The Diterpenoid 7-Keto-Sempervirol,Derived from Lycium chinense,Displays Anthelmintic Activity against both Schistosoma mansoni and Fasciola hepatica

BackgroundTwo platyhelminths of biomedical and commercial significance are Schistosoma mansoni (blood fluke) and Fasciola hepatica (liver fluke). These related trematodes are responsible for the chronic neglected tropical diseases schistosomiasis and fascioliasis, respectively. As no vaccine is currently available for anti-flukicidal immunoprophylaxis, current treatment is mediated by mono-chemical chemotherapy in the form of mass drug administration (MDA) (praziquantel for schistosomiasis) or drenching (triclabendazole for fascioliasis) programmes. This overreliance on single chemotherapeutic classes has dramatically limited the number of novel chemical entities entering anthelmintic drug discovery pipelines, raising significant concerns for the future of sustainable blood and liver fluke control.

Methodology/ Principle Findings

Here we demonstrate that 7-keto-sempervirol, a diterpenoid isolated from Lycium chinense, has dual anthelmintic activity against related S. mansoni and F. hepatica trematodes. Using a microtiter plate-based helminth fluorescent bioassay (HFB), this activity is specific (Therapeutic index = 4.2, when compared to HepG2 cell lines) and moderately potent (LD₅₀ = 19.1 μM) against S. mansoni schistosomula cultured in vitro. This anti-schistosomula effect translates into activity against both adult male and female schistosomes cultured in vitro where 7-keto-sempervirol negatively affects motility/behaviour, surface architecture (inducing tegumental holes, tubercle swelling and spine loss/shortening), oviposition rates and egg morphology. As assessed by the HFB and microscopic phenotypic scoring matrices, 7-keto-sempervirol also effectively kills in vitro cultured F. hepatica newly excysted juveniles (NEJs, LD₅₀ = 17.7 μM). Scanning electron microscopy (SEM) evaluation of adult F. hepatica liver flukes co-cultured in vitro with 7-keto-sempervirol additionally demonstrates phenotypic abnormalities including breaches in tegumental integrity and spine loss.

Conclusions/ Significance

7-keto-sempervirol negatively affects the viability and phenotype of two related pathogenic trematodes responsible for significant human and animal infectious diseases. This plant-derived, natural product is also active against both larval and adult developmental forms. As such, the data collectively indicate that 7-keto-sempervirol is an important starting point for anthelmintic drug development. Medicinal chemistry optimisation of more potent 7-keto-sempervirol analogues could lead to the identification of novel chemical entities useful for future combinatorial or replacement anthelmintic control.     

Ion Channel Blockers as Antimicrobial Agents,Efflux Inhibitors,and Enhancers of Macrophage Killing Activity against Drug Resistant Mycobacterium tuberculosis

Given the ability of M. tuberculosis to survive as an intracellular pathogen and its propensity to develop resistance to the existing antituberculosis drugs, its treatment requires new approaches. Here the antimycobacterial properties of verapamil, thioridazine, chlorpromazine, flupenthixol and haloperidol were investigated against a panel of drug resistant M. tuberculosis strains, both in vitro and on human-infected macrophages. These compounds are efflux inhibitors that share among them the characteristic of being ion channel blockers. In vitro, all compounds exhibited synergistic inhibitory activities when combined with isoniazid and rifampicin, and were able to inhibit active efflux, demonstrating their role as efflux inhibitors. Gene expression analysis showed that M. tuberculosis efflux genes were overexpressed in response to antibiotic exposure, in vitro and within macrophages, irrespective of their resistance pattern. These compounds displayed a rapid and high killing activity against M. tuberculosis, associated with a decrease in intracellular ATP levels demonstrating that the bactericidal action of the ion channel blockers against M. tuberculosis clinical strains is associated with their interference with energy metabolism. The compounds led to a decrease in the intracellular mycobacterial load by increasing phagosome acidification and activating lysosomal hydrolases. The results presented in this study enable us to propose the following mechanism of action for these compounds: a) in the bacteria, the compounds generate a cascade of events involving the inhibition of the respiratory chain complexes and energy production for efflux activity. Indirectly, this reduce the resistance level to antituberculosis drugs potentiating their activity; b) on the host cell, the treatment with the ion channel blockers increases phagosome acidification and induces the expression of phagosomal hydrolases, leading to bacterial growth restriction irrespective of their resistance pattern. This work highlights the potential value ion channel blockers as adjuvants of tuberculosis chemotherapy, in particular for the development of new therapeutic strategies, with strong potential for treatment shortening against drug susceptible and resistant forms of tuberculosis. Medicinal chemistry studies are now needed to improve the properties of these compounds, increasing their M. tuberculosis efflux-inhibition and killing-enhancement activity and reduce their toxicity for humans, therefore optimizing their potential for clinical usage.     

