Pyrvinium Pamoate and Structural Analogs Are Early Macrofilaricide Leads.

Onchocerciasis and lymphatic filariasis are neglected tropical diseases caused by infection with filarial worms. Annual or biannual mass drug administration with microfilaricidal drugs that kill the microfilarial stages of the parasites has helped reduce infection rates and thus prevent transmission of both infections. However, success depends on high population coverage that is maintained for the duration of the adult worm's lifespan. Given that these filarial worms can live up to 14 years in their human hosts, a macrofilaricidal drug would vastly accelerate elimination efforts. Here, we have evaluated the repurposed drug pyrvinium pamoate as well as newly synthesized analogs of pyrvinium for their efficacy against filarial worms in vitro and in vivo. We found that pyrvinium pamoate, tetrahydropyrvinium and one of the analogs were highly potent in inhibiting worms in in vitro whole-worm screening assays, and that all three compounds reduced female worm fecundity and inhibited embryogenesis in the Brugia pahangi-gerbil in vivo model of infection.

Macrofilaricidal Benzimidazole-Benzoxaborole Hybrids as an Approach to the Treatment of River Blindness: Part 2. Ketone Linked Analogs.

The optimization of a series of benzimidazole-benzoxaborole hybrid molecules linked via a ketone that exhibit good activity against Onchocerca volvulus, a filarial nematode responsible for the disease onchocerciasis, also known as river blindness, is described. The lead identified in this series, 21 (AN15470), was found to have acceptable pharmacokinetic properties to enable an evaluation following oral dosing in an animal model of onchocerciasis. Compound 21was effective in killing worms implanted in Mongolian gerbils when dosed orally as a suspension at 100 mg/kg/day for 14 days but not when dosed orally at 100 mg/kg/day for 7 days.

Macrofilaricidal Benzimidazole-Benzoxaborole Hybrids as an Approach to the Treatment of River Blindness: Part 1. Amide Linked Analogs.

A series of benzimidazole-benzoxaborole hybrid molecules linked via an amide linker are described that exhibit good in vitro activity against Onchocerca volvulus, a filarial nematode responsible for the disease onchocerciasis, also known as river blindness. The lead identified in this series, 8a (AN8799), was found to have acceptable pharmacokinetic properties to enable evaluation in animal models of human filariasis. Compound 8a was effective in killing Brugia malayi, B. pahangi, and Litomosoides sigmodontis worms present in Mongolian gerbils when dosed subcutaneously as a suspension at 100 mg/kg/day for 14 days but not when dosed orally at 100 mg/kg/day for 28 days. The measurement of plasma levels of 8a at the end of the dosing period and at the time of sacrifice revealed an interesting dependence of activity on the extended exposure for both 8a and the positive control, flubendazole.

Proteomic identification of IPSE/alpha-1 as a major hepatotoxin secreted by Schistosoma mansoni eggs.

BACKGROUND: Eggs deposited in the liver of the mammalian host by the blood fluke parasite, Schistosoma mansoni, normally drive a T-helper-2 (Th2)-mediated granulomatous response in immune-competent mice. By contrast, in mice deprived of T-cells and incapable of producing granulomata, egg-secreted proteins (ESP) induce acute hepatic injury and death. Previous work has shown that one such ESP, the T2 ribonuclease known as omega-1, is hepatotoxic in vivo in that specific antisera to omega-1 prevent hepatocyte damage. METHODOLOGY/PRINCIPAL FINDINGS: Using an in vitro culture system employing mouse primary hepatocytes and alanine transaminase (ALT) activity as a marker of heptocyte injury, we demonstrated that S. mansoni eggs, egg-secreted proteins (ESP), soluble-egg antigen (SEA), and omega-1 are directly hepatotoxic and in a dose-dependent manner. Depletion of omega-1 using a monoclonal antibody abolished the toxicity of pure omega-1 and diminished the toxicity in ESP and SEA by 47 and 33%, respectively. Anion exchange chromatography of ESP yielded one predominant hepatotoxic fraction. Proteomics of that fraction identified the presence of IPSE/alpha-1 (IL-4 inducing principle from S. mansoni eggs), a known activator of basophils and inducer of Th2-type responses. Pure recombinant IPSE/alpha-1 also displayed a dose-dependent hepatotoxicity in vitro. Monoclonal antibody depletion of IPSE/alpha-1 abolished the latter's toxicity and diminished the total toxicity of ESP and SEA by 32 and 35%, respectively. Combined depletion of omega-1 and IPSE/alpha-1 diminished hepatotoxicity of ESP and SEA by 60 and 58% respectively. CONCLUSIONS: We identified IPSE/alpha-1 as a novel hepatotoxin and conclude that both IPSE/alpha-1 and omega-1 account for the majority of the hepatotoxicity secreted by S. mansoni eggs.

