillus spp., its therapeutic efficacy in an acute mouse model for IA was less convincing. Given the limited therapeutic options in the current antifungal market for invasive infections, OlPC activity should be assessed in a less stringent in vivo model, potentially in combination treatment with other already marketed antifungal drugs.

Leishmaniasis has been treated for decades with pentavalent antimony preparations until the emergence of antimony resistance has forced a switch in hyperendemic areas towards alternative therapeutics, such as miltefosine, amphotericin B and paromomycin. The use of miltefosine and amphotericin B has particularly been encouraged as first-line therapy for visceral leishmaniasis, however, all are increasingly confronted with treatment failures and/or the emergence of drug resistance. This chapter provides a concise overview of the mode-of-action of the current anti-leishmanial drugs and links this to the different resistance mechanisms that have been proposed over the past years. The pivotal importance of proactive drug-resistance research is highlighted with reference to the most commonly used laboratory methods.

Human African trypanosomiasis (HAT), also known as sleeping sickness is a parasitic disease transmitted by the bite of the 'Glossina' insect, commonly known as the tsetse fly. This disease affects mostly poor populations living in remote rural areas of Africa. Untreated, it is usually fatal. Currently, safe and effective treatments against this disease are lacking. Phenotypic screening of triazine non-nucleoside HIV-1 reverse transcriptase inhibitors (monomers) resulted in potent and selective antitrypanosomal compounds. This serendipitous discovery and the presence of dimers in many compounds active against these neglected tropical diseases prompted us to investigate antitrypanosomal activity of triazine dimers. Optimization of the triazine dimers resulted in 3,3'-(((ethane-1,2-diylbis(azanediyl))bis(4-(mesityloxy)-1,3,5-triazine-6,2-diyl))bis(azanediyl))dibenzonitrile (compound 38), a compound with very potent in vitro and moderate in vivo antitrypanosomal activity.

Graphical abstract, A 900 compound nitroimidazole-based library derived from our pretomanid backup program with TB Alliance was screened for utility against human African trypanosomiasis (HAT) by the Drugs for Neglected Diseases initiative. Potent hits included 2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]thiazine 8-oxides, which surprisingly displayed good metabolic stability and excellent cell permeability. Following comprehensive mouse pharmacokinetic assessments on four hits and determination of the most active chiral form, a thiazine oxide counterpart of pretomanid (24) was identified as the best lead. With once daily oral dosing, this compound delivered complete cures in an acute infection mouse model of HAT and increased survival times in a stage 2 model, implying the need for more prolonged CNS exposure. In preliminary SAR findings, antitrypanosomal activity was reduced by removal of the benzylic methylene but enhanced through a phenylpyridine-based side chain, providing important direction for future studies.

Respiratory syncytial virus (RSV) infections remain a major cause of respiratory disease and hospitalizations among infants. Infection recurs frequently and establishes a weak and short-lived immunity. To date, RSV immunoprophylaxis and vaccine research is mainly focused on the RSV fusion (F) protein, but a vaccine remains elusive. The RSV F protein is a highly conserved surface glycoprotein and is the main target of neutralizing antibodies induced by natural infection. Here, we analyzed an internalization process of antigen-antibody complexes after binding of RSV-specific antibodies to RSV antigens expressed on the surface of infected cells. The RSV F protein and attachment (G) protein were found to be internalized in both infected and transfected cells after the addition of either RSV-specific polyclonal antibodies (PAbs) or RSV glycoprotein-specific monoclonal antibodies (MAbs), as determined by indirect immunofluorescence staining and flow-cytometric analysis. Internalization experiments with different cell lines, well-differentiated primary bronchial epithelial cells (WD-PBECs), and RSV isolates suggest that antibody internalization can be considered a general feature of RSV. More specifically for RSV F, the mechanism of internalization was shown to be clathrin dependent. All RSV F-targeted MAbs tested, regardless of their epitopes, induced internalization of RSV F. No differences could be observed between the different MAbs, indicating that RSV F internalization was epitope independent. Since this process can be either antiviral, by affecting virus assembly and production, or beneficial for the virus, by limiting the efficacy of antibodies and effector mechanism, further research is required to determine the extent to which this occurs in vivo and how this might impact RSV replication. IMPORTANCE Current research into the development of new immunoprophylaxis and vaccines is mainly focused on the RSV F protein since, among others, RSV F-specific antibodies are able to protect infants from severe disease, if administered prophylactically. However, antibody responses established after natural RSV infections are poorly protective against reinfection, and high levels of antibodies do not always correlate with protection. Therefore, RSV might be capable of interfering, at least partially, with antibody-induced neutralization. In this study, a process through which surface-expressed RSV F proteins are internalized after interaction with RSV-specific antibodies is described. One the one hand, this antigen-antibody complex internalization could result in an antiviral effect, since it may interfere with virus particle formation and virus production. On the other hand, this mechanism may also reduce the efficacy of antibody-mediated effector mechanisms toward infected cells.