Using C. elegans Forward and Reverse Genetics to Identify New Compounds with Anthelmintic Activity

BackgroundThe lack of new anthelmintic agents is of growing concern because it affects human health and our food supply, as both livestock and plants are affected. Two principal factors contribute to this problem. First, nematode resistance to anthelmintic drugs is increasing worldwide and second, many effective nematicides pose environmental hazards. In this paper we address this problem by deploying a high throughput screening platform for anthelmintic drug discovery using the nematode Caenorhabditis elegans as a surrogate for infectious nematodes. This method offers the possibility of identifying new anthelmintics in a cost-effective and timely manner.Conclusions/SignificanceThe challenge of anthelmintic drug discovery is exacerbated by several factors; including, 1) the biochemical similarity between host and parasite genomes, 2) the geographic location of parasitic nematodes and 3) the rapid development of resistance. Accordingly, an approach that can screen large compound collections rapidly is required. C. elegans as a surrogate parasite offers the ability to screen compounds rapidly and, equally importantly, with specificity, thus reducing the potential toxicity of these compounds to the host and the environment. We believe this approach will help to replenish the pipeline of potential nematicides.     

In vitro antioxidant,antimicrobial and antiproliferative studies of four different extracts of Orthosiphon stamineus,Gynura procumbens and Ficus deltoidea

BackgroundMedicinal plants are important source of drugs with pharmacological activities. Therefore, there is always rising demands to discover more therapeutic agents from various species. Orthosiphon stamineus, Gynura procumbens and Ficus deltoidea are high valued medicinal plants of Malaysia contain rich source of phenolic and flavonoid compounds. The aims of the present study were to evaluate anti-oxidant, antimicrobial and anti-proliferative effects on A549, HePG2 and MCF7 cell lines of four different extracts of Orthosiphon stamineus, Gynura procumbens and Ficus deltoidea.MethodologyThe leaves of all selected plants were extracted with methanol, chloroform, ethyl acetate and butanol separately with simple cold maceration. Antioxidant activity of all crude extracts were quantitatively measured against DPPH and Ferric Reducing Assay. Antimicrobial evaluation was done by Microdilution and MTT assay and antipoliferative activity of all extracts of selected plant were evaluated against A549, HePG2 and MCF7 cell lines.ResultsResults showed that methanol extract exhibited highest percentage free radical scavenging activity of almost all extracts of selected plants. Antimicrobials results showed chloroform and methanol extracts of O. stamineus extract were the two most active extracts against resistant MRSA but not S. aureus. Only methanol extract of G. procumbens showed antimicrobial activity against the tested pathogens. Chloroform and methanol extracts of F. deltoidea elicited antimicrobial activity against S. aureus but not MRSA. Antiproliferative activity against three tested cell lines results showed that ethyl acetate extract of O. stamineus showed good effect whereas methanol extract of F. deltoidea and G. procumbens exhibited good antiproliferative activity.ConclusionsThe results of the present investigation demonstrated significant variations in the antioxidant, antimicrobial and antiproliferative effects of different solvent extracts. These data could be helpful in isolation of pure potent compounds with good biological activities from the extracts of plants.     

