Crystal structures of Schistosoma mansoni histone deacetylase 8 reveal a novel binding site for allosteric inhibitors.

Parasitic diseases cause significant global morbidity and mortality particularly in the poorest regions of the world. Schistosomiasis, one of the most widespread neglected tropical diseases, affects more than 200 million people worldwide. Histone deacetylase (HDAC) inhibitors are prominent epigenetic drugs that are being investigated in the treatment of several diseases, including cancers and parasitic diseases. Schistosoma mansoni HDAC8 (SmHDAC8) is highly expressed in all life cycle stages of the parasite, and selective inhibition is required in order to avoid undesirable off-target effects in the host. Herein, by X-ray crystal structures of SmHDAC8-inhibitor complexes, biochemical and phenotypic studies, we found two schistosomicidal spiroindoline derivatives binding a novel site, next to Trp198, on the enzyme surface. We determined that by acting on this site, either by mutation of the Trp198 or by compound binding, a decrease in the activity of the enzyme is achieved. Remarkably, this allosteric site differs from the human counterpart; rather, it is conserved in all Schistosoma species, as well as Rhabidoptera and Trematoda classes, thus paving the way for the design of HDAC8-selective allosteric inhibitors with improved properties.

Gas-phase ion migration spectrum analysis of the volatile flavors of large yellow croaker oil after different storage periods.

The large yellow croaker, a species of fish found in the northwestern Pacific, is favored by consumers because of its prevalence in saltwater bodies, golden yellow abdomen, high calcium content, high protein, high fat content, and a flavor that originates from its lipids and volatile components. Volatile organic compounds significantly affect the aroma of food. In this work, electronic nose and headspace gas chromatography-ion mobility spectrometry were applied to analyze the flavor differences in fish oil durations. Through electronic nose system analysis, sensors W1C, W3S, W6S, and W2S directly affected fish oil flavor, and their flavor components were different. Gas chromatography-ion mobility spectrometry identified 26 volatile components (19 aldehydes, 3 ketones, 2 alcohols, 1 furan, and 1 olefin). (E,E)-2,4-hexadienal (D), (E,E)-2,4-hexadienal (M), 2,4-heptadienal (M), (E)-2-octenal, 2-propanone, 2-heptanone (M), 3-pentanone (D), and 1-octen-3-ol were the key flavor components of the fish oil. In conclusion, the combination of GC-IMS and PCA can identify the differences in flavor changes of large yellow croaker oil during 0-120 days storage. After 60 days storage, the types and signals of 2-propanone, 2-heptanone (M) components increase significantly. When 120 days storage, at this time, (E,E)-2,4-hexadienal (D), (E,E)-2,4-hexadienal (M), 2,4-heptadienal (M), (E)-2-octenal,(E)-2-octenal significantly. It has become the main flavor substance of fish oil. In summary, as the storage period increases, the components increase, and the oxidizing substances will increase, resulting in the deterioration of fish oil.

Drug effects on metabolic profiles of Schistosoma mansoni adult male parasites detected by 1H-NMR spectroscopy.

Schistosomiasis is one of the most devastating neglected tropical parasitic diseases caused by trematodes of the genus Schistosoma. Praziquantel (PZQ) is today the only drug used in humans and animals for the treatment of schistosomiasis but unfortunately it is poorly effective on larval and juvenile stages of the parasite. Therefore, it is urgent the discovery of new drug targets and compounds. We have recently showed that the anti-anginal drug perhexiline maleate (PHX) is very active on multiple developmental stages of Schistosoma mansoni in vitro. It is well known that PHX impacts the lipid metabolism in mammals, but the final target on schistosomes still remains unknown. The aim of this study was to evaluate the ability of 1H nuclear magnetic resonance (NMR) spectroscopy in revealing metabolic perturbations due to PHX treatment of S. mansoni adult male worms. The effects of PHX were compared with the ones induced by vehicle and gambogic acid, in order to detect different metabolic profiles and specificity of the PHX action. Remarkably a list of metabolites associated to PHX-treatment was identified with enrichment in several connected metabolic pathways including also the Kennedy pathway mediating the glycerophospholipid metabolism. Our study represents the first 1H-NMR metabolomic approach to characterize the response of S. mansoni to drug treatment. The obtained "metabolic fingerprint" associated to PHX treatment could represent a strategy for displaying cellular metabolic changes for any given drug and to compare compounds targeting similar or distinct biochemical pathways.

