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Background Drugs currently used for controlling onchocerciasis and lymphatic filariasis (LF) are mainly microfilaricidal, with minimal or no effect on the adult worms. For efficient management of these diseases, it is necessary to search for new drugs with macrofilaricidal activities that can be used singly or in combination with existing ones. Daniellia oliveri and Psorospermum febrifugum are two plants commonly used in the local management of these infections in Bambui, a township in the North West Region of Cameroon, but there is currently no documented scientific evidence to support their claimed anthelmintic efficacy and safety. The aim of this study was to provide evidence in support of the search for means to eliminate these diseases by screening extracts and chromatographic fractions isolated from these plants for efficacy against the parasitic roundworms Onchocerca ochengi and Brugia pahangi. Methods The viability of O. ochengi adult worms was assessed using the MTT/formazan assay. Fully confluent monkey kidney epithelial cells (LLC-MK2) served as the feeder layer for the O. ochengi microfilariae (mfs) assays. Viability of the mfs was assessed by microscopic examination for mean motility scoring (relative to the negative control) every 24 h post addition of an extract. The Worminator system was used to test the effects of the extracts on adult B. pahangi motility, and mean motility units were determined for each worm. Cytotoxicity of the active extracts on N27 cells was assessed using the MTS assay. Results Extracts from D. oliveri and P. febrifugum were effective against the adult roundworms O. ochengi and B. pahangi. Interestingly, extracts showing macrofilaricidal activities against O. ochengi also showed activity against O. ochengi mfs. The hexane stem bark extract of D. oliveri (DOBHEX) was more selective for adult O. ochengi than for mfs, with a half maximal and 100% inhibitory concentration (IC50 and IC100, respectively) against adult O. ochengi of 13.9 and 31.3 μg/ml, respectively. The in vitro cytotoxicity of all active extracts on N27 cells showed selective toxicity for parasites (selectivity index > 1). Bioassay-guided fractionation of the extracts yielded fractions with activity against adult B. pahangi, thus confirming the presence of bioactive principles in the plant extracts. Conclusions Our study supports the use of D. oliveri and P. febrifugum in the traditional treatment of onchocerciasis and LF. The further purification of active extracts from these plants could yield lead compounds for filarial drug discovery and development. Graphical Abstract

Helminths represent a diverse category of parasitic organisms that can thrive within a host for years, if not decades, in the absence of treatment. As such, they must establish mechanisms to subsist off their hosts, evade the immune system, and develop a niche among the other cohabiting microbial communities. The complex interplay of biologically small molecules (collectively known as the metabolome) derived from, utilized by, or in response to the presence of helminths within a host is an emerging field of study. In this Perspective, we briefly summarize the current existing literature, categorize key host–pathogen–microbiome interfaces that could be studied in the context of the metabolome, and provide background on mass spectrometry-based metabolomic methodology. Overall, we hope to provide a comprehensive guide for utilizing metabolomics in the context of helminthic disease.

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.

The quinazolines CBR417 and CBR490 were previously shown to be potent anti-wolbachials that deplete Wolbachia endosymbionts of filarial nematodes and present promising pre-clinical candidates for human filarial diseases such as onchocerciasis. In the present study we tested both candidates in two models of chronic filarial infection, namely the Litomosoides sigmodontis and Brugia pahangi jird model and assessed their long-term effect on Wolbachia depletion, microfilariae counts and filarial embryogenesis 16−18 weeks after treatment initiation (wpt). Once per day (QD) oral treatment with CBR417 (50 mg/kg) for 4 days or twice per day (BID) with CBR490 (25 mg/kg) for 7 days during patent L. sigmodontis infection reduced the Wolbachia load by >99% and completely cleared peripheral microfilaremia from 10–14 wpt. Similarly, 7 days of QD treatments (40 mg/kg) with CBR417 or CBR490 cleared >99% of Wolbachia from B. pahangi and reduced peritoneal microfilariae counts by 93% in the case of CBR417 treatment. Transmission electron microscopy analysis indicated intensive damage to the B. pahangi ovaries following CBR417 treatment and in accordance filarial embryogenesis was inhibited in both models after CBR417 or CBR490 treatment. Suboptimal treatment regimens of CBR417 or CBR490 did not lead to a maintained reduction of the microfilariae and Wolbachia load. In conclusion, CBR417 or CBR490 are pre-clinical candidates for filarial diseases, which achieve long-term clearance of Wolbachia endosymbionts of filarial nematodes, inhibit filarial embryogenesis and clear microfilaremia with treatments as short as 7 days., Graphical abstract Image 1, Highlights • CBR417 and CBR490 provide long-term effects in 2 chronic filaria jird models. • CBR417 and CBR490 deplete >99% Wolbachia in B. pahangi and L. sigmodontis filariae. • CBR417 and CBR490 clear L. sigmodontis microfilariae after 10–14 weeks. • CBR417 and CBR490 inhibit filarial embryogenesis in both models. • Suboptimal doses do not maintain reduction of microfilariae and Wolbachia.

Confirmed diagnosis of chronic Chagas disease (CD) requires positive results by two different IgG serology tests. Variable sensitivity has been reported among tests and in different geographic regions. Inadequate specificity presents a particular challenge in low-prevalence settings such as the United States. This study provides a direct comparison of the latest-generation IgG serology assays with four previously assessed FDA-cleared tests. Seven hundred ten blood donor plasma specimens were evaluated by Wiener Lisado and Wiener v.4.0 enzyme-linked immunosorbent assays (ELISAs) and Abbott PRISM Chagas chemiluminescent assay (ChLIA). Sensitivity and specificity were assessed relative to infection status as determined by the original blood donation testing algorithm. All three latest-generation assays demonstrated 100% specificity (95% confidence interval [CI], 98.6 to 100.0). Wiener Lisado, Wiener v.4.0, and Abbott PRISM had sensitivities of 97.1% (95% CI, 95.1 to 98.4), 98.9% (95% CI, 97.4 to 99.6), and 95.5% (95% CI, 93.2 to 97.3), respectively. As with previously evaluated FDA-cleared tests, all three assays had the highest reactivity and sensitivity in samples from donors born in South America and lowest reactivity and sensitivity in specimens from those born in Mexico, with intermediate results in specimens from Central American donors. Wiener v.4.0 had the highest diagnostic sensitivity in all comparisons. Our findings suggest that the latest-generation CD serology tests could improve diagnostic sensitivity without affecting specificity.

Several issues have been identified with the current programs for the elimination of onchocerciasis that target only transmission by using mass drug administration (MDA) of the drug ivermectin. Alternative and/or complementary treatment regimens as part of a more comprehensive strategy to eliminate onchocerciasis are needed. We posit that the addition of “prophylactic” drugs or therapeutic drugs that can be utilized in a prophylactic strategy to the toolbox of present microfilaricidal drugs and/or future macrofilaricidal treatment regimens will not only improve the chances of meeting the elimination goals but may hasten the time to elimination and also will support achieving a sustained elimination of onchocerciasis. These “prophylactic” drugs will target the infective third- (L3) and fourth-stage (L4) larvae of Onchocerca volvulus and consequently prevent the establishment of new infections not only in uninfected individuals but also in already infected individuals and thus reduce the overall adult worm burden and transmission. Importantly, an effective prophylactic treatment regimen can utilize drugs that are already part of the onchocerciasis elimination program (ivermectin), those being considered for MDA (moxidectin), and/or the potential macrofilaricidal drugs (oxfendazole and emodepside) currently under clinical development. Prophylaxis of onchocerciasis is not a new concept. We present new data showing that these drugs can inhibit L3 molting and/or inhibit motility of L4 at IC50 and IC90 that are covered by the concentration of these drugs in plasma based on the corresponding pharmacological profiles obtained in human clinical trials when these drugs were tested using various doses for the therapeutic treatments of various helminth infections.