Repurposing Salicylanilide Anthelmintic Drugs to Combat Drug Resistant Staphylococcus aureus

Staphylococcus aureus is a Gram-positive bacterium that has become the leading cause of hospital acquired infections in the US. Repurposing Food and Drug Administration (FDA) approved drugs for antimicrobial therapy involves lower risks and costs compared to de novo development of novel antimicrobial agents. In this study, we examined the antimicrobial properties of two commercially available anthelmintic drugs. The FDA approved drug niclosamide and the veterinary drug oxyclozanide displayed strong in vivo and in vitro activity against methicillin resistant S. aureus (minimum inhibitory concentration (MIC): 0.125 and 0.5 μg/ml respectively; minimum effective concentration: ≤ 0.78 μg/ml for both drugs). The two drugs were also effective against another Gram-positive bacteria Enterococcus faecium (MIC 0.25 and 2 μg/ml respectively), but not against the Gram-negative species Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter aerogenes. The in vitro antimicrobial activity of niclosamide and oxyclozanide were determined against methicillin, vancomycin, linezolid or daptomycin resistant S. aureus clinical isolates, with MICs at 0.0625-0.5 and 0.125-2 μg/ml for niclosamide and oxyclozanide respectively. A time-kill study demonstrated that niclosamide is bacteriostatic, whereas oxyclozanide is bactericidal. Interestingly, oxyclozanide permeabilized the bacterial membrane but neither of the anthelmintic drugs exhibited demonstrable toxicity to sheep erythrocytes. Oxyclozanide was non-toxic to HepG2 human liver carcinoma cells within the range of its in vitro MICs but niclosamide displayed toxicity even at low concentrations. These data show that the salicylanilide anthelmintic drugs niclosamide and oxyclozanide are suitable candidates for mechanism of action studies and further clinical evaluation for treatment of staphylococcal infections.     

Interactions of anthelmintic drugs in Caenorhabditis elegans neuro-muscular ion channel mutants

Due to the increasing development of anthelmintic resistance in nematodes worldwide, it is important to search for anthelmintic compounds with new modes of action and also to investigate the possibility to combine compounds with possible synergistic effects. There might also be the chance to take advantage of the fact that nematode populations which have developed resistance against one anthelmintic class might respond hypersusceptibly to another drug class. The aim of this study was to investigate responses of Caenorhabditis elegans populations with mutations in neuro-muscular ion channels to different anthelmintic classes. Furthermore, potential synergistic effects between two anthelmintic compounds from different classes, i.e. emodepside and tribendimidine, were studied. Although there was neither a synergistic nor an antagonistic effect between emodepside and tribendimidine, other types of interactions could be identified. The C. elegans GABA_A-receptor (GABA_A-R) unc-49 mutants, showing decreased emodepside susceptibility, were more susceptible to tribendimidine than wild-type C. elegans. In contrast, the reverse phenomenon – hypersusceptibility to emodepside in tribendimidine resistant acetylcholine-receptor (AChR) loss of function mutants – was not observed. Moreover, the slo-1 mutant strain (completely emodepside resistant) also showed hypersusceptibility to piperazine. Interestingly, neither the GABA_A-R unc-49 mutants nor the AChR mutants showed decreased susceptibility against piperazine, although there were some studies that indicated an involvement of GABA_A-R or AChR in the piperazine mode of action. In conclusion, the present study provides evidence suggesting that interactions between commercially available anthelmintic drugs with different modes of action might be a relatively common phenomenon but this has to be carefully worked out for each anthelmintic and each anthelmintic drug combination. Moreover, results obtained in C. elegans will have to be confirmed using parasitic nematodes in the future.     