Luminescence-Based, Low- and Medium-Throughput Assays for Drug Screening in Schistosoma mansoni Larval Stage.

Schistosomiasis is one of the major parasitic diseases with more than  200 million people infected worldwide every year. Praziquantel is the drug of choice against the schistosomiasis although the use of a single drug to treat such a large amount of infected people appears particularly worrisome. For this reason, the search of new schistosomicidal compounds is viewed as an urgent goal and a number of screening campaigns have been carried out in the past years. The larval stage of Schistosoma (schistosomula) has been widely used in order to identify new compounds against the parasite. Here we describe detailed practical procedures for a luminescence-based assay proven to be highly effective for the selection of schistosomicidal compounds on small and medium-high scale. The assay is based on the quantitation of the parasite ATP, a good indicator of metabolically active cells, as measure of schistosomula viability. This assay is fast and reproducible, and it is suitable either for manual or for semiautomated screenings.

Discovery by organism based high-throughput screening of new multi-stage compounds affecting Schistosoma mansoni viability, egg formation and production.

Schistosomiasis, one of the most prevalent neglected parasitic diseases affecting humans and animals, is caused by the Platyhelminthes of the genus Schistosoma. Schistosomes are the only trematodes to have evolved sexual dimorphism and the constant pairing with a male is essential for the sexual maturation of the female. Pairing is required for the full development of the two major female organs, ovary and vitellarium that are involved in the production of different cell types such as oocytes and vitellocytes, which represent the core elements of the whole egg machinery. Sexually mature females can produce a large number of eggs each day. Due to the importance of egg production for both life cycle and pathogenesis, there is significant interest in the search for new strategies and compounds not only affecting parasite viability but also egg production. Here we use a recently developed high-throughput organism-based approach, based on ATP quantitation in the schistosomula larval stage of Schistosoma mansoni for the screening of a large compound library, and describe a pharmacophore-based drug selection approach and phenotypic analyses to identify novel multi-stage schistosomicidal compounds. Interestingly, worm pairs treated with seven of the eight compounds identified show a phenotype characterized by defects in eggshell assemblage within the ootype and egg formation with degenerated oocytes and vitelline cells engulfment in the uterus and/or oviduct. We describe promising new molecules that not only impair the schistosomula larval stage but also impact juvenile and adult worm viability and egg formation and production in vitro.

Discovery and Characterization of Novel Anti-schistosomal Properties of the Anti-anginal Drug, Perhexiline and Its Impact on Schistosoma mansoni Male and Female Reproductive Systems.

BACKGROUND: Schistosomiasis, one of the world's greatest human neglected tropical diseases, is caused by parasitic trematodes of the genus Schistosoma. A unique feature of schistosome biology is that the induction of sexual maturation as well as the maintenance of the differentiation status of female reproductive organs and egg production, necessary for both disease transmission and pathogenesis, are strictly dependent on the male. The treatment and most control initiatives of schistosomiasis rely today on the long-term application of a single drug, praziquantel (PZQ), mostly by campaigns of mass drug administration. PZQ, while very active on adult parasites, has much lower activity against juvenile worms. Monotherapy also favors the selection of drug resistance and, therefore, new drugs are urgently needed. METHODS AND FINDINGS: Following the screening of a small compound library with an ATP-based luminescent assay on Schistosoma mansoni schistosomula, we here report the identification and characterization of novel antischistosomal properties of the anti-anginal drug perhexiline maleate (PHX). By phenotypic worm survival assays and confocal microscopy studies we show that PHX, in vitro, has a marked lethal effect on all S. mansoni parasite life stages (newly transformed schistosomula, juvenile and adult worms) of the definitive host. We further demonstrate that sub-lethal doses of PHX significantly impair egg production and lipid depletion within the vitellarium of adult female worms. Moreover, we highlighted tegumental damage in adult male worms and remarkable reproductive system alterations in both female and male adult parasites. The in vivo study in S. mansoni-patent mice showed a notable variability of worm burdens in the individual experiments, with an overall minimal schistosomicidal effect upon PHX treatment. The short PHX half-life in mice, together with its very high rodent plasma proteins binding could be the cause of the modest efficacy of PHX in the schistosomiasis murine model. CONCLUSIONS/SIGNIFICANCE: Overall, our data indicate that PHX could represent a promising starting point for novel schistosomicidal drug discovery programmes.