Filarial worms cause multiple debilitating diseases in millions of people worldwide, including river blindness. Currently available drugs reduce transmission by killing larvae (microfilariae), but there are no effective cures targeting the adult parasites (macrofilaricides) which survive and reproduce in the host for very long periods. To identify effective macrofilaricides, we carried out phenotypic screening of a library of 2121 approved drugs for clinical use against adult Brugia pahangi and prioritized the hits for further studies by integrating those results with a computational prioritization of drugs and associated targets. This resulted in the identification of 18 hits with anti-macrofilaricidal activity, of which two classes, azoles and aspartic protease inhibitors, were further expanded upon. Follow up screening against Onchocerca spp. (adult Onchocerca ochengi and pre-adult O. volvulus) confirmed activity for 13 drugs (the majority having IC50 &lt, 10 &mu, M), and a counter screen of a subset against L. loa microfilariae showed the potential to identify selective drugs that prevent adverse events when co-infected individuals are treated. Stage specific activity was also observed. Many of these drugs are amenable to structural optimization, and also have known canonical targets, making them promising candidates for further optimization that can lead to identifying and characterizing novel anti-macrofilarial drugs.

Neglected parasitic diseases remain a major public health issue worldwide, especially in tropical and subtropical areas. Human parasite diversity is very large, ranging from protozoa to worms. In most cases, more effective and new drugs are urgently needed. Previous studies indicated that the gold(I) drug auranofin (Ridaura&reg, ) is effective against several parasites. Among new gold(I) complexes, the phosphole-containing gold(I) complex {1-phenyl-2,5-di(2-pyridyl)phosphole}AuCl (abbreviated as GoPI) is an irreversible inhibitor of both purified human glutathione and thioredoxin reductases. GoPI-sugar is a novel 1-thio-&beta, d-glucopyranose 2,3,4,6-tetraacetato-S-derivative that is a chimera of the structures of GoPI and auranofin, designed to improve stability and bioavailability of GoPI. These metal-ligand complexes are of particular interest because of their combined abilities to irreversibly target the essential dithiol/selenol catalytic pair of selenium-dependent thioredoxin reductase activity, and to kill cells from breast and brain tumors. In this work, screening of various parasites&mdash, protozoans, trematodes, and nematodes&mdash, was undertaken to determine the in vitro killing activity of GoPI-sugar compared to auranofin. GoPI-sugar was found to efficiently kill intramacrophagic Leishmania donovani amastigotes and adult filarial and trematode worms.

Antibiotic treatment has emerged as a promising strategy to sterilize and kill filarial nematodes due to their dependence on their endosymbiotic bacteria, Wolbachia. Several studies have shown that novel and FDA-approved antibiotics are efficacious at depleting the filarial nematodes of their endosymbiont, thus reducing female fecundity. However, it remains unclear if antibiotics can permanently deplete Wolbachia and cause sterility for the lifespan of the adult worms. Concerns about resistance arising from mass drug administration necessitate a careful exploration of potential Wolbachia recrudescence. In the present study, we investigated the long-term effects of the FDA-approved antibiotic, rifampicin, in the Brugia pahangi jird model of infection. Initially, rifampicin treatment depleted Wolbachia in adult worms and simultaneously impaired female worm fecundity. However, during an 8-month washout period, Wolbachia titers rebounded and embryogenesis returned to normal. Genome sequence analyses of Wolbachia revealed that despite the population bottleneck and recovery, no genetic changes occurred that could account for the rebound. Clusters of densely packed Wolbachia within the worm’s ovarian tissues were observed by confocal microscopy and remained in worms treated with rifampicin, suggesting that they may serve as privileged sites that allow Wolbachia to persist in worms while treated with antibiotic. To our knowledge, these clusters have not been previously described and may be the source of the Wolbachia rebound., Author summary Onchocerciasis (river blindness) and lymphatic filariasis (elephantiasis) are two neglected tropical diseases caused by filarial nematodes, which harbor the endosymbiotic bacteria, Wolbachia. Major efforts to discover new drugs to treat these diseases have led to the discovery of novel compounds including those that target Wolbachia. We investigated the long-term effects of rifampicin on the filarial nematode, Brugia pahangi, and its endosymbiont, Wolbachia, in an in vivo rodent model of infection. Initially, Wolbachia titers were significantly reduced by 95% and female fecundity was impaired shortly after treatment. 8 months later however, Wolbachia rebounded and embryogenesis returned to normal. Sequence analysis of the Wolbachia genome revealed that despite the population bottleneck and recovery, no genetic changes occurred that could account for the rebound. Clusters of Wolbachia were observed within the ovaries of female worms throughout the entire 8-month study. These clusters may sequester Wolbachia and allow the bacteria to persist during antibiotic treatment, thereby enabling them to repopulate ovarian tissues and ensuring their vertical transmission to future generations of microfilariae.

Efforts to identify new drugs for therapeutic and preventive treatments against parasitic nematodes have gained increasing interest with expanding pathogen omics databases and drug databases from which new anthelmintic compounds might be identified. Here, a novel approach focused on integrating a pan-Nematoda multi-omics data targeted to a specific nematode organ system (the intestinal tract) with evidence-based filtering and chemogenomic screening was undertaken. Based on de novo computational target prioritization of the 3,564 conserved intestine genes in A. suum, exocytosis was identified as a high priority pathway, and predicted inhibitors of exocytosis were tested using the large roundworm (Ascaris suum larval stages), a filarial worm (Brugia pahangi adult and L3), a whipworm (Trichuris muris adult), and the non-parasitic nematode Caenorhabditis elegans. 10 of 13 inhibitors were found to cause rapid immotility in A. suum L3 larvae, and five inhibitors were effective against the three phylogenetically diverse parasitic nematode species, indicating potential for a broad spectrum anthelmintics. Several distinct pathologic phenotypes were resolved related to molting, motility, or intestinal cell and tissue damage using conventional and novel histologic methods. Pathologic profiles characteristic for each inhibitor will guide future research to uncover mechanisms of the anthelmintic effects and improve on drug designs. This progress firmly validates the focus on intestinal cell biology as a useful resource to develop novel anthelmintic strategies., Author summary The intestinal cells of parasitic nematodes are not known to regenerate, therefore disruption of essential processes that cause irreparable damage to intestinal cells is expected to promote worm expulsion. To facilitate improved methods of therapy we need to better understand the basic intestinal cell and tissue functions of this critical organ. To that end we have undertaken a comprehensive analysis of multi-omics omics data and identify and prioritize intestinal genes/pathways with essential functions and associated drugs and established a foundational model of the STH intestinal system using the large roundworm Ascaris suum to test and validate inhibitors of these functions. We found 10 inhibitors that impacted motility, and seven of those showed severe pathology and an apparent irreparable damage to intestinal cells. Furthermore, five inhibitors were effective against the three phylogenetically diverse parasitic nematode species, indicating potential for broad spectrum anthelmintics. Our results firmly validate the focus on intestinal cell biology as a useful resource to develop novel anthelmintic strategies.