Background Since miltefosine monotherapy against visceral leishmaniasis (VL) caused by Leishmania donovani has been discontinued in the Indian subcontinent due to an increase in the number of treatment failures, single dose liposomal amphotericin B is now advocated as a treatment option of choice. Paromomycin-miltefosine combination therapy can be used as substitute first-line treatment in regions without cold-chain potential. Previous laboratory studies in the closely related species Leishmania infantum have demonstrated that paromomycin monotherapy fairly rapidly selects for resistance producing a phenotype with increased fitness. Given the possible clinical implications of these findings for the current field situation, the present study aimed to identify the potential hazards of paromomycin-miltefosine combination therapy. Principal findings Drug interaction studies using the fixed-ratio isobologram method revealed an indifferent interaction between paromomycin and miltefosine. In hamsters infected with L. infantum, the combination resulted in cumulative efficacy in reducing parasite burdens in the liver, spleen and bone marrow. Selected resistant lines against the single drugs did not display cross-resistance. When the intracellular amastigote stage was repeatedly exposed to the paromomycin-miltefosine combination, either in vitro or in vivo, no significant susceptibility decrease towards either drug was noted. Conclusions These results suggest that implementation of paromomycin-miltefosine combination therapy indeed could represent a safe and affordable treatment option for L. donovani VL as miltefosine appears to overrule the anticipated rapid development of PMM resistance., Author summary Liposomal amphotericin B is presently being used as first-line treatment option against visceral leishmaniasis in the Indian subcontinent. However, the need for temperature-controlled transport and storage limits its widespread use in rural areas. Previous studies already suggested that paromomycin-miltefosine combination therapy could be a valuable alternative, side passing some of the disadvantages associated with monotherapy, such as development of drug resistance. As the first reports of miltefosine resistant clinical isolates have already surfaced and paromomycin resistance could be easily induced under laboratory conditions, it remains essential to assess the risk of developing resistance against both drugs upon combination therapy. This study evaluated the efficacy of combined therapy against a Leishmania species closely related to the agent found in the Indian subcontinent, using both in vitro and in vivo models with the aim to select multidrug-resistant species by simultaneous exposure to paromomycin and miltefosine. The combination of both drugs in the hamster model resulted in a cumulative efficacy but did not lead to a significant susceptibility decrease, indicating that paromomycin-miltefosine combination therapy may represent a safe and affordable treatment option for visceral leishmaniasis.

Background and Aims: Kickxia ramosissima (Wall.) Janch (or Nanorrhinum ramosissimum (Wall.) Betsche is a well-known medicinal plant in Pakistan that is traditionally used in diabetic and inflammatory conditions. Because little information is available on its phytochemical composition, a range of constituents were isolated and evaluated in vitro in assays related to the traditional use. Methods: Dried whole plant material was extracted and chromatographically fractionated. Isolated constituents were evaluated in silico and in vitro in assays related to the traditional use against diabetes (inhibition of α-glucosidase activity; inhibition of Advanced Glycation Endproducts - AGEs) and in inflammatory conditions (inhibition of AAPH induced linoleic acid peroxidation, inhibition of 15-LOX, antimicrobial activity). Results: Phytochemical analysis of the extracts and fractions led to isolation of 7 compounds, including the iridoids kickxiasine (being a new compound), mussaenosidic acid, mussaenoside and linarioside; the flavonoids pectolinarigenin and pectolinarin; and 4-hydroxy-benzoic acid methyl ester. The iridoids showed weak antiglycation activity. The flavonoids, however, showed interesting results as pectolinarigenin was highly active compared to pectolinarin. In the α-glucosidase inhibition assay, only weak activity was observed for the iridoids. However, the flavonoid pectolinarigenin showed good activity, followed by pectolinarin. In the 15-LOX experiment, moderate inhibition was recorded for most compounds, the iridoids mussaenosidic acid and mussaenoside being the most active. In the AAPH assay, weak or no inhibition was recorded for all compounds. The in silico assays for the α-glucosidase and 15-LOX assays confirmed the results of respective in vitro assays. Pectolinarigenin showed moderate antimicrobial activity against Staphylococcus aureus, Plasmodium falciparum K1 and Trypanosoma cruzi, but it was not cytotoxic on a human MRC-5 cell line. Conclusions: Based on our findings the tradtional use of K. ramosissima could at least in part be confirmed.

