Infection with Fasciola hepatica (liver flukes) causes fascioliasis in humans and fasciolosis in ruminants. The current strategy for controlling fascioliasis/fasciolosis is predominantly mediated by chemotherapy with triclabendazole (TCBZ). As this flukicide targets newly excysted juveniles (NEJs), immature flukes and mature liver flukes, it’s use as the frontline chemotherapy for over four decades has led to widespread drug resistance. New chemotherapeutics are, therefore, urgently required. Here, continuing our studies of flukicidal phytochemicals derived from Hedera helix (common ivy), we investigated the anthelmintic properties of three ivy fruit saponins, including α-hederin ( IVL-11 ), against F. hepatica . During ex vivo culture, IVL-11 was as effective as TCBZ in killing NEJs and immature flukes. However, this saponin acted more quickly than TCBZ when tested against mature flukes. Microscopic examination of IVL-11 treated liver flukes revealed surface integrity breaches, actin disorganisation and cell membrane permeabilisation. Upon in vivo studies, using a novel method to isolate IVL-11 in large quantities from H. helix leaf, oral delivery of IVL-11 (up to 250 mg/kg bodyweight) to Ovis aries (sheep) was found to be safe, well-tolerated and detectable in the liver, peripheral blood, hepatic portal blood and bile. IVL-11 (250 mg/kg bodyweight) treatment of O. aries infected with F. hepatica led to a significant reduction in fluke body sizes as well as a delay and decrease in fecundity during in vivo efficacy studies. Together, our results confirm that IVL-11 exhibits flukicidal characteristics suitable for progression as a renewably obtained, natural product for treating fasciolosis.

Infection with Fasciola hepatica (liver fluke) causes fasciolosis (or fascioliasis) and poses a considerable economic as well as welfare burden to both the agricultural and animal health sectors. Here, we explore the ex vivo anthelmintic potential of synthetic derivatives of hederagenin, isolated in bulk from Hedera helix. Thirty-six compounds were initially screened against F. hepatica newly excysted juveniles (NEJs) of the Italian strain. Eleven of these compounds were active against NEJs and were selected for further study, using adult F. hepatica derived from a local abattoir (provenance unknown). From these eleven compounds, six demonstrated activity and were further assessed against immature liver flukes of the Italian strain. Subsequently, the most active compounds (n = 5) were further evaluated in ex vivo dose response experiments against adult Italian strain liver flukes. Overall, MC042 was identified as the most active molecule and the EC50 obtained from immature and adult liver fluke assays (at 24 h post co-culture) are estimated as 1.07 & mu;M and 13.02 & mu;M, respectively. When compared to the in vitro cytotoxicity of MDBK bovine cell line, MC042 demonstrated the highest anthelmintic selectivity (44.37 for immature and 3.64 for adult flukes). These data indicate that modified hederagenins display properties suitable for further investigations as candidate flukicides.

Infecting the stomach of almost 50 % of people, Helicobacter pylori is a causative agent of gastritis, peptic ulcers and stomach cancers. Interactions between bacterial membrane-bound lectin, Blood group Antigen Binding Adhesin (BabA), and human blood group antigens are key in the initiation of infection. Herein, the synthesis of a B-antigen hexasaccharide (B6) and a B-Lewis b heptasaccharide (BLeb7) and Bovine Serum Albumin glycoconjugates thereof is reported to assess the binding properties and preferences of BabA from different strains. From a previously reported trisaccharide acceptor a versatile key Lacto-N-tetraose tetrasaccharide intermediate was synthesized, which allowed us to explore various routes to the final targets, either via initial introduction of fucosyl residues followed by introduction of the B-determinant or vice versa. The first approach proved unsuccessful, whereas the second afforded the target structures in good yields. Protein conjugation using isothiocyanate methodology allowed us to reach high glycan loadings (up to 23 per protein) to mimic multivalent displays encountered in Nature. Protein glycoconjugate inhibition binding studies were performed with H. pylori strains displaying high or low affinity for Lewis b hexasaccharide structures showing that the binding to the high affinity strain was reduced due to the presence of the B-determinant in the Bleb7-conjugates and further reduced by the absence of the Lewis fucose residue in the B6-conjugate.