The enormous prevalence of infections caused by parasitic nematodes worldwide, coupled to the rapid emergence of their resistance to commonly used anthelmintic drugs, presents an urgent need for the discovery of new drugs. Herein, we have identified several classes of small molecules with broad spectrum activity against these pathogens. Previously, we reported the identification of carnitine palmitoyltransferases (CPTs) as a representative class of enzymes as potential targets for metabolic chokepoint intervention that was elucidated from a combination of chemogenomic screening and experimental testing in nematodes. Expanding on these previous findings, we have discovered that several chemical classes of known small molecule inhibitors of mammalian CPTs have potent activity as anthelmintics. Cross-clade efficacy against a broad spectrum of adult parasitic nematodes was demonstrated for multiple compounds from different series. Several analogs of these initial hit compounds were designed and synthesized. The compounds we report represent a good starting point for further lead identification and optimization for development of new anthelmintic drugs with broad spectrum activity and a novel mechanism of action.

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.

Chagas disease affects an estimated 300,000 individuals in the United States. Diagnosis in the chronic phase requires positive results from two different IgG serological tests. Three enzyme-linked immunosorbent assays (ELISAs) (Hemagen, Ortho, and Wiener) and one rapid test (InBios) are FDA cleared, but comparative data in U.S. populations are sparse. We evaluated 500 seropositive and 300 seronegative blood donor plasma samples., Chagas disease affects an estimated 300,000 individuals in the United States. Diagnosis in the chronic phase requires positive results from two different IgG serological tests. Three enzyme-linked immunosorbent assays (ELISAs) (Hemagen, Ortho, and Wiener) and one rapid test (InBios) are FDA cleared, but comparative data in U.S. populations are sparse. We evaluated 500 seropositive and 300 seronegative blood donor plasma samples. Country of birth was known for 255 seropositive specimens, which were grouped into regions as follows: Mexico (n = 94), Central America (n = 88), and South America (n = 73). Specimens were tested by the four FDA-cleared IgG serological assays. Test performance was evaluated by two comparators and latent class analysis. InBios had the highest sensitivity (97.4% to 99.3%) but the lowest specificity (87.5% to 92.3%). Hemagen had the lowest sensitivity (88.0% to 92.0%) but high specificity (99.0% to 100.0%). The level of sensitivity was intermediate for Ortho (92.4% to 96.5%) and Wiener (94.0% to 97.1%); both had high specificity (98.8% to 100.0% and 96.7% to 99.3%, respectively). The levels of antibody reactivity and clinical sensitivity were lowest in donors from Mexico, intermediate in those from Central America, and highest in those from South America. Our findings provide an initial evidence base to improve laboratory diagnosis of Chagas disease in the United States. The best current testing algorithm would employ a high-sensitivity screening test followed by a high-specificity confirmatory test.

BackgroundChagas disease affects an estimated 300,000 individuals in the US. Diagnosis in the chronic phase requires positive results by two different IgG serological tests. Three ELISAs (Hemagen, Ortho, Wiener) and one rapid test (InBios) are FDA-cleared, but comparative data in US populations are sparse.MethodsWe evaluated 500 seropositive and 300 seronegative blood donor plasma samples. Country of birth was known for 255 seropositive specimens and grouped into regions: Mexico (n=94), Central America (n=88) and South America (n=73). Specimens were tested by the four FDA-cleared IgG serological assays. Test performance was evaluated by two comparators and latent class analysis.ResultsInBios had the highest sensitivity (97.4-99.3%), but lowest specificity (87.5-92.3%). Hemagen had the lowest sensitivity (88.0-92.0%), but high specificity (99.0-100.0%). Sensitivity was intermediate for Ortho (92.4-96.5%) and Wiener (94.0-97.1%); both had high specificity (98.8-100.0% and 96.7-99.3%, respectively). Antibody reactivity and clinical sensitivity was lowest in donors from Mexico, intermediate in those from Central America and highest in those from South America.ConclusionsOur findings provide an initial evidence base to improve laboratory diagnosis of Chagas disease in the US. The best current testing algorithm would employ a high sensitivity screening test followed by a high specificity confirmatory test.

Background Onchocerciasis currently afflicts an estimated 15 million people and is the second leading infectious cause of blindness world-wide. The development of a macrofilaricide to cure the disease has been hindered by the lack of appropriate small laboratory animal models. This study therefore, was aimed at developing and validating the Mongolian gerbil, as an Onchocerca ochengi (the closest in phylogeny to O. volvulus) adult male worm model. Methodology/Principal findings Mongolian gerbils (Meriones unguiculatus) were each implanted with 20 O. ochengi male worms (collected from infected cattle), in the peritoneum. Following drug or placebo treatments, the implanted worms were recovered from the animals and analyzed for burden, motility and viability. Worm recovery in control gerbils was on average 35%, with 89% of the worms being 100% motile. Treatment of the gerbils implanted with male worms with flubendazole (FBZ) resulted in a significant reduction (p = 0.0021) in worm burden (6.0% versus 27.8% in the control animals); all recovered worms from the treated group had 0% worm motility versus 91.1% motility in control animals. FBZ treatment had similar results even after four different experiments. Using this model, we tested a related drug, oxfendazole (OFZ), and found it to also significantly (p = 0.0097) affect worm motility (22.7% versus 95.0% in the control group). Conclusions/Significance We have developed and validated a novel gerbil O. ochengi adult male worm model for testing new macrofilaricidal drugs in vivo. It was also used to determine the efficacy of oxfendazole in vivo., Author summary Onchocerciasis is a neglected tropical disease caused by the filarial nematode Onchocerca volvulus. The only drug currently used for the treatment of this disease and reduction of transmission is ivermectin. However, this drug kills only the microfilarial stage of the parasite, and not the adult worm, and cases of parasite resistance to the drug have been recorded. Therefore, there is an urgent need to find an efficacious macrofilaricidal drug that targets the adult worms. To identify an effective macrofilaricide, suitable small laboratory animal models are needed for routine drug screening and/or validation in vivo. To develop such a model, we implanted Mongolian gerbils with adult O. ochengi male worms; O. ochengi is the closest filarial relative of O. volvulus. Gerbils appeared to be permissive to O. ochengi male worms and when treated with flubendazole, a known macrofilaricidal drug, the drug significantly decreased worm burden, motility and viability when compared to vehicle-treated animals. This model was also used to test the efficacy of oxfendazole, a drug belonging to same class as flubendazole, and it also showed potent activity on O. ochengi male worms. The gerbil O. ochengi male worm model is now available to screen new candidate macrofilaricides.