Cyclopeptide alkaloids are polyamidic, macrocyclic compounds, containing a 13-, 14-, or 15-membered ring. The ring system consists of a hydroxystyrylamine moiety, an amino acid, and a β-hydroxy amino acid; attached to the ring is a side chain, comprised of one or two more amino acid moieties. In vitro antiplasmodial activity was shown before for several compounds belonging to this class, and in this paper the antiplasmodial and cytotoxic activities of ten more cyclopeptide alkaloids are reported. Combining these results and the IC50 values that were reported by our group previously, a library consisting of 19 cyclopeptide alkaloids was created. A qualitative SAR (structure-activity relationship) study indicated that a 13-membered macrocyclic ring is preferable over a 14-membered one. Furthermore, the presence of a β-hydroxy proline moiety could correlate with higher antiplasmodial activity, and methoxylation (or, to a lesser extent, hydroxylation) of the styrylamine moiety could be important for displaying antiplasmodial activity. In addition, QSAR (quantitative structure-activity relationship) models were developed, using PLS (partial least squares regression) and MLR (multiple linear regression). On the one hand, these models allow for the indication of the most important descriptors (molecular properties) responsible for the antiplasmodial activity. Additionally, predictions made for interesting structures did not contradict the expectations raised in the qualitative SAR study.

With the aim of increasing the structural diversity on the early antimalarial drug plasmodione, an efficient and versatile procedure to prepare a series of biaryl- and N-arylalkylamines as plasmodione analogues is described. Using the naturally occurring and commercially available menadione as starting material, a 2-step sequence using a Kochi-Anderson reaction and subsequent Pd-catalyzed Suzuki-Miyaura coupling was developed to prepare three representative biphenyl derivatives in good yields for antimalarial evaluation. In addition, synthetic methodologies to afford 3-benzylmenadione derivatives bearing a terminal -N(Me)2 or -N(Et)2 in different positions (ortho, meta and para) on the aryl ring of the benzylic chain of plasmodione were investigated through reductive amination was used as the optimal route to prepare these protonable N-arylalkylamine privileged scaffolds. The antimalarial activities were evaluated and discussed in light of their physicochemical properties. Among the newly synthesized compounds, the para-position of the substituent remains the most favourable position on the benzyl chain and the carbamate -NHBoc was found active both in vitro (42 nM versus 29 nM for plasmodione) and in vivo in Plasmodium berghei-infected mice. The measured acido-basic features of these new molecules support the cytosol-food vacuole shuttling properties of non-protonable plasmodione derivatives essential for redox-cycling. These findings may be useful in antimalarial drug optimization.

Caesalpinia benthamiana is widely used as antimalarial in Guinean traditional medicine. Leaf extracts of the plant were tested for their in vitro antiprotozoal activity against Trypanosoma brucei brucei and T. cruzi and the chloroquine-sensitive Ghana strain of Plasmodium falciparum along with their cytotoxicity on MRC-5 cells. The methanolic extract showed the strongest antiprotozoal activity against P. falciparum (IC50 4 μg/ml), a good activity against T. brucei (IC50 13 μg/ml), and a moderate activity against T. cruzi (IC50 31 μg/ml) along with an IC50 on human MRC-5 cells of 32 μg/ml. Bioassay-guided fractionation from the methanolic extract led to antiplasmodially active subfractions. A prospective, placebo-controlled ethnotherapeutic trial assessed the antimalarial effectiveness and tolerability of C. benthamiana syrup administered orally to children with uncomplicated malaria as compared with chloroquine syrup. Phytochemical screening of the leaf extracts indicated the presence of flavonoids, terpenoids, tannins, saponins, and iridoids.

The emergence of antibiotic-resistant Helicobacter pylori strains impacts the efficacy of eradication therapy and promotes the development of alternative treatment strategies. Apocynin inhibits neutrophil NADPH oxidase and hence may decrease reactive oxygen species-mediated tissue damage in H. pylori-infected stomach tissue. Apocynin was tested in vitro for its cytotoxic and direct antibacterial effects. The therapeutic efficacy of orally administered apocynin (100 mg/kg/day through drinking water or 200 mg/kg/day through combined administration of drinking water and slow-release formulation) was assessed at 9 weeks after infection in the Mongolian gerbil model. Bacterial burdens were quantified by viable plate count and quantitative PCR. Histopathological evaluation of antrum and pylorus provided insight into mucosal inflammation and injury. Apocynin showed no cytotoxic or direct antibacterial effects in vitro or in vivo. Nine weeks of apocynin treatment at 200 mg/kg/day reduced active H. pylori gastritis as neutrophil infiltration in the mucous neck region and pit abscess formation decreased significantly. In our gerbil model, prolonged high-dose apocynin treatment significantly improved H. pylori-induced pit abscess formation without indications of drug toxicity and thus further investigation of the dosage regimen and formulation and the long-term impact on neoplastic development should be carried out.