Egg excretion indicators for the measurement of soil-transmitted helminth response to treatment

BackgroundPeriodic administration of anthelmintic drugs is a cost-effective intervention for morbidity control of soil-transmitted helminth (STH) infections. However, with programs expanding, drug pressure potentially selecting for drug-resistant parasites increases. While monitoring anthelmintic drug efficacy is crucial to inform country control program strategies, different factors must be taken into consideration that influence drug efficacy and make it difficult to standardize treatment outcome measures. We aimed to identify suitable approaches to assess and compare the efficacy of different anthelmintic treatments.MethodologyWe built an individual participant-level database from 11 randomized controlled trials and two observational studies in which subjects received single-agent or combination therapy, or placebo. Eggs per gram of stool were calculated from egg counts at baseline and post-treatment. Egg reduction rates (ERR; based on mean group egg counts) and individual-patient ERR (iERR) were utilized to express drug efficacy and analyzed after log-transformation with a linear mixed effect model. The analyses were separated by follow-up duration (14–21 and 22–45 days) after drug administration.Principal findingsThe 13 studies enrolled 5,759 STH stool-positive individuals; 5,688 received active medication or placebo contributing a total of 11,103 STH infections (65% had two or three concurrent infections), of whom 3,904 (8,503 infections) and 1,784 (2,550 infections) had efficacy assessed at 14–21 days and 22–45 days post-treatment, respectively. Neither the number of helminth co-infections nor duration of follow-up affected ERR for any helminth species. The number of participants treated with single-dose albendazole was 689 (18%), with single-dose mebendazole 658 (17%), and with albendazole-based co-administrations 775 (23%). The overall mean ERR assessed by day 14–21 for albendazole and mebendazole was 94.5% and 87.4%, respectively on Ascaris lumbricoides, 86.8% and 40.8% on hookworm, and 44.9% and 23.8% on Trichuris trichiura. The World Health Organization (WHO) recommended criteria for efficacy were met in 50%, 62%, and 33% studies of albendazole for A. lumbricoides, T. trichiura, and hookworm, respectively and 25% of mebendazole studies. iERR analyses showed similar results, with cure achieved in 92% of A. lumbricoides-infected subjects treated with albendazole and 93% with mebendazole; corresponding figures for hookworm were 70% and 17%, and for T. trichiura 22% and 20%.Conclusions/significanceCombining the traditional efficacy assessment using group averages with individual responses provides a more complete picture of how anthelmintic treatments perform. Most treatments analyzed fail to meet the WHO minimal criteria for efficacy based on group means. Drug combinations (i.e., albendazole-ivermectin and albendazole-oxantel pamoate) are promising treatments for STH infections.     

Marine Sediment-Derived Streptomyces Bacteria from British Columbia,Canada Are a Promising Microbiota Resource for the Discovery of Antimicrobial Natural Products

Representatives of the genus Streptomyces from terrestrial sources have been the focus of intensive research for the last four decades because of their prolific production of chemically diverse and biologically important compounds. However, metabolite research from this ecological niche had declined significantly in the past years because of the rediscovery of the same bioactive compounds and redundancy of the sample strains. More recently, a new picture has begun to emerge in which marine-derived Streptomyces bacteria have become the latest hot spot as new source for unique and biologically active compounds. Here, we investigated the marine sediments collected in the temperate cold waters from British Columbia, Canada as a valuable source for new groups of marine-derived Streptomyces with antimicrobial activities. We performed culture dependent isolation from 49 marine sediments samples and obtained 186 Streptomyces isolates, 47 of which exhibited antimicrobial activities. Phylogenetic analyses of the active isolates resulted in the identification of four different clusters of bioactive Streptomyces including a cluster with isolates that appear to represent novel species. Moreover, we explored whether these marine-derived Streptomyces produce new secondary metabolites with antimicrobial properties. Chemical analyses revealed structurally diverse secondary metabolites, including four new antibacterial novobiocin analogues. We conducted structure-activity relationships (SAR) studies of these novobiocin analogues against methicillin-resistant Staphylococcus aureus (MRSA). In this study, we revealed the importance of carbamoyl and OMe moieties at positions 3” and 4” of novobiose as well as the hydrogen substituent at position 5 of hydroxybenzoate ring for the anti-MRSA activity. Changes in the substituents at these positions dramatically impede or completely eliminate the inhibitory activity of novobiocins against MRSA.     