River blindness and lymphatic filariasis are two filarial diseases that globally affect millions of people mostly in impoverished countries. Current mass drug administration programs rely on drugs that primarily target the microfilariae, which are released from adult female worms. The female worms can live for several years, releasing millions of microfilariae throughout the course of infection. Thus, to stop transmission of infection and shorten the time to elimination of these diseases, a safe and effective drug that kills the adult stage is needed. The benzimidazole anthelmintic flubendazole (FBZ) is 100% efficacious as a macrofilaricide in experimental filarial rodent models but it must be administered subcutaneously (SC) due to its low oral bioavailability. Studies were undertaken to assess the efficacy of a new oral amorphous solid dispersion (ASD) formulation of FBZ on Brugia pahangi infected jirds (Meriones unguiculatus) and compare it to a single or multiple doses of FBZ given subcutaneously. Results showed that worm burden was not significantly decreased in animals given oral doses of ASD FBZ (0.2–15 mg/kg). Regardless, doses as low as 1.5 mg/kg caused extensive ultrastructural damage to developing embryos and microfilariae (mf). SC injections of FBZ in suspension (10 mg/kg) given for 5 days however, eliminated all worms in all animals, and a single SC injection reduced worm burden by 63% compared to the control group. In summary, oral doses of ASD formulated FBZ did not significantly reduce total worm burden but longer treatments, extended takedown times or a second dosing regimen, may decrease female fecundity and the number of mf shed by female worms., Author summary Safe and effective macrofilaricidal drugs are critically needed to treat onchocerciasis and lymphatic filariasis, which affect over 54 million people worldwide. Flubendazole (FBZ) in its current commercial formulations is an effective anthelminthic for intestinal soil transmitted helminth (STH) infections but not for filarial infections due to its low bioavailability. The purpose of this study was to assess the efficacy of a new amorphous solid dispersion (ASD) formulation of FBZ given orally to jirds (Meriones unguiculatus) infected with the filarial nematode Brugia pahangi and compare it to FBZ (in suspension) given subcutaneously as a single or multiple dose. Our results indicated that treatment with ASD FBZ did not significantly reduce the total number of worms. However, doses as low as 1.5 mg/kg caused ultrastructural damage to the early stages of developing embryos. No worms were recovered from jirds given 10 mg/kg SC injections of FBZ (suspension) for 5 days when necropsied 68 days after the first-dose, and the number of mf at necropsy was significantly decreased compared to the control group. Animals given only a single 10 mg/kg SC injection had a 63% decrease in the number of adult worms. These animals also had fewer female worms and mf compared to the control group suggesting that even a single SC injection of FBZ had an effect on female survival and fecundity. TEM of worms recovered from jirds given low doses of the ASD formulated FBZ (1.5 mg/kg) suggested that female fecundity and mf production were reduced, but longer treatments, longer takedown times or a second dosing regimen of ASD FBZ may be needed to significantly decrease the total worm burden.

Balamuthia mandrillaris is responsible for hundreds of reported cases of amoebic encephalitis, the majority of which have been fatal. Despite being an exceptionally deadly pathogen, B. mandrillaris is understudied, leaving many open questions regarding epidemiology, diagnosis, and treatment. Due to the lack of effective drugs to fight B. mandrillaris infections, mortality rates remain high even for patients receiving intensive care. This report addresses the need for new treatment options through a drug repurposing screen to identify novel B. mandrillaris inhibitors. The most promising candidate identified was the quinoline antibiotic nitroxoline, which has a long history of safe use in humans. We show that nitroxoline kills B. mandrillaris at pharmacologically relevant concentrations and exhibits greater potency and selectivity than drugs commonly used in the current standard of care. The findings that we present demonstrate the potential of nitroxoline to be an important new tool in the treatment of life-threatening B. mandrillaris infections., Balamuthia mandrillaris is a pathogenic free-living amoeba that causes a rare but almost always fatal infection of the central nervous system called granulomatous amoebic encephalitis (GAE). Two distinct forms of B. mandrillaris—a proliferative trophozoite form and a nonproliferative cyst form, which is highly resistant to harsh physical and chemical conditions—have been isolated from environmental samples worldwide and are both observed in infected tissue. Patients suffering from GAE are typically treated with aggressive and prolonged multidrug regimens that often include the antimicrobial agents miltefosine and pentamidine isethionate. However, survival rates remain low, and studies evaluating the susceptibility of B. mandrillaris to these compounds and other potential therapeutics are limited. To address the need for more-effective treatments, we screened 2,177 clinically approved compounds for in vitro activity against B. mandrillaris. The quinoline antibiotic nitroxoline (8-hydroxy-5-nitroquinoline), which has safely been used in humans to treat urinary tract infections, was identified as a lead compound. We show that nitroxoline inhibits both trophozoites and cysts at low micromolar concentrations, which are within a pharmacologically relevant range. We compared the in vitro efficacy of nitroxoline to that of drugs currently used in the standard of care for GAE and found that nitroxoline is the most potent and selective inhibitor of B. mandrillaris tested. Furthermore, we demonstrate that nitroxoline prevents B. mandrillaris-mediated destruction of host cells in cultured fibroblast and primary brain explant models also at pharmacologically relevant concentrations. Taken together, our findings indicate that nitroxoline is a promising candidate for repurposing as a novel treatment of B. mandrillaris infections.

Balamuthia mandrillarisis a pathogenic free-living amoeba that causes a rare but almost always fatal infection of the central nervous system called granulomatous amoebic encephalitis (GAE). Two distinct forms ofB. mandrillaris– a proliferative trophozoite form and a non-proliferative cyst form, which is highly resistant to harsh physical and chemical conditions – have been isolated from environmental samples worldwide and are both observed in infected tissue. Patients suffering from GAE are typically treated with aggressive and prolonged multi-drug regimens often including the antimicrobial agents miltefosine and pentamidine isethionate. However, survival rates remain low and studies evaluating the susceptibility ofB. mandrillaristo these compounds and other potential therapeutics are limited. To address the need for more effective treatments, we screened 2,177 clinically-approved compounds forin vitroactivity againstB. mandrillaris. The quinoline antibiotic nitroxoline, which has safely been used in humans to treat urinary tract infections, was identified as a lead compound. We show that nitroxoline inhibits both trophozoites and cysts at low micromolar concentrations, which are within a physiologically relevant range. We compare thein vitroefficacy of nitroxoline to drugs currently used in the standard of care for GAE and find that nitroxoline is the most potent and selective inhibitor ofB. mandrillaristested. Furthermore, we demonstrate that nitroxoline preventsB. mandrillaris-mediateddestruction of host cells in cultured fibroblast and primary brain explant models also at physiologically relevant concentrations. Together, our findings indicate that nitroxoline is a promising candidate for repurposing as a novel treatment ofB. mandrillarisinfections.ImportanceBalamuthia mandrillarisis responsible for hundreds of reported cases of amoebic encephalitis, the majority of which have been fatal. Despite being an exceptionally deadly pathogen,B. mandrillarisis understudied, leaving many open questions regarding epidemiology, diagnosis, and treatment. Due to the lack of effective drugs to fightB. mandrillarisinfections, mortality rates remain high even for patients receiving intensive care. This study addresses the need for new anti-amoebic drugs using a high-throughput screening approach to identify novelB. mandrillarisinhibitors. The most promising candidate identified was the quinoline antibiotic nitroxoline, which has a long history of safe use in humans. We show that nitroxoline killsB. mandrillarisat physiologically relevant concentrations and exhibits greater potency and selectivity than drugs commonly used in the current standard of care. The findings we present demonstrate the potential of nitroxoline to be an important new tool in the treatment of life threateningB. mandrillarisinfections.

The last half-century has provided all of the (few) drugs currently used to treat human helminthiases. Concern regarding the long-term utility of these drugs, given how readily resistance evolves in the veterinary-agricultural sector, spurs the discovery of new chemical entities. We review the approaches and technologies in use to identify anthelmintics and discuss a number of drug discovery paradigms that may prove pivotal to the next half-century of anthelmintic development.

