Alessandra Guidi

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.

Genetic and Molecular Basis of Drug Resistance and Species-Specific Drug Action in Schistosome Parasites

Blood Fluke Resistance The larval stages of the blood fluke Schistosoma mansoni are disseminated via a replicative cycle in freshwater snails. When people come into contact with contaminated water, the larvae attach to and penetrate the skin. The resulting disease, bilharzia or schistosomiasis, afflicts approximately 67 million people in Africa and South America. Unfortunately, the parasite is showing resistance to one of the available therapeutic drugs, oxamniquine, which means that schistosome control relies on a single drug, praziquantel. Valentim et al. (p. 1385, published online 21 November) analyzed the genetic and molecular basis of resistance to oxamniquine through a combination of genetic linkage mapping, genome sequencing, functional genomics analysis, and x-ray crystallography. Mutations in a distinctive sulfotransferase are responsible for oxamniquine resistance in a human blood fluke. Oxamniquine resistance evolved in the human blood fluke (Schistosoma mansoni) in Brazil in the 1970s. We crossed parental parasites differing ~500-fold in drug response, determined drug sensitivity and marker segregation in clonally derived second-generation progeny, and identified a single quantitative trait locus (logarithm of odds = 31) on chromosome 6. A sulfotransferase was identified as the causative gene by using RNA interference knockdown and biochemical complementation assays, and we subsequently demonstrated independent origins of loss-of-function mutations in field-derived and laboratory-selected resistant parasites. These results demonstrate the utility of linkage mapping in a human helminth parasite, while crystallographic analyses of protein-drug interactions illuminate the mode of drug action and provide a framework for rational design of oxamniquine derivatives that kill both S. mansoni and S. haematobium, the two species responsible for >99% of schistosomiasis cases worldwide.

Whole Organism High-Content Screening by Label-Free, Image-Based Bayesian Classification for Parasitic Diseases

Sole reliance on one drug, Praziquantel, for treatment and control of schistosomiasis raises concerns about development of widespread resistance, prompting renewed interest in the discovery of new anthelmintics. To discover new leads we designed an automated label-free, high content-based, high throughput screen (HTS) to assess drug-induced effects on in vitro cultured larvae (schistosomula) using bright-field imaging. Automatic image analysis and Bayesian prediction models define morphological damage, hit/non-hit prediction and larval phenotype characterization. Motility was also assessed from time-lapse images. In screening a 10,041 compound library the HTS correctly detected 99.8% of the hits scored visually. A proportion of these larval hits were also active in an adult worm ex-vivo screen and are the subject of ongoing studies. The method allows, for the first time, screening of large compound collections against schistosomes and the methods are adaptable to other whole organism and cell-based screening by morphology and motility phenotyping.

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.

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.

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.

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.

Structural and Functional Characterization of the Enantiomers of the Antischistosomal Drug Oxamniquine.

Background For over two decades, a racemic mixture of oxamniquine (OXA) was administered to patients infected by Schistosoma mansoni, but whether one or both enantiomers exert antischistosomal activity was unknown. Recently, a ~30 kDa S. m ansoni sulfotransferase (SmSULT) was identified as the target of OXA action. Methodology/Principal Findings Here, we separate the OXA enantiomers using chromatographic methods and assign their optical activities as dextrorotary [(+)-OXA] or levorotary [(-)-OXA]. Crystal structures of the parasite enzyme in complex with optically pure (+)-OXA and (-)-OXA) reveal their absolute configurations as S- and R-, respectively. When tested in vitro, S-OXA demonstrated the bulk of schistosomicidal activity, while R-OXA had antischistosomal effects when present at relatively high concentrations. Crystal structures R-OXA•SmSULT and S-OXA•SmSULT complexes reveal similarities in the modes of OXA binding, but only the S-OXA enantiomer is observed in the structure of the enzyme exposed to racemic OXA. Conclusions/Significance Together the data suggest the higher schistosomicidal activity of S-OXA is correlated with its ability to outcompete R-OXA binding the sulfotransferase active site. These findings have important implications for the design, syntheses, and dosing of new OXA-based antischistosomal compounds.

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.