RNA interference in Schistosoma mansoni schistosomula: selectivity, sensitivity and operation for larger-scale screening.

BACKGROUND: The possible emergence of resistance to the only available drug for schistosomiasis spurs drug discovery that has been recently incentivized by the availability of improved transcriptome and genome sequence information. Transient RNAi has emerged as a straightforward and important technique to interrogate that information through decreased or loss of gene function and identify potential drug targets. To date, RNAi studies in schistosome stages infecting humans have focused on single (or up to 3) genes of interest. Therefore, in the context of standardizing larger RNAi screens, data are limited on the extent of possible off-targeting effects, gene-to-gene variability in RNAi efficiency and the operational capabilities and limits of RNAi. METHODOLOGY/PRINCIPAL FINDINGS: We investigated in vitro the sensitivity and selectivity of RNAi using double-stranded (ds)RNA (approximately 500 bp) designed to target 11 Schistosoma mansoni genes that are expressed in different tissues; the gut, tegument and otherwise. Among the genes investigated were 5 that had been previously predicted to be essential for parasite survival. We employed mechanically transformed schistosomula that are relevant to parasitism in humans, amenable to screen automation and easier to obtain in greater numbers than adult parasites. The operational parameters investigated included defined culture media for optimal parasite maintenance, transfection strategy, time- and dose-dependency of RNAi, and dosing limits. Of 7 defined culture media tested, Basch Medium 169 was optimal for parasite maintenance. RNAi was best achieved by co-incubating parasites and dsRNA (standardized to 30 µg/ml for 6 days); electroporation provided no added benefit. RNAi, including interference of more than one transcript, was selective to the gene target(s) within the pools of transcripts representative of each tissue. Concentrations of dsRNA above 90 µg/ml were directly toxic. RNAi efficiency was transcript-dependent (from 40 to >75% knockdown relative to controls) and this may have contributed to the lack of obvious phenotypes observed, even after prolonged incubations of 3 weeks. Within minutes of their mechanical preparation from cercariae, schistosomula accumulated fluorescent macromolecules in the gut indicating that the gut is an important route through which RNAi is expedited in the developing parasite. CONCLUSIONS: Transient RNAi operates gene-selectively in S. mansoni newly transformed schistosomula yet the sensitivity of individual gene targets varies. These findings and the operational parameters defined will facilitate larger RNAi screens.

Biolistic transformation of Schistosoma mansoni: Studies with modified reporter-gene constructs containing regulatory regions of protease genes.

Biolistics of the flatworm parasite Schistosoma mansoni facilitates the accurate spatial expression of transgenes under the control of gene-specific promoter elements. To improve transgene expression, either in the number of positive worms and/or an increased transgene signal per worm, we tested plasmid constructs incorporating 5' and 3' gene-specific genomic fragments, and parts of the open reading frame for two S. mansoni proteases, cathepsins F and D (SmCF and SmCD). GFP-expression was gut-localized, a novel finding for SmCD and consistent with previous data for SmCF. The mCherry fluorescent protein can also operate as a reporter. Though certain constructs imparted stronger and better distributed signals per positive worm, the low yields throughout (1-5% positive per experiment) precluded further quantifications of improvement. Electroporation of the same constructs was also weakly efficient (1-10% positives per experiment). However, reporter signals were found in tissues other than the gut, which may represent dysregulated transcription.

Drug discovery for schistosomiasis: hit and lead compounds identified in a library of known drugs by medium-throughput phenotypic screening.