Cysteine proteinases play important roles in the pathogenesis of several parasitic infections and have been proposed as targets for the structure-based approach of drug design. As the first step toward applying this strategy to design inhibitors as antiparasitic agents for Clonorchis sinensis, we overexpressed and characterized the 24-kDa cysteine proteinase from adult worms. First, the partial cysteine proteinase gene from C. sinensis was cloned by performing reverse transcription polymerase chain reaction (RT-PCR) with degenerate oligonucleotide primers derived from conserved cysteine proteinase sequences. The 5' and the 3' regions of the cysteine proteinase gene were amplified using the PCR protocol for the rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The cDNA has an open reading frame of 981 bp, and the deduced amino acid sequence shares similarity with the cathepsin L-like cysteine proteinases from Schistosoma mansoni, Paragonimus westermani metacercaria, Fasciola hepatica, and human cathepsin L by 52%, 47%, 34%, and 29%, respectively. The cysteine proteinase was then overexpressed in the yeast Pichia pastoris as an active enzyme on a large-scale basis (19.7 mg/L). The active recombinant enzyme was purified from culture media using a Ni2+-NTA-agarose affinity column and gel filtration chromatography. This 24-kDa recombinant protein exhibited a substrate preference for Z-Phe-Arg-AMC (benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amino-4-methyl-coumarin) compared with Z-Arg-Arg-AMC, and the activity was inhibited by E-64 (L-trans-epoxysuccinylleucylamido(4-quanidino)butane).

The mature form of the cathepsin B-like protease of Leishmania major (LmajcatB) is a 243 amino acid protein belonging to the papain family of cysteine proteases and is 54% identical to human-liver cathepsin B. Despite the high identity and structural similarity with cathepsin B, LmajcatB does not readily hydrolyse benzyloxycarbonyl-Arg-Arg-7-amino-4-methyl coumarin (Z-Arg-Arg-AMC), which is cleaved by cathepsin B enzymes. It does, however, hydrolyse Z-Phe-Arg-AMC, a substrate typically cleaved by cathepsin L and B enzymes. Based upon computer generated protein models of LmajcatB and mammalian cathepsin B, it was predicted that this variation in substrate specificity was attributed to Gly234 at the S2 subsite of LmajcatB, which forms a larger, more hydrophobic pocket compared with mammalian cathepsin B. To test this hypothesis, recombinant LmajcatB was expressed in the Pichia pastoris yeast expression system. The quality of the recombinant enzyme was confirmed by kinetic characterization, N-terminal sequencing, and Western blot analysis. Alteration of Gly234 to Glu, which is found at the corresponding site in mammalian cathepsin B, increased recombinant LmajcatB (rLmajcatB) activity toward Z-Arg-Arg-AMC 8-fold over the wild-type recombinant enzyme (kcat/Km = 3740±413 M-1·s-1 versus 472±72.4 M-1·s-1). The results of inhibition assays of rLmajcatB with an inhibitor of cathepsin L enzymes, K11002 (morpholine urea-Phe-homoPhe-vinylsulphonylphenyl, kinact/Ki = 208200±36000 M-1·s-1), and a cathepsin B specific inhibitor, CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-ʟ-isoleucyl-L-proline, kinact/Ki = 199200±32900 M-1·s-1], support the findings that this protozoan protease has the P2 specificity of cathepsin L-like enzymes while retaining structural homology to mammalian cathepsin B.

Lu, C-C., Nguyen, T., Morris, S., Hill, D., and Sakanari, J. A. 1998. Anisakis simplex: Mutation bursts in the Reactive site centers of serine protease inhibitors from an ascarid nematode. Experimental Parasitology 89 , 257–261.

Cysteine proteases play important roles in the pathogenesis of several parasitic infections and have been proposed as targets for the structure-based strategy of drug design. As a first step toward applying this strategy to design inhibitors as antiparasitic agents for leishmaniasis, we have isolated and sequenced the full-length clones of two cysteine protease genes from Leishmania major. One of the genes is structurally similar to the cathepsin L-like family and the other is similar to the cathepsin B-like family of cysteine proteases. The L. major cathepsin L-like sequence has a proregion that shares high sequence similarity with other cathepsin L sequences but not cathepsin B sequences and has a proline/threonine-rich C-terminal extension. The cathepsin L-like gene occurs in multiple copies, whereas there may be only one copy of the cathepsin B-like gene. Northern blot analyses show that both genes are expressed in the promastigote and amastigote stages, and pulse field gel electrophoresis revealed that the cathepsin L- and B-like genes are each found on two nonhomologous chromosomes. The L. major L-like amino acid sequence is 75% identical to the L. mexicana sequence, 74% identical to the L. pifanoi sequence, 47% identical with the Trypanosoma cruzi sequence, 47% identical with the T. congolense sequence, and 45% identical with the T. brucei sequence. L. major is one of two trypanosomatid species for which a cathepsin B-like gene has been identified and sequenced; its amino acid sequence is 82% identical to the one from L. mexicana. Tree inference based on distance and parsimony methods of kinetoplastid cathepsin L proteins yielded independent support for phylogenetic hypotheses inferred from analyses of ribosomal RNA genes. Because the cathepsin L locus has a high level of phylogenetic signal with respect to trypanosomatid taxa, this locus has great potential utility for investigating the evolutionary history of trypanosomatids and related organisms.

Cryptosporidiosis, caused by the protozoan parasite Cryptosporidium parvum , can stunt infant growth and can be lethal in immunocompromised individuals. The most widely used drugs for treating cryptosporidiosis are nitazoxanide and paromomycin, although both exhibit limited efficacy. To investigate an alternative approach to therapy, we demonstrate that the clan CA cysteine protease inhibitor N -methyl piperazine-Phe-homoPhe-vinylsulfone phenyl (K11777) inhibits C. parvum growth in mammalian cell lines in a concentration-dependent manner. Further, using the C57BL/6 gamma interferon receptor knockout (IFN-γR-KO) mouse model, which is highly susceptible to C. parvum , oral or intraperitoneal treatment with K11777 for 10 days rescued mice from otherwise lethal infections. Histologic examination of untreated mice showed intestinal inflammation, villous blunting, and abundant intracellular parasite stages. In contrast, K11777-treated mice (210 mg/kg of body weight/day) showed only minimal inflammation and no epithelial changes. Three putative protease targets (termed cryptopains 1 to 3, or CpaCATL-1, -2, and -3) were identified in the C. parvum genome, but only two are transcribed in infected mammals. A homology model predicted that K11777 would bind to cryptopain 1. Recombinant enzymatically active cryptopain 1 was successfully targeted by K11777 in a competition assay with a labeled active-site-directed probe. K11777 exhibited no toxicity in vitro and in vivo , and surviving animals remained free of parasites 3 weeks after treatment. The discovery that a cysteine protease inhibitor provides potent anticryptosporidial activity in an animal model of infection encourages the investigation and development of this biocide class as a new, and urgently needed, chemotherapy for cryptosporidiosis.