Control of liver fluke infections remains a significant challenge in the livestock sector due to widespread distribution of drug resistant parasite populations. In particular, increasing prevalence and economic losses due to infection with Fasciola hepatica is a direct result of drug resistance to the gold standard flukicide, triclabendazole. Sustainable control of this significant zoonotic pathogen, therefore, urgently requires the identification of new anthelmintics. Plants represent a source of molecules with potential flukicidal effects and, amongst their secondary metabolites, the diterpenoid abietic acids can be isolated in large quantities. In this study, nineteen (19) chemically modified abietic acid analogues (MC_X) were first evaluated for their anthelmintic activities against F. hepatica newly excysted juveniles (NEJs, from the laboratory-derived Italian strain); from this, 6 analogues were secondly evaluated for their anthelmintic activities against adult wild strain flukes. One analogue, MC010, was progressed further against 8-week immature- and 12-week mature Italian strain flukes. Here, MC010 demonstrated moderate activity against both of these intra-mammalian fluke stages (with an adult fluke EC50 = 12.97 mu M at 72 h post culture). Overt mammalian cell toxicity of MC010 was inferred from the Madin-Darby bovine kidney (MDBK) cell line (CC50 = 17.52 mu M at 24 h post culture) and demonstrated that medicinal chemistry improvements are necessary before abietic acid analogues could be considered as potential anthelmintics against liver fluke pathogens

Leishmaniasis is a neglected tropical disease caused by insect-vector borne protozoan parasites of the,Leishmania species. Whilst infection threatens and affects millions of the global poor, vaccines are absent and drug therapy limited. Extensive efforts have recently been made to discover new leads from small molecule synthetic compound libraries held by industry; however, the number of new chemical entities identified and entering development as anti-leishmanials has been very low. This has led to increased interest in the possibility of discovering naturally derived compounds with potent antileishmanial activity which may be developed towards clinical applications. Plant-derived triterpenoid and steroidal saponins have long been considered as anti-microbials and here we describe an investigation of a library of 137 natural (9) and semi-synthetic saponins (128) for activity againstLeishmania mexicana, a causative agent of cutaneous leishmaniasis. The triterpenoid sapogenin, hederagenin, readily obtained in large quantities fromHedera helix(common ivy), was converted into a range of 128 derivatives. These semi-synthetic compounds, as well as saponins isolated from ivy, were examined with a phenotypic screening approach to identify potent and selective anti-leishmanial hits. This led to the identification of 12 compounds, including the natural saponin gypsogenin, demonstrating high potency (ED50< 10.5 mu M) against axenicL. mexicanaamastigotes, the mammalian pathogenic form. One of these, hederagenin disuccinate, was sufficiently non-toxic to the macrophage host cell to facilitate further analyses, selectivity index (SI) > 10. Whilst this was not active in an infected cell model, the anti-leishmanial properties of hederagenin-derivatives have been demonstrated, and the possibility of improving the selectivity of natural hederagenin through chemical modification has been established.

Steroidal and triterpenoid saponins are attractive for their wide-ranging pharmacological properties. The triterpenoid saponins Anemoclemoside A and B are root constituents of the Chinese folk medicinal plant Anemoclema glaucifolium (Ranunculaceae). Both compounds feature an unusual cyclic acetal linkage to the carbohydrate l-arabinose in its open chain form rather than the typical glycosidic bond present in normal saponins. The straightforward and scalable syntheses of both saponins starting from l-arabinose as well as l-lyxose and l-rhamnose are described.

While(13)C-labelled proteins are common tools in NMR studies, lack of access to(13)C-labelled carbohydrate structures has restricted their use.l-Fucose is involved in a wide range of physiological and pathophysiological processes in mammalian organisms. Here,l-[U-C-13(6)]-Fuc labelled type I Lewis b (Le(b)) structures have been synthesised for use in NMR binding studies with the Blood-group Antigen Binding Adhesin (BabA), a membrane-bound protein from the bacteriumHelicobacter pylori. As part of this work, an efficient synthesis of a benzylatedl-[U-C-13(6)]-Fuc thioglycoside donor froml-[U-C-13(6)]-Gal has been developed. The design and synthesis of an orthogonally protected tetrasaccharide precursor enabled controlled introduction of one or two(13)C-labelled or non-labelled fucosyl residues prior to global deprotection. NMR analysis showed that it is straightforward to assign the anomeric centres as well as the H-5 positions to the individual fucosyl residues which are relevant for NMR binding studies.