BACKGROUND: Praziquantel (PZQ) is the only widely available drug to treat schistosomiasis. Given the potential for drug resistance, it is prudent to search for novel therapeutics. Identification of anti-schistosomal chemicals has traditionally relied on phenotypic (whole organism) screening with adult worms in vitro and/or animal models of disease-tools that limit automation and throughput with modern microtiter plate-formatted compound libraries. METHODS: A partially automated, three-component phenotypic screen workflow is presented that utilizes at its apex the schistosomular stage of the parasite adapted to a 96-well plate format with a throughput of 640 compounds per month. Hits that arise are subsequently screened in vitro against adult parasites and finally for efficacy in a murine model of disease. Two GO/NO GO criteria filters in the workflow prioritize hit compounds for tests in the animal disease model in accordance with a target drug profile that demands short-course oral therapy. The screen workflow was inaugurated with 2,160 chemically diverse natural and synthetic compounds, of which 821 are drugs already approved for human use. This affords a unique starting point to 'reposition' (re-profile) drugs as anti-schistosomals with potential savings in development timelines and costs. FINDINGS: Multiple and dynamic phenotypes could be categorized for schistosomula and adults in vitro, and a diverse set of 'hit' drugs and chemistries were identified, including anti-schistosomals, anthelmintics, antibiotics, and neuromodulators. Of those hits prioritized for tests in the animal disease model, a number of leads were identified, one of which compares reasonably well with PZQ in significantly decreasing worm and egg burdens, and disease-associated pathology. Data arising from the three components of the screen are posted online as a community resource. CONCLUSIONS: To accelerate the identification of novel anti-schistosomals, we have developed a partially automated screen workflow that interfaces schistosomula with microtiter plate-formatted compound libraries. The workflow has identified various compounds and drugs as hits in vitro and leads, with the prescribed oral efficacy, in vivo. Efforts to improve throughput, automation, and rigor of the screening workflow are ongoing.

Differential use of protease families for invasion by schistosome cercariae.

Schistosomes are parasitic platyhelminths (flatworms) of birds and mammals. As a parasitic disease of humans, schistosomiasis ranks second only to malaria in global importance. Schistosome larvae (cercariae) must invade and penetrate skin as an initial step to successful infection of the vertebrate host. Proteolytic enzymes secreted from the acetabular glands of cercariae contribute significantly to the invasion process. In this comparative study, we analyzed protease activities secreted by cercariae of Schistosoma mansoni, Schistosoma japonicum and Schistosomatium douthitti. Using protease-family specific, irreversible active-site probes, fluorogenic peptidyl substrates, immuno-histochemistry and high-resolution mass spectrometry, considerable species differences were noted in the quantity and character of proteases. Serine proteases, the most abundant enzymes secreted by S. mansoni cercariae, were not identified in S. japonicum. In contrast, the acetabular gland contents of S. japonicum cercariae had a 40-fold greater cathepsin B-like activity than those of S. mansoni. Based on the present data and previous reports, we propose that cysteine proteases represent an archetypal tool for tissue invasion among primitive metazoa and the use of serine proteases arose later in schistosome evolution. Computational analysis of serine protease phylogeny revealed an extraordinarily distant relationship between S. mansoni serine proteases and other members of the Clan PA family S1 proteases.

Schistosomiasis mansoni: novel chemotherapy using a cysteine protease inhibitor.

BACKGROUND: Schistosomiasis is a chronic, debilitating parasitic disease infecting more than 200 million people and is second only to malaria in terms of public health importance. Due to the lack of a vaccine, patient therapy is heavily reliant on chemotherapy with praziquantel as the World Health Organization-recommended drug, but concerns over drug resistance encourage the search for new drug leads. METHODS AND FINDINGS: The efficacy of the vinyl sulfone cysteine protease inhibitor K11777 was tested in the murine model of schistosomiasis mansoni. Disease parameters measured were worm and egg burdens, and organ pathology including hepato- and splenomegaly, presence of parasite egg-induced granulomas in the liver, and levels of circulating alanine aminotransferase activity as a marker of hepatocellular function. K11777 (25 mg/kg twice daily [BID]), administered intraperitoneally at the time of parasite migration through the skin and lungs (days 1-14 postinfection [p.i.]), resulted in parasitologic cure (elimination of parasite eggs) in five of seven cases and a resolution of other disease parameters. K11777 (50 mg/kg BID), administered at the commencement of egg-laying by mature parasites (days 30-37 p.i.), reduced worm and egg burdens, and ameliorated organ pathology. Using protease class-specific substrates and active-site labeling, one molecular target of K11777 was identified as the gut-associated cathepsin B1 cysteine protease, although other cysteine protease targets are not excluded. In rodents, dogs, and primates, K11777 is nonmutagenic with satisfactory safety and pharmacokinetic profiles. CONCLUSIONS: The significant reduction in parasite burden and pathology by this vinyl sulfone cysteine protease inhibitor validates schistosome cysteine proteases as drug targets and offers the potential of a new direction for chemotherapy of human schistosomiasis.