Background Cercarial elastase is the major invasive larval protease in Schistosoma mansoni, a parasitic blood fluke, and is essential for host skin invasion. Genome sequence analysis reveals a greatly expanded family of cercarial elastase gene isoforms in Schistosoma mansoni. This expansion appears to be unique to S. mansoni, and it is unknown whether gene duplication has led to divergent protease function. Methods Profiling of transcript and protein expression patterns reveals that cercarial elastase isoforms are similarly expressed throughout the S. mansoni life cycle. Computational modeling predicts key differences in the substrate-binding pockets of various cercarial elastase isoforms, suggesting a diversification of substrate preferences compared with the ancestral gene of the family. In addition, active site labeling of SmCE reveals that it is activated prior to exit of the parasite from its intermediate snail host. Conclusions The expansion of the cercarial gene family in S. mansoni is likely to be an example of gene dosage. In addition to its critical role in human skin penetration, data presented here suggests a novel role for the protease in egress from the intermediate snail host. This study demonstrates how enzyme activity-based analysis complements genomic and proteomic studies, and is key in elucidating proteolytic function., Author Summary Schistosome parasites are a major cause of disease in the developing world. The larval stage of the parasite transitions between an intermediate snail host and a definitive human host in a dramatic fashion, burrowing out of the snail and subsequently penetrating human skin. This process is facilitated by secreted proteases. In Schistosoma mansoni, cercarial elastase is the predominant secreted protease and essential for host skin invasion. Genomic analysis reveals a greatly expanded cercarial elastase gene family in S. mansoni. Despite sequence divergence, SmCE isoforms show similar expression profiles throughout the S. mansoni life cycle and have largely similar substrate specificities, suggesting that the majority of protease isoforms are functionally redundant and therefore their expansion is an example of gene dosage. However, activity-based profiling also indicates that a subset of SmCE isoforms are activated prior to the parasite's exit from its intermediate snail host, suggesting that the protease may also have a role in this process.

Lymphatic filariasis is caused by filarial nematode parasites, including Brugia malayi. Adult worms live in the lymphatic system and cause a strong immune reaction that leads to the obstruction of lymph vessels and swelling of the extremities. Chronic disease leads to the painful and disfiguring condition known as elephantiasis. Current drug therapy is effective against the microfilariae (larval stage) of the parasite, but no drugs are effective against the adult worms. One of the major stumbling blocks toward developing effective macrofilaricides to kill the adult worms is the lack of a high throughput screening method for candidate drugs. Current methods utilize systems that measure one well at a time and are time consuming and often expensive. We have developed a low-cost and simple visual imaging system to automate and quantify screening entire plates based on parasite movement. This system can be applied to the study of many macroparasites as well as other macroscopic organisms., Author Summary The World Health Organization estimates that there are approximately 37 million people who are afflicted by Onchocerca volvulus (the parasitic worm that causes river blindness) and over 120 million people afflicted by the filarial worms Wuchereria and Brugia spp. (causative agents of lymphatic filariasis or elephantiasis). Current mass drug administration includes albendazole and either diethylcarbamazine or ivermectin. These drugs, however, are effective at killing the early larval stage (microfilariae) released from adult female worms but they do not kill the adult worms. Adult worms can live up to 10 or more years, releasing thousands of microfilariae per day. It is essential therefore to treat infected individuals with macrofilaricides in order to prevent the adult parasites from producing microfiliariae for the duration of the infection and to treat the disease. In order to screen candidate drugs for use as macrofilaricides, we have developed an inexpensive system and simple method for quantifying the effectiveness of drugs on parasite movement. The apparatus uses a commodity video camera, a computer and a newly developed free and open source software application to provide automated and quantitative measurements of parasite motility on each plate of worms. This system is not only useful for high throughput screening of macroparasites but can also be applied to the study of other macroscopic organisms as well.

A serine protease and a serine protease inhibitor were purified from infective larvae of the parasitic nematode Anisakis simplex. The serine protease was found to be trypsin-like and preferentially cleaved substrates with the basic amino acid arginine at the P1 position (Z-Gly-Pro-Arg-AMC (where Z is benzyloxycarbonyl), Km = 0.019 mM, and Z-Phe-Pro-Arg-AMC, Km = 0.013 mM) at rates similar to those determined for trypsin (0.002 mM and 0.006 mM, respectively). However, the presence of a bulky hydrophobic residue at the P2 position (Z-Phe-Arg-AMC, Km = 13.3 mM, and Z-Ile-Leu-Val-Arg-AMC, Km = 24.7 mM) greatly decreased the rate of substrate hydrolysis. Internal amino acid sequence information was obtained from three endo Lys-C digestion fragments of the purified enzyme. These sequences were > 89% (33:37) identical with that of porcine trypsin. A second serine protease 85% (11:13) identical with that of a secreted tissue-destructive serine protease from the pathogenic bacterium Dichelobacter nodosus was also identified. The serine protease inhibitor was found to inhibit trypsin, elastase, and the Anisakis serine protease stoichiometrically, but did not inhibit chymotrypsin. The amino acid sequence of the amino terminus as well as two internal endo Lys-C fragments were determined. Approximately 96% (47:49) of the residues were identical with soybean trypsin inhibitor, indicating that this inhibitor belongs to the Kunitz-type family of inhibitors.

The gastrointestinal invasive stages of two parasitic nematodes, Ancylostoma caninum and Anisakis simplex, were each found to release in vitro a hydrolytic enzyme that degrades the glycosaminoglycan hyaluronic acid. The parasite hyaluronidases were partially purified by ion-exchange chromatography and biochemically characterized. The hyaluronidase from A. caninum adult worms had a molecular weight of 65,000 and a pH optimum of 6 with activity at neutral pH, while the hyaluronidase from A. simplex larvae had a molecular weight of 40,000 and a pH optimum of 4 with no activity at neutral pH. Both parasite hyaluronidases also degraded the glycosaminoglycan chondroitin sulfate A. Cupric sulfate and high concentrations of sodium chloride were inhibitory. The nematode hyaluronidases are postulated to have a role in tissue histolysis and mucosal invasion; their distinct biochemical properties have relevance to the pathogenesis of the zoonoses anisakiasis and eosinophilic enteritis.

D. immitis third-stage larvae (L3) were cultured with fluoromethyl ketone cysteine protease inhibitors. By Day 5 in culture, none of the larvae cultured with 0.1, 0.2, 0.6, or 1.0 mM benzyloxycarbonyl–Phe–Ala–CH2F (Z– Phe–Ala–CH2F) has molted, while 63.2% of larvae in media without inhibitor had molted. At the two lower concentrations of inhibitor more larvae had initiated, but not completed, the molt. In addition to Z–Phe–Ala–CH2F, four other fluoromethyl ketone derivatives, Z–Phe– Arg–CH2F, amorpholine urea–(Mu)–Leu–Phe–CH2F, Mu–Tyr–Phe–CH2F, and Mu–Phe– Phe–CH2F, were tested to determine their effects on L3 in culture. All fluoromethyl ketones tested except Z–Phe–Arg–CH2F inhibited molting. Larvae cultured in inhibitors were determined to be alive as judged qualitatively by motility and quantitatively by reduction of 3-(4,5-diethylthiazol-2-yl)-2.5-diphenyltetrazolium. Electron microscopy demonstrated that L3 which were unable to molt after being cultured in a fluoromethyl ketone derivative had synthesized the new fourth-stage (L4) cuticle but had not shed the L3 cuticle. The same fluoromethyl ketone derivative that did not inhibit molting, Z–Phe–Arg–CH2F, was a slightly less effective inhibitor of larval extract-initiated hydrolysis of the synthetic peptide substrate, Z–Val–Leu–Arg–7-amino-4-methyl coumarin. L3 were also cultured through the molt in media containing the synthetic peptide substrate Z–Val–Leu–Arg–4-methoxy-B-naphthylamide to examine cysteine protease activity in situ. Fuorescence as seen on Days 0–4 during the molting process was first observed on the anterior tip of the larvae, and subsequently in the pharynx, with progression down the L4 as it shed the L3 cuticle.