Subfamily Limoniinae Speiser, 1909 (Diptera,Limoniidae) from Baltic Amber (Eocene): The Genus Elephantomyia Osten Sacken, 1860

A revision of the genus Elephantomyia Osten Sacken (Diptera: Limoniidae) from Baltic amber (Eocene) is presented. Four species—E. baltica Alexander, E. brevipalpa Loew, E. longirostris Loew, and E. pulchella Loew—are redescribed and documented with photographs and drawings. In addition, two new species of the genus are described: Elephantomyia bozenae sp. nov., and Elephantomyia irinae sp. nov. All these fossil species are placed within the subgenus Elephantomyia. A key to the extinct species of Elephantomyia is provided, and the genus’ ecological pattern and evolutionary aspects are discussed.     

The Emergence of Resistance to the Benzimidazole Anthlemintics in Parasitic Nematodes of Livestock Is Characterised by Multiple Independent Hard and Soft Selective Sweeps

Anthelmintic resistance is a major problem for the control of parasitic nematodes of livestock and of growing concern for human parasite control. However, there is little understanding of how resistance arises and spreads or of the “genetic signature” of selection for this group of important pathogens. We have investigated these questions in the system for which anthelmintic resistance is most advanced; benzimidazole resistance in the sheep parasites Haemonchus contortus and Teladorsagia circumcincta. Population genetic analysis with neutral microsatellite markers reveals that T. circumcincta has higher genetic diversity but lower genetic differentiation between farms than H. contortus in the UK. We propose that this is due to epidemiological differences between the two parasites resulting in greater seasonal bottlenecking of H. contortus. There is a remarkably high level of resistance haplotype diversity in both parasites compared with drug resistance studies in other eukaryotic systems. Our analysis suggests a minimum of four independent origins of resistance mutations on just seven farms for H. contortus, and even more for T. circumincta. Both hard and soft selective sweeps have occurred with striking differences between individual farms. The sweeps are generally softer for T. circumcincta than H. contortus, consistent with its higher level of genetic diversity and consequent greater availability of new mutations. We propose a model in which multiple independent resistance mutations recurrently arise and spread by migration to explain the widespread occurrence of resistance in these parasites. Finally, in spite of the complex haplotypic diversity, we show that selection can be detected at the target locus using simple measures of genetic diversity and departures from neutrality. This work has important implications for the application of genome-wide approaches to identify new anthelmintic resistance loci and the likelihood of anthelmintic resistance emerging as selection pressure is increased in human soil-transmitted nematodes by community wide treatment programs.     

Drosophila Food-Associated Pheromones: Effect of Experience,Genotype and Antibiotics on Larval Behavior

Animals ubiquitously use chemical signals to communicate many aspects of their social life. These chemical signals often consist of environmental cues mixed with species-specific signals—pheromones—emitted by conspecifics. During their life, insects can use pheromones to aggregate, disperse, choose a mate, or find the most suitable food source on which to lay eggs. Before pupariation, larvae of several Drosophila species migrate to food sources depending on their composition and the presence of pheromones. Some pheromones derive from microbiota gut activity and these food-associated cues can enhance larval attraction or repulsion. To explore the mechanisms underlying the preference (attraction/repulsion) to these cues and clarify their effect, we manipulated factors potentially involved in larval response. In particular, we found that the (i) early exposure to conspecifics, (ii) genotype, and (iii) antibiotic treatment changed D. melanogaster larval behavior. Generally, larvae—tested either individually or in groups—strongly avoided food processed by other larvae. Compared to previous reports on larval attractive pheromones, our data suggest that such attractive effects are largely masked by food-associated compounds eliciting larval aversion. The antagonistic effect of attractive vs. aversive compounds could modulate larval choice of a pupariation site and impact the dispersion of individuals in nature.     