Proteomic analysis of Schistosoma mansoni cercarial secretions.

Schistosomiasis is a global health problem caused by several species of schistosome blood flukes. The initial stage of infection is invasion of human skin by a multicellular larva, the cercaria. We identified proteins released by cercariae when they are experimentally induced to exhibit invasive behavior. Comparison of the proteome obtained from skin lipid-induced cercariae (the natural activator), a cleaner mechanical induction procedure, and an uninduced proteomic control allowed identification of protein groups contained in cercarial acetabular gland secretion versus other sources. These included a group of proteins involved in calcium binding, calcium regulation, and calcium-activated functions; two proteins (paramyosin and SPO-1) implicated in immune evasion; and protease isoforms implicated in degradation of host skin barriers. Several other protein families, traditionally found as cytosolic proteins, appeared concentrated in secretory cells. These included proteins with chaperone activity such as HSP70, -86, and -60. Comparison of the three experimental proteomes also allowed identification of protein contaminants from the environment that were identified because of the high sensitivity of the MS/MS system used. These included proteins from the intermediate host snail in which cercariae develop, the investigator, and the laboratory environment. Identification of proteins secreted by invasive larvae provides important new information for validation of models of skin invasion and immune evasion and aids in rational development of an anti-schistosome vaccine.

SmCB2, a novel tegumental cathepsin B from adult Schistosoma mansoni.

Papain-like cysteine endopeptidases have been recognized as potential targets for chemotherapy and serodiagnostic reagents in infections with the human parasitic helminth Schistosoma. A novel cathepsin B endopeptidase from adult S. mansoni has been isolated and characterized. The enzyme is termed SmCB2 to distinguish it from the first recorded schistosome cathepsin B, SmCB1, also known as Sm31. A rapid and convenient protocol involving anion exchange and affinity chromatography is described for the isolation of SmCB1 and SmCB2 from the same parasite starting material. SmCB2 has been functionally expressed in and purified from Pichia pastoris. Both native and recombinant SmCB2 migrate similarly (33 kDa) by SDS-PAGE. Both display strict acidic pH activity profiles and similar K(m) and k(cat) for dipeptidyl amidomethylcoumarin substrates. We conclude that the recombinant enzyme is properly folded. The S(2) subsite specificity of recombinant SmCB2 exhibits the preferences Phe>Leu>Val>>Arg. By immunoblotting with anti-SmCB2 IgG, a 33 kDa protein was identified in soluble extracts of male schistosomes. By immunohistochemistry, SmCB2 was localized in the tegumental tubercles and parenchyma of males with less product being visualized in the parenchyma of females. The enzyme may be lysosomal and function at the host parasite-interface.

Cercarial elastase is encoded by a functionally conserved gene family across multiple species of schistosomes.

Water borne cercaria(ae) of the trematode genus Schistosoma rapidly penetrate host skin. A single serine protease activity, cercarial elastase, is deposited in advance of the invading parasite by holocytosis of vesicles from ten large acetabular gland cells. Cercarial elastase activity is a composite of multiple isoforms. Genes coding for the isoforms can be divided into two classes by amino acid and promoter sequence homology. Two of the five genes identified in Schistosoma mansoni account for over 90% of the activity and protein released. The remaining genes produce little protein or are silent. Positional scanning synthetic combinatorial substrate libraries demonstrate that the two major isoforms have similar substrate specificities and are, therefore, isoenzymes. The closely related Schistosoma hematobium and the distantly related Schistosomatium douthitti also contain multiple orthologous cercarial elastase genes suggesting that gene duplication may have occurred after speciation in Schistosoma evolution and that this duplication has been conserved.