Ecotin, a serine protease inhibitor found in the periplasm of Escherichia coli, is unusual in its ability to inhibit chymotrypsin, trypsin, and elastase. To address the structural basis of its broad specificity, the gene for ecotin has been cloned and its sequence determined. A promoter of the 17-base pair spacing class was identified, and the probable transcriptional start site lies 18 base pairs upstream from a ribosome binding locus. The gene is followed by a series of conserved repetitive extragenic palindromic sequences. Ecotin has a signal peptide of 20 amino acids which confirms its periplasmic localization. Sequence analyses by Edman degradation and mass spectrometry confirmed 71% of the deduced protein sequence of calculated monomeric molecular mass 16,096 Da. Comparisons of the primary structure for the 142-amino acid protein with the major classes of serine protease inhibitors suggest that ecotin is a novel inhibitor. The reactive site of ecotin was determined to be Met84 for its complexes with chymotrypsin, trypsin, and elastase. The scissile Met84-Met85 bond lies within a disulfide-bonded protein segment similar to other classes of inhibitors.

Giardia lamblia is a protozoan parasite and the earliest branching clade of eukaryota. The Giardia life cycle alternates between an asexually replicating vegetative form and an infectious cyst form. Encystation and excystation are crucial processes for the survival and transmission of Giardia. Cysteine proteases in Giardia have been implicated in proteolytic processing events that enable the continuance of the life cycle throughout encystation and excystation. Using quantitative real-time PCR, the expression of twenty-seven clan CA cysteine protease genes in the Giardia genome was measured during both vegetative growth and encystation. Giardia cysteine protease 2 was the most highly expressed cysteine protease during both life cycle stages measured, with a dramatic expression increase during encystation. The mRNA transcript for Giardia cysteine protease 2 was 7-fold up-regulated during encystation and was greater than 3-fold higher than any other Giardia protease gene product. Recombinant Giardia cysteine protease 2 was expressed, purified, and biochemically characterized. The activity of the recombinant cysteine protease 2 protein was confirmed to be identical to the dominant cysteine protease activity found in G. lamblia lysates. Giardia cysteine protease 2 was co-localized with cyst wall protein in encystation-specific vesicles during encystation and processed cyst wall protein 2 to the size found in Giardia cyst walls. These data suggest that Giardia cysteine protease 2 is not only the major cysteine endoprotease expressed in Giardia, but is also central to the encystation process.

Anisakiasis is a disease caused by the ingestion of anisakid nematodes in raw or improperly prepared fish dishes. Invading larvae penetrate the mucosa and submucosa of the gastrointestinal tract and produce lesions characterized by a marked inflammatory response. Judy Sakanari describes the biochemical and molecular studies on the proteins excreted and/or secreted by Anisakis larvae that are currently underway to help elucidate the role these proteins may play in the invasion process and the pathogenesis of anisakiasis.

Two major human diseases caused by filariid nematodes are onchocerciasis, or river blindness, and lymphatic filariasis, which can lead to elephantiasis. The drugs ivermectin, diethylcarbamazine (DEC), and albendazole are used in control programs for these diseases, but are mainly effective against the microfilarial stage and have minimal or no effect on adult worms. Adult Onchocerca volvulus and Brugia malayi worms (macrofilariae) can live for up to 15 years, reproducing and allowing the infection to persist in a population. Therefore, to support control or elimination of these two diseases, effective macrofilaricidal drugs are necessary, in addition to current drugs. In an effort to identify macrofilaricidal drugs, we screened an FDA-approved library with adult worms of Brugia spp. and Onchocerca ochengi, third-stage larvae (L3s) of Onchocerca volvulus, and the microfilariae of both O. ochengi and Loa loa. We found that auranofin, a gold-containing drug used for rheumatoid arthritis, was effective in vitro in killing both Brugia spp. and O. ochengi adult worms and in inhibiting the molting of L3s of O. volvulus with IC50 values in the low micromolar to nanomolar range. Auranofin had an approximately 43-fold higher IC50 against the microfilariae of L. loa compared with the IC50 for adult female O. ochengi, which may be beneficial if used in areas where Onchocerca and Brugia are co-endemic with L. loa, to prevent severe adverse reactions to the drug-induced death of L. loa microfilariae. Further testing indicated that auranofin is also effective in reducing Brugia adult worm burden in infected gerbils and that auranofin may be targeting the thioredoxin reductase in this nematode., Author Summary Onchocerciasis or river blindness, and lymphatic filariasis, which can lead to disfiguring elephantiasis, are two neglected tropical diseases that affect millions of people, primarily in developing countries. Both diseases are caused by filariid nematodes; onchocerciasis is caused by Onchocerca volvulus and lymphatic filariasis is caused by Brugia malayi, B. timori, and Wuchereria bancrofti. Currently, there are no drugs available that are highly efficacious against adult worms; existing drugs mainly kill the first-stage larvae (microfilariae). While these drugs can reduce the transmission of infections in a population, the adult filariids (macrofilariae) can continue to produce microfilariae and perpetuate the cycle of infection. Finding a drug that could kill the adult worms would be an important tool in eliminating onchocerciasis and lymphatic filariasis. To identify potential macrofilaricidal drugs, we developed a high throughput screening method to test FDA-approved drugs on adult Brugia spp., which serves as a model for O. volvulus. Using this screening method, we identified a drug called auranofin that kills adult Onchocerca and adult Brugia spp. in vitro, inhibits the molting of O. volvulus L3s, and reduces the worm burden in an in vivo gerbil-B. pahangi model system. Auranofin is known to inhibit a critical enzyme called thioredoxin reductase in some parasite species, and subsequent testing of the effects of auranofin on the thioredoxin reductase of Brugia indicates that this may be auranofin’s mode of action in this nematode as well.

Individual specimens of Anisakis, Pseudoterranova, and Contracaecum collected from marine mammals inhabiting northern Pacific waters were used for comparative diagnostic and molecular phylogenetic analyses. Forty-eight new sequences were obtained for this study of 14 Anisakis taxa, 8 Pseudoterranova taxa, 4 Contracaecum taxa, and 4 outgroup species. Partial 28S (LSU) and complete internal transcribed spacer (ITS-1, 5.8S, ITS-2) ribosomal DNA was amplified by the polymerase chain reaction and sequenced. Sequences of ITS indicated that Pseudoterranova specimens from Zalophus californianus (California sea lion), Mirounga angustirostris (northern elephant seal), Phoca vitulina (harbor seal), Enhydra lutris (sea otter), and Eumetopias jubatus (Steller's sea lion) exactly matched P. decipiens s. str., extending the host and geographic range of this species. Anisakis from northern Pacific marine mammals were most closely related to members of the A. simplex species complex. Comparison of Anisakis ITS sequences diagnosed the presence of A. simplex C in 2 M. angustirostris hosts, which is a new host record. Anisakis specimens from Phocoena phocoena (harbor porpoise), Lissodelphis borealis (Pacific rightwhale porpoise), and E. jubatus included 3 ITS sequences that did not match any known species. Contracaecum adults obtained from Z. californianus were most closely related to C. ogmorhini s.l. and C. rudolphii, but ITS sequences of these Contracaecum specimens did not match C. ogmorhini s. str. or C. margolisi. These novel Anisakis and Contracaecum ITS sequences may represent previously uncharacterized species. Phylogenetic analysis of LSU sequences revealed strong support for the monophyly of Anisakinae, Contracaecum plus Phocascaris, Pseudoterranova, and Anisakis. Phylogenetic trees inferred from ITS sequences yielded robustly supported relationships for Pseudoterranova and Anisakis species that are primarily consistent with previously published phenograms based on multilocus electrophoretic data.