Radiation-Sensitive Mutants of CAENORHABDITIS ELEGANS

Nine rad (for abnormal radiation sensitivity) mutants hypersensitive to ultraviolet light were isolated in the small nematode Caenorhabditis elegans. The mutations are recessive to their wild-type alleles, map to four of the six linkage groups in C. elegans and define nine new games named rad-1 through rad-9. Two of the mutants—rad-1 and rad-2—are very hypersensitive to X rays, and three—rad-2, rad-3 and rad-4—are hypersensitive to methyl methanesulfonate under particular conditions of exposure. The hypersensitivity of these mutants to more than one DNA-damaging agent suggests that they may be abnormal in DNA repair. One mutant—rad-5, a temperature-sensitive sterile mutant—shows an elevated frequency of spontaneous mutation at more than one locus; rad-4, which shows a cold-sensitive embryogenesis, reduces meiotic X-chromosome nondisjunction tenfold and partially suppresses some but not all mutations that increase meiotic X-chromosome nondisjunction; the viability of rad-6 hermaphrodites is half that of rad-6 males at 25°; and newly mature (but not older) rad-8 hermaphrodites produce many inviable embryo progeny. Meiotic recombination frequencies were measured for seven rad mutants and found to be close to normal.     

The narrow-spectrum anthelmintic oxantel is a potent agonist of a novel acetylcholine receptor subtype in whipworms

In the absence of efficient alternative strategies, the control of parasitic nematodes, impacting human and animal health, mainly relies on the use of broad-spectrum anthelmintic compounds. Unfortunately, most of these drugs have a limited single-dose efficacy against infections caused by the whipworm, Trichuris. These infections are of both human and veterinary importance. However, in contrast to a wide range of parasitic nematode species, the narrow-spectrum anthelmintic oxantel has a high efficacy on Trichuris spp. Despite this knowledge, the molecular target(s) of oxantel within Trichuris is still unknown. In the distantly related pig roundworm, Ascaris suum, oxantel has a small, but significant effect on the recombinant homomeric Nicotine-sensitive ionotropic acetylcholine receptor (N-AChR) made up of five ACR-16 subunits. Therefore, we hypothesized that in whipworms, a putative homolog of an ACR-16 subunit, can form a functional oxantel-sensitive receptor. Using the pig whipworm T. suis as a model, we identified and cloned a novel ACR-16-like subunit and successfully expressed the corresponding homomeric channel in Xenopus laevis oocytes. Electrophysiological experiments revealed this receptor to have distinctive pharmacological properties with oxantel acting as a full agonist, hence we refer to the receptor as an O-AChR subtype. Pyrantel activated this novel O-AChR subtype moderately, whereas classic nicotinic agonists surprisingly resulted in only minor responses. We observed that the expression of the ACR-16-like subunit in the free-living nematode Caenorhabditis elegans conferred an increased sensitivity to oxantel of recombinant worms. We demonstrated that the novel Tsu-ACR-16-like receptor is indeed a target for oxantel, although other receptors may be involved. These finding brings new insight into the understanding of the high sensitivity of whipworms to oxantel, and highlights the importance of the discovery of additional distinct receptor subunit types within Trichuris that can be used as screening tools to evaluate the effect of new synthetic or natural anthelmintic compounds.     

The Discovery,Distribution, and Evolution of Viruses Associated with Drosophila melanogaster

Drosophila melanogaster is a valuable invertebrate model for viral infection and antiviral immunity, and is a focus for studies of insect-virus coevolution. Here we use a metagenomic approach to identify more than 20 previously undetected RNA viruses and a DNA virus associated with wild D. melanogaster. These viruses not only include distant relatives of known insect pathogens but also novel groups of insect-infecting viruses. By sequencing virus-derived small RNAs, we show that the viruses represent active infections of Drosophila. We find that the RNA viruses differ in the number and properties of their small RNAs, and we detect both siRNAs and a novel miRNA from the DNA virus. Analysis of small RNAs also allows us to identify putative viral sequences that lack detectable sequence similarity to known viruses. By surveying >2,000 individually collected wild adult Drosophila we show that more than 30% of D. melanogaster carry a detectable virus, and more than 6% carry multiple viruses. However, despite a high prevalence of the Wolbachia endosymbiont—which is known to be protective against virus infections in Drosophila—we were unable to detect any relationship between the presence of Wolbachia and the presence of any virus. Using publicly available RNA-seq datasets, we show that the community of viruses in Drosophila laboratories is very different from that seen in the wild, but that some of the newly discovered viruses are nevertheless widespread in laboratory lines and are ubiquitous in cell culture. By sequencing viruses from individual wild-collected flies we show that some viruses are shared between D. melanogaster and D. simulans. Our results provide an essential evolutionary and ecological context for host–virus interaction in Drosophila, and the newly reported viral sequences will help develop D. melanogaster further as a model for molecular and evolutionary virus research.     