Application of RNAi to Genomic Drug Target Validation in Schistosomes.

Concerns over the possibility of resistance developing to praziquantel (PZQ), has stimulated efforts to develop new drugs for schistosomiasis. In addition to the development of improved whole organism screens, the success of RNA interference (RNAi) in schistosomes offers great promise for the identification of potential drug targets to initiate drug discovery. In this study we set out to contribute to RNAi based validation of putative drug targets. Initially a list of 24 target candidates was compiled based on the identification of putative essential genes in schistosomes orthologous of C. elegans essential genes. Knockdown of Calmodulin (Smp_026560.2) (Sm-Calm), that topped this list, produced a phenotype characterised by waves of contraction in adult worms but no phenotype in schistosomula. Knockdown of the atypical Protein Kinase C (Smp_096310) (Sm-aPKC) resulted in loss of viability in both schistosomula and adults and led us to focus our attention on other kinase genes that were identified in the above list and through whole organism screening of known kinase inhibitor sets followed by chemogenomic evaluation. RNAi knockdown of these kinase genes failed to affect adult worm viability but, like Sm-aPKC, knockdown of Polo-like kinase 1, Sm-PLK1 (Smp_009600) and p38-MAPK, Sm-MAPK p38 (Smp_133020) resulted in an increased mortality of schistosomula after 2-3 weeks, an effect more marked in the presence of human red blood cells (hRBC). For Sm-PLK-1 the same effects were seen with the specific inhibitor, BI2536, which also affected viable egg production in adult worms. For Sm-PLK-1 and Sm-aPKC the in vitro effects were reflected in lower recoveries in vivo. We conclude that the use of RNAi combined with culture with hRBC is a reliable method for evaluating genes important for larval development. However, in view of the slow manifestation of the effects of Sm-aPKC knockdown in adults and the lack of effects of Sm-PLK-1 and Sm-MAPK p38 on adult viability, these kinases may not represent suitable drug targets.

Development and validation of a luminescence-based, medium-throughput assay for drug screening in Schistosoma mansoni.

BACKGROUND: Schistosomiasis, one of the world's greatest neglected tropical diseases, is responsible for over 280,000 human deaths per annum. Praziquantel, developed in the 1970s, has high efficacy, excellent tolerability, few and transient side effects, simple administration procedures and competitive cost and it is currently the only recommended drug for treatment of human schistosomiasis. The use of a single drug to treat a population of over 200 million infected people appears particularly alarming when considering the threat of drug resistance. Quantitative, objective and validated methods for the screening of compound collections are needed for the discovery of novel anti-schistosomal drugs. METHODOLOGY/PRINCIPAL FINDINGS: The present work describes the development and validation of a luminescence-based, medium-throughput assay for the detection of schistosomula viability through quantitation of ATP, a good indicator of metabolically active cells in culture. This validated method is demonstrated to be fast, highly reliable, sensitive and automation-friendly. The optimized assay was used for the screening of a small compound library on S. mansoni schistosomula, showing that the proposed method is suitable for a medium-throughput semi-automated screening. Interestingly, the pilot screening identified hits previously reported to have some anti-parasitic activity, further supporting the validity of this assay for anthelminthic drug discovery. CONCLUSIONS: The developed and validated schistosomula viability luminescence-based assay was shown to be successful and suitable for the identification of novel compounds potentially exploitable in future schistosomiasis therapies.