Lectins with a beta-propeller fold bind glycans on the cell surface through multivalent binding sites and appropriate directionality. These proteins are formed by repeats of short domains, raising questions about evolutionary duplication. However, these repeats are difficult to detect in translated genomes and seldom correctly annotated in sequence databases. To address these issues, we defined the blade signature of the five types of beta-propellers using 3D-structural data. With these templates, we predicted 3,887 beta-propeller lectins in 1,889 species and organized this information in a searchable online database. The data reveal a widespread distribution of beta-propeller lectins across species. Prediction also emphasizes multiple architectures and led to the discovery of a beta-propeller assembly scenario. This was confirmed by producing and characterizing a predicted protein coded in the genome of Kordia zhangzhouensis. The crystal structure uncovers an intermediate in the evolution of beta-propeller assembly and demonstrates the power of our tools.

The antiprotozoal effect of saponins is transitory, as when saponins are deglycosylated to the sapogenin by rumen microorganisms they become inactive. We postulated that the substitution of the sugar moiety of the saponin with small polar residues would produce sapogen-like analogs which might be resistant to degradation in the rumen as they would not be enzymatically cleaved, allowing the antiprotozoal effect to persist over time. In this study, we used an acute assay based on the ability of protozoa to break down [14C] leucine-labeled Streptococcus bovis and a longer term assay based on protozoal motility over 24 h to evaluate both the antiprotozoal effect and the stability of this effect with fifteen hederagenin bis-esters esterified with two identical groups, and five cholesterol and cholic acid based derivatives carrying one to three succinate residues. The acute antiprotozoal effect of hederagenin derivatives was more pronounced than that of cholesterol and cholic acid derivatives. Modifications in the structure of hederagenin, cholesterol, and cholic acid derivatives resulted in compounds with different biological activities in terms of acute effect and stability, although those which were highly toxic to protozoa were not always the most stable over time. Most of the hederagenin bis-esters, and in particular hederagenin bis-succinate (TSB24), hederagenin bis-betainate dichloride (TSB37) and hederagenin bis-adipate (TSB47) had a persistent effect against rumen protozoa in vitro, shifting the fermentation pattern toward higher propionate and lower butyrate. These chemically modified triterpenes could potentially be used in ruminant diets as an effective defaunation agent to, ultimately, increase nitrogen utilization, decrease methane emissions, and enhance animal production. Further trials in vivo or in long term rumen simulators are now needed to confirm the in vitro observations presented.

The antiprotozoal effect of saponins is transitory, as when saponins are deglycosylated to sapogenins by rumen microorganisms they become inactive. We hypothesised that the combination of saponins with glycosidase-inhibiting iminosugars might potentially increase the effectiveness of saponins over time by preventing their deglycosylation in the rumen. Alternatively, modifying the structure of the saponins by substituting the sugar moiety with other small polar residues might maintain their activity as the sugar substitute would not be enzymatically cleaved. The aim of this in vitro study was to evaluate the acute antiprotozoal effect and the stability of this effect over a 24 h incubation period using ivy saponins, a stevia extract rich in iminosugars, ivy saponins with stevia extract, and a chemically modified ivy saponin, hederagenin bis-succinate (HBS). The effects on fermentation parameters and rumen bacterial communities were also studied. Ivy saponins with stevia and HBS had a greater antiprotozoal effect than ivy saponins, and this effect was maintained after 24 h of incubation (P<0.001). The combination of ivy and stevia extracts was more effective in shifting the fermentation pattern towards higher propionate (+39%) and lower butyrate (-32%) and lower ammonia concentration (-64%) than the extracts incubated separately. HBS caused a decrease in butyrate (-45%) and an increase in propionate (+43%) molar proportions. However, the decrease in ammonia concentration (-42%) observed in the presence of HBS was less than that caused by ivy saponins, either alone or with stevia. Whereas HBS and stevia impacted on bacterial population in terms of community structure, only HBS had an effect in terms of biodiversity (P<0.05). It was concluded that ivy saponins with stevia and the modified saponin HBS had a strong antiprotozoal effect, although they differed in their effects on fermentation parameters and bacteria communities. Ivy saponins combined with an iminosugar-rich stevia extract and/or HBS should be evaluated to determine their antiprotozoal effect in vivo.