Discovery of Anthelmintic Drug Targets and Drugs Using Chokepoints in Nematode Metabolic Pathways

Parasitic roundworm infections plague more than 2 billion people (1/3 of humanity) and cause drastic losses in crops and livestock. New anthelmintic drugs are urgently needed as new drug resistance and environmental concerns arise. A “chokepoint reaction” is defined as a reaction that either consumes a unique substrate or produces a unique product. A chokepoint analysis provides a systematic method of identifying novel potential drug targets. Chokepoint enzymes were identified in the genomes of 10 nematode species, and the intersection and union of all chokepoint enzymes were found. By studying and experimentally testing available compounds known to target proteins orthologous to nematode chokepoint proteins in public databases, this study uncovers features of chokepoints that make them successful drug targets. Chemogenomic screening was performed on drug-like compounds from public drug databases to find existing compounds that target homologs of nematode chokepoints. The compounds were prioritized based on chemical properties frequently found in successful drugs and were experimentally tested using Caenorhabditis elegans. Several drugs that are already known anthelmintic drugs and novel candidate targets were identified. Seven of the compounds were tested in Caenorhabditis elegans and three yielded a detrimental phenotype. One of these three drug-like compounds, Perhexiline, also yielded a deleterious effect in Haemonchus contortus and Onchocerca lienalis, two nematodes with divergent forms of parasitism. Perhexiline, known to affect the fatty acid oxidation pathway in mammals, caused a reduction in oxygen consumption rates in C. elegans and genome-wide gene expression profiles provided an additional confirmation of its mode of action. Computational modeling of Perhexiline and its target provided structural insights regarding its binding mode and specificity. Our lists of prioritized drug targets and drug-like compounds have potential to expedite the discovery of new anthelmintic drugs with broad-spectrum efficacy.     

Agaricus subrufescens: A review

Medicinal mushrooms have currently become a hot issue due to their various therapeutic properties. Of these, Agaricus subrufescens, also known as the “almond mushroom”, has long been valued by many societies (i.e., Brazil, China, France, and USA). Since its discovery in 1893, this mushroom has been cultivated throughout the world, especially in Brazil where several strains of A. subrufescens have been developed and used as health food and alternative medicine. This article presents up-to-date information on this mushroom including its taxonomy and health promoting benefits. Medicinal properties of A. subrufescens are emphasized in several studies which are reviewed here. In addition, safety issues concerning the use of this fungus will be discussed.     

The Antibacterial Properties of 4, 8, 4′, 8′-Tetramethoxy (1,1′-biphenanthrene) -2,7,2′,7′-Tetrol from Fibrous Roots of Bletilla striata

Biphenanthrene compound, 4, 8, 4′, 8′-tetramethoxy (1, 1′-biphenanthrene)—2, 7, 2′, 7′-tetrol (LF05), recently isolated from fibrous roots of Bletilla striata, exhibits antibacterial activity against several Gram-positive bacteria. In this study, we investigated the antibacterial properties, potential mode of action and cytotoxicity. Minimum inhibitory concentrations (MICs) tests showed LF05 was active against all tested Gram-positive strains, including methicillin-resistant Staphylococcus aureus (MRSA) and staphylococcal clinical isolates. Minimum bactericidal concentration (MBC) tests demonstrated LF05 was bactericidal against S. aureus ATCC 29213 and Bacillus subtilis 168 whereas bacteriostatic against S. aureus ATCC 43300, WX 0002, and other strains of S. aureus. Time-kill assays further confirmed these observations. The flow cytometric assay indicated that LF05 damaged the cell membrane of S. aureus ATCC 29213 and B. subtilis 168. Consistent with this finding, 4 × MIC of LF05 caused release of ATP in B. subtilis 168 within 10 min. Checkerboard test demonstrated LF05 exhibited additive effect when combined with vancomycin, erythromycin and berberine. The addition of rat plasma or bovine serum albumin to bacterial cultures caused significantly loss in antibacterial activity of LF05. Interestingly, LF05 was highly toxic to several tumor cells. Results of these studies indicate that LF05 is bactericidal against some Gram-positive bacteria and acts as a membrane structure disruptor. The application of biphenanthrene in the treatment of S. aureus infection, especially local infection, deserves further study.     