Schistosomiasis control: praziquantel forever?

Since no vaccine exists against schistosomiasis and the molluscs acting as intermediate hosts are not easy to attack, chemotherapy is the main approach for schistosomiasis control. Praziquantel is currently the only available antischistosomal drug and it is distributed mainly through mass administration programs to millions of people every year. A number of positive features make praziquantel an excellent drug, especially with regard to safety, efficacy, cost and ease of distribution. A major flaw is its lack of efficacy against the immature stages of the parasite. In view of its massive and repeated use on large numbers of individuals, the development of drug resistance is a much feared possibility. The mechanism of action of praziquantel is still unclear, a fact that does not favor the development of derivatives or alternatives. A large number of compounds have been tested as potential antischistosomal agents. Some of them are promising, but none so far represents a suitable substitute or adjunct to praziquantel. The research of new antischistosomal compounds is an imperative and urgent matter.

Activated retinal glia mediated axon regeneration in experimental glaucoma.

Glaucoma, a leading cause of blindness, is a neurodegenerative disease characterized by progressive loss of retinal ganglion cell axons in the optic nerve and their cell bodies in the retina. Reactive retinal glial changes have been observed in glaucoma but the role of such glial changes in the pathogenesis of the condition remains unclear. In the present study we found that retinal ganglion cells in an experimental animal model of glaucoma have an increased axon regenerative potential. Regeneration of adult rat retinal ganglion cell axons after optic nerve crush was significantly increased in vivo when combined with intraocular pressure-induced experimental glaucoma. This enhanced axon regeneration response was correlated with a significant increase in activation of glial fibrillary acidic protein+retinal glia. Using a dissociated retinal ganglion cell culture model we showed that reducing the number of activated retinal glia with a glial specific toxin, α-Aminoadipic acid, significantly reduced the growth potential of retinal ganglion cells from glaucomatous rat eyes, suggesting that activated retinal glia mediate, at least in part, the growth promoting effect. This was shown to be mediated by both membrane-bound and soluble glial-derived factors. Neurotrophin and ciliary neurotrophic/leukemia inhibitory factor blockers did not affect the regenerative potential, excluding these growth factors as principal mediators of the enhanced growth response occurring in glaucomatous retinal cultures. These observations are the first to reveal that retinal ganglion cells from glaucomatous rat eyes have an enhanced regenerative capacity. Furthermore, our results suggest that activated retinal glia mediate at least part of this response. Further work to understand and enhance the regeneration-promoting effect of activated retinal glia is required to determine if this approach could be useful as part of a therapeutic strategy to encourage optic nerve regeneration in glaucoma.

Somatotropic gene response to recombinant growth hormone treatment in buffalo leucocytes.

The use of recombinant bovine growth hormone (rbGH) to increase milk yield in cows is banned in some countries. In others, where it is authorised, it has triggered harsh debates on labelling of dairy products. If many studies have been performed on bovines, there is a lack of information on buffaloes, which are sometimes treated with rbGH and re-present an important economical resource for dairy products in some countries. Analytical methods with legal value for surveillance of rbGH treatments do not yet exist. Research on gene expression biomarkers is one of the most promising approaches to this purpose. For this reason, we treated five buffaloes for 10 weeks with a sustained-release formulation of rbGH and analysed the response of 20 somatotropic axis genes in leucocytes by real-time polymerase chain reaction. Overall changes in gene expression levels were of low magnitude and sometimes affected by the 'time' factor. Only the IGFBP-1 gene showed a significant under-expression (about two-fold; p <0.001) in treated animals. Taken together, these results give evidence that expression analysis of the somatotropic axis genes in leucocytes is little helpful for discrimination of rbGH-treated buffaloes, but do not exclude that another array of genes could provide useful patterns of variation.

Genetic analysis of decreased praziquantel sensitivity in a laboratory strain of Schistosoma mansoni.