Gold compounds form a new class of promising metal-based drugs with a number of potential therapeutic applications, particularly in the fields of anticancer and antimicrobial treatments. Previous research revealed that a group of structurally diverse gold compounds cause conspicuous inhibition of the protease activities of the human proteasome. Given the pharmacological importance of protease inhibition, the present study further explored whether these gold compounds might inhibit a few other proteases that are accepted druggable targets for disease treatment. In particular, four distinct cysteine proteases were considered here: cathepsin B and L that play a primary role in tumor-cell invasion and metastasis; rhodesain, the major cathepsin L-like cysteine protease of Trypanosoma brucei rhodesiense and CPB2.8 Delta CTE, a Leishmania mexicana mature cysteine protease. Based on the encouraging results obtained for some of the tested gold compounds on the two parasitic cysteine proteases, especially against CPB2.8 Delta CTE, with IC50s in the micromolar range, we next evaluated whether those gold compounds might contrast effectively the growth of the respective protozoa and indeed important antiprotozoal properties were disclosed; on the other hand a certain lack of selectivity was highlighted. Also, no direct or clear correlation could be established between the in vitro antiprotozoal properties and the level of protease inhibition. The implications of these results are discussed in relation to possible pharmaceutical applications.

UNLABELLED The viable plate count (VPC) is considered as the reference method for bacterial enumeration in periodontal microbiology but shows some important limitations for anaerobic bacteria. As anaerobes such as Porphyromonas gingivalis are difficult to culture, VPC becomes time-consuming and less sensitive. Hence, efficient normalization of experimental data to bacterial cell count requires alternative rapid and reliable quantification methods. This study compared the performance of VPC with that of turbidity measurement and real-time PCR (qPCR) in an experimental context using highly concentrated bacterial suspensions. Our TaqMan-based qPCR assay for P. gingivalis 16S rRNA proved to be sensitive and specific. Turbidity measurements offer a fast method to assess P. gingivalis growth, but suffer from high variability and a limited dynamic range. VPC was very time-consuming and less repeatable than qPCR. Our study concludes that qPCR provides the most rapid and precise approach for P. gingivalis quantification. Although our data were gathered in a specific research context, we believe that our conclusions on the inferior performance of VPC and turbidity measurements in comparison to qPCR can be extended to other research and clinical settings and even to other difficult-to-culture micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY Various clinical and research settings require fast and reliable quantification of bacterial suspensions. The viable plate count method (VPC) is generally seen as 'the gold standard' for bacterial enumeration. However, VPC-based quantification of anaerobes such as Porphyromonas gingivalis is time-consuming due to their stringent growth requirements and shows poor repeatability. Comparison of VPC, turbidity measurement and TaqMan-based qPCR demonstrated that qPCR possesses important advantages regarding speed, accuracy and repeatability.

The presence of a structural recognition motif for the nucleoside P2 transporter in a library of pyrimidine and triazine non-nucleoside HIV-1 reverse transcriptase inhibitors, prompted for the evaluation of antitrypanosomal activity. It was demonstrated that the structureactivity relationship for anti-HIV and antitrypanosomal activity was different. Optimization in the diaryl triazine series led to 6-(mesityloxy)-N2-phenyl-1,3,5-triazine-2,4-diamine (69), a compound with potent in vitro and moderate in vivo antitrypanosomal activity.

Objective: To investigate the leishmanicidal effect of the Bixa orellana crude seed extract and its fractions against Leishmania amazonensis. Methods: Four main fractions (BO-A, BO-B, BO-C and BO-D) were obtained by exhaustion with solvent with increased polarity from the Bixa orellana crude seed extract and 28 sub-fractions. The antileishmanial activity was evaluated in intracellular amastigotes and the cytotoxicity was assessed in murine intraperitoneal macrophages. Results: The BO-A and BO-B fractions showed a good antileishmanial activity with IC50 values of (12.9±4.1) and (12.4±0.3) μg/mL, respectively. The sub-fractions BO-B1 (IC50=(11.8±3.8) μg/mL) and BO-B3 [IC50=(13.6±4.7) μg/mL] also proved to have a good leishmanicidal effect. In general, the sub-fractions showed a lower toxicity than the crude extract. A selectivity index of 9 indicated a moderate selectivity of the BO-A, BO-B and BO-C fractions and BO-B1 sub-fraction. Conclusions: Potential of this plant against cutaneous leishmaniasis should be further investigated.