A Four-Point Screening Method for Assessing Molecular Mechanism of Action (MMOA) Identifies Tideglusib as a Time-Dependent Inhibitor of Trypanosoma brucei GSK3β

BackgroundNew therapeutics are needed for neglected tropical diseases including Human African trypanosomiasis (HAT), a progressive and fatal disease caused by the protozoan parasites Trypanosoma brucei gambiense and T. b. rhodesiense. There is a need for simple, efficient, cost effective methods to identify new molecules with unique molecular mechanisms of action (MMOAs). The mechanistic features of a binding mode, such as competition with endogenous substrates and time-dependence can affect the observed inhibitory IC₅₀, and differentiate molecules and their therapeutic usefulness. Simple screening methods to determine time-dependence and competition can be used to differentiate compounds with different MMOAs in order to identify new therapeutic opportunities.Conclusions/SignificanceAltogether this work demonstrates a straightforward method for determining molecular mechanisms of action and its application for mechanistic differentiation of two potent TbGSK3β inhibitors. The four point MMOA method identified tideglusib as a mechanistically differentiated TbGSK3β inhibitor. Tideglusib was shown to inhibit parasite growth in this work, and has been reported to be well tolerated in one year of dosing in human clinical studies. Consequently, further supportive studies on the potential therapeutic usefulness of tideglusib for HAT are justified.     

Bioassay-guided isolation of three anthelmintic compounds from Warburgia ugandensis Sprague subspecies ugandensis, and the mechanism of action of polygodial

Parasitic helminths continue to pose problems in human and veterinary medicine, as well as in agriculture. Resistance to current anthelmintics has prompted the search for new drugs. Anthelmintic metabolites from medicinal plants could be good anthelmintic drug candidates. However, the compounds active against nematodes have not been identified in most medicinal plants with anthelmintic activity. In this study, we aimed to identify the active compounds against helminths in Warburgia ugandensis Sprague subspecies ugandensis (Canellaceae) and study the underlying mechanism of action. A bioassay-guided isolation of anthelmintic compounds from the plant was performed using a Caenorhabditis elegans (C. elegans) test model with a WMicrotracker instrument to monitor motility. Three active compounds were purified and identified by nuclear magnetic resonance and high resolution MS: warburganal (IC₅₀: 28.2?±?8.6?μM), polygodial (IC₅₀: 13.1?±?5.3?μM) and alpha-linolenic acid (ALA, IC₅₀: 70.1?±?17.5?μM). A checkerboard assay for warburganal and ALA as well as polygodial and ALA showed a fractional inhibitory concentration index of 0.41 and 0.37, respectively, suggesting that polygodial and ALA, as well as warburganal and ALA, have a synergistic effect against nematodes. A preliminary structure–activity relationship study for polygodial showed that the α,β-unsaturated 1,4-dialdehyde structural motif is essential for the potent activity. None of a panel of C. elegans mutant strains, resistant against major anthelmintic drug classes, showed significant resistance to polygodial, implying that polygodial may block C. elegans motility through a mechanism which differs from that of currently marketed drugs. Further measurements showed that polygodial inhibits mitochondrial ATP synthesis of C. elegans in a dose-dependent manner (IC₅₀: 1.8?±?1.0?μM). Therefore, we believe that the underlying mechanism of action of polygodial is probably inhibition of mitochondrial ATP synthesis. In conclusion, polygodial could be a promising anthelmintic drug candidate worth considering for further development.