A laboratory strain of Schistosoma mansoni subjected to repeated in vivo praziquantel (PZQ) treatments for several generations has been previously found to have lesser sensitivity to the drug than the original unselected strain. In this study we have collected evidence on the mode of inheritance of the partial insensitivity exhibited by the PZQ-selected schistosomes. A single male and a single female worm of the two strains, assorted in the four possible combinations, were introduced into the mesenteric veins of mice and the eggs produced by each pair were used as the source of the F(1) progeny. PZQ sensitivity was assessed using both in vivo and in vitro methods. In the first approach, the PZQ ED(50) was determined by infecting mice with cercariae of the strains to be tested, treating at seven weeks with different drug doses and counting the number of surviving worms three weeks later. For the in vitro approach, adult schistosomes kept in culture were exposed overnight to different PZQ concentrations and their survival was monitored during the subsequent 7 days. Results from both approaches lead to the conclusion that hybrid schistosomes of the F(1) generation have a drug sensitivity intermediate between those of the two parental strains and are thus suggestive of a pattern of partial dominance for the trait under study.

Schistosoma mansoni: lack of correlation between praziquantel-induced intra-worm calcium influx and parasite death.

The schistosomicidal activity of praziquantel (PZQ) is accompanied by a large influx of calcium into the worms, suggesting that this phenomenon could be the source of the observed muscular contraction, surface disruption and eventual death of the parasite. We have incubated live adult schistosomes in a medium containing radioactive calcium and we were able to confirm that PZQ does indeed stimulate calcium entry into the parasite. An even higher calcium uptake, however, occurred in schistosomes exposed to PZQ after pre-incubation with cytochalasin D, a condition that suppresses PZQ schistosomicidal effects and allows the complete survival of the parasites. The calcium blockers nicardipine and nifedipine also failed to prevent the calcium influx induced by PZQ. Similarly, a large calcium influx occurred in 28-day-old worms exposed to PZQ, in spite of the fact that these immature worms are largely insensitive to the schistosomicidal effects of the drug. Schistosomes incubated overnight with radioactive calcium and PZQ and then returned to normal medium, retained a calcium content higher than worms pre-incubated with cytochalasin D, but the difference could be a consequence--rather than a cause--of schistosomicidal effects. These results suggest that calcium accumulation by itself, at least as measured in whole parasites maintained in vitro, may not represent an exhaustive explanation for the schistosomicidal effects of PZQ.

The schistosome enzyme that activates oxamniquine has the characteristics of a sulfotransferase

Available evidence suggests that the antischistosomal drug oxamniquine is converted to a reactive ester by a schistosome enzyme that is missing in drug-resistant parasites. This study presents data supporting the idea that the active ester is a sulfate and the activating enzyme is a sulfotransferase. Evidence comes from the fact that the parasite extract loses its activating capability upon dialysis, implying the requirement of some dialyzable cofactor. The addition of the sulfate donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) restored activity of the dialyzate, a strong indication that a sulfotransferase is probably involved. Classical sulfotransferase substrates like beta-estradiol and quercetin competitively inhibited the activation of oxamniquine. Furthermore, these substrates could be sulfonated in vitro using an extract of sensitive (but not resistant) schistosomes. Gel filtration analysis showed that the activating factor eluted in a fraction corresponding to a molecular mass of about 32 kDa, which is the average size of typical sulfotransferase subunits. Ion exchange and affinity chromatography confirmed the sulfotransferase nature of the enzyme. Putative sulfotransferases present in schistosome databases are being examined for their possible role as oxamniquine activators.

Unsuspected role of the brain morphogenetic gene Otx1 in hematopoiesis.

Otx1 belongs to the paired class of homeobox genes and plays a pivotal role in brain development. Here, we show that Otx1 is expressed in hematopoietic pluripotent and erythroid progenitor cells. Moreover, bone marrow cells from mice lacking Otx1 exhibit a cell-autonomous impairment of the erythroid compartment. In agreement with these results, molecular analysis revealed decreased levels of erythroid genes that include the SCL and GATA-1 transcription factors. Accordingly, a gain of function of SCL rescues the erythroid deficiency in Otx1-/- mice. Taken together, our findings indicate a function for Otx1 in the regulation of blood cell production.