Park, H., Hong, K-M., Sakanari, J. A., Choi, J-H., Park, S-K., Kim, K-Y., Hwang, H-A., Paik, M-K., Yun, K-J., Shin, C-H., Lee, J-B., Ryu, J-S., Min, D-Y. 2001. Paragonimus westermani: Cloning of a cathepsin F-like cysteine proteinase from the adult worm. Experimental Parasitology98, 223 –227.

A cysteine proteinase gene homologous to cathepsins L genes was isolated from a B. microplus cDNA library. The precursor protein deduced from the nucleotide sequence contains 332 amino acid residues consisting of a signal sequence (pre-region), a pro-region and a mature proteinase. The DNA fragment coding for the proenzyme was cloned and expressed using the E. coli expression vector pMAL-p. The recombinant protein (MBP+PROCP) once activated is able to hydrolyze synthetic substrates as well as protein substrates like hemoglobin, vitellin and gelatin. Its optimal enzymatic activity on both fluorogenic and protein substrates was found to occur at an acidic pH. Expression of the proteinase gene was tested by RT-PCR with tick larvae RNA. Detection of amplified sequences indicates that the gene is expressed at this stage of the tick life cycle and the molecule is therefore potentially a target for chemotherapy or an immunogen in a vaccine.

Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture orin vivo. We now report that specific cysteine protease inhibitors killLeishmaniaparasitesin vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition ofLeishmaniacysteine protease activity was accompanied by defects in the parasite’s lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-protease antibodies with biotinylated surface proteins and accumulation of undigested debris and protease in the flagellar pocket of treated parasites were consistent with a pathway of protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stablein vivoto ameliorate the pathology associated with a mouse model ofLeishmaniainfection.

Two elastase inhibitors, ASPI-1 and ASPI-2, from the parasitic nematode Anisakis simplex , have been isolated and characterized. Because these inhibitors are similar in size (60 amino acids in length) and primary sequence (52 and 47% identical) to the Ascaris suum chymotrypsin/elastase inhibitor-1 (AsC/E-1), we suggest that these Anisakis elastase inhibitors belong to the same unique class of canonical inhibitors formed by the family of Ascaris inhibitors (Huang K, Strynadka NCJ, Bernard VD, Peanasky RJ, James MG. Structure 1994;2:679–689). To compare ASPI-1 with AsC/E-1, we expressed both inhibitors in Pichia pastoris and found that: (1) the association constant of rASPI-1 with porcine pancreatic elastase (PPE) is similar to native inhibitor ( K a =4.5×10 9 and 6.4×10 9 M −1 , respectively); (2) rASPI-1 is a potent inhibitor of PPE and human leukocyte elastase ( K a =1.6×10 9 M −1 ); and (3) it is only a very weak inhibitor of chymotrypsin (CHYM) ( K a =1.2×10 6 M −1 ). In contrast to the Anisakis inhibitor, however, rAsC/E inhibitor-1 is a very strong inhibitor of both PPE ( K a =3.5×10 10 M −1 ) and CHYM ( K a =3.6×10 12 M −1 ). We also found that the determined reactive sites (P 1 –P 1 ′) of rASPI-1 and rAsC/E-1, as recognized by PPE, are Ala 28-Met 29 and Leu 31-Met 32, respectively. These P 1 –P 1 ′ residues of AsC/E-1 constitute the same reactive site as that also recognized by CHYM (Peanasky RJ, Bentz Y, Homandberg GA, Minor ST, Babin DR. Arch Biochem Biophys 1994;232:135–142). The difference in specificities of ASPI-1 and AsC/E-1 toward their cognate serine proteases may be attributed to the P 1 and P 3 ′ residues in the inhibitors. Elastase, which recognizes both alanine and leucine, can accommodate both ascarid inhibitors, whereas chymotrypsin, which prefers bulky, hydrophobic residues, only recognizes the Ascaris C/E inhibitor-1.

The crystal structures of papain, cruzain, and human liver cathepsin B were used to build homology-based enzyme models of a cathepsin L-like cysteine protease (cpL) and a cathepsin B-like cysteine protease (cpB) from the protozoan parasite Leishmania major. Although structurally a member of the cathepsin B subfamily, the L. major cpB is not able to cleave synthetic substrates having an arginine in position P2. This biochemical property correlates with the prediction of a glycine instead of a glutamic acid at position 205 (papain numbering). The modeled active sites of the L. major cpB and cpL were used to screen the Available Chemicals Directory (a database of about 150,000 commercially available compounds) for potential cysteine protease inhibitors, using DOCK3.5. Based on both steric and force field considerations, 69 compounds were selected. Of these, 18 showed IC50's between 50 and 100 microM and 3 had IC50's below 50 microM. A secondary library of compounds, originally derived from a structural screen against the homologous protease of Plasmodium falciparum (falcipain), and subsequently expanded by combinatorial chemistry, was also screened. Three inhibitors were identified which were not only effective against the L. major protease but also inhibited parasite growth at 5-50 microM.

Background Skin invasion is the initial step in infection of the human host by schistosome blood flukes. Schistosome larvae have the remarkable ability to overcome the physical and biochemical barriers present in skin in the absence of any mechanical trauma. While a serine peptidase with activity against insoluble elastin appears to be essential for this process in one species of schistosomes, Schistosoma mansoni, it is unknown whether other schistosome species use the same peptidase to facilitate entry into their hosts. Methods Recent genome sequencing projects, together with a number of biochemical studies, identified alternative peptidases that Schistosoma japonicum or Trichobilharzia regenti could use to facilitate migration through skin. In this study, we used comparative proteomic analysis of human skin treated with purified cercarial elastase, the known invasive peptidase of S. mansoni, or S. mansoni cathespin B2, a close homolog of the putative invasive peptidase of S. japonicum, to identify substrates of either peptidase. Select skin proteins were then confirmed as substrates by in vitro digestion assays. Conclusions This study demonstrates that an S. mansoni ortholog of the candidate invasive peptidase of S. japonicum and T. regenti, cathepsin B2, is capable of efficiently cleaving many of the same host skin substrates as the invasive serine peptidase of S. mansoni, cercarial elastase. At the same time, identification of unique substrates and the broader species specificity of cathepsin B2 suggest that the cercarial elastase gene family amplified as an adaptation of schistosomes to human hosts., Author Summary Schistosome parasites are a major cause of disease in the developing world, but the mechanism by which these parasites first infect their host has been studied at the molecular level only for S. mansoni. In this paper, we have mined recent genome annotations of S. mansoni and S. japonicum, a zoonotic schistosome species, to identify differential expansion of peptidase gene families that may be involved in parasite invasion and subsequent migration through skin. Having identified a serine peptidase gene family in S. mansoni and a cysteine peptidase gene family in S. japonicum, we then used a comparative proteomic approach to identify potential substrates of representative members of both classes of enzymes from S. mansoni in human skin. The results of this study suggest that while these species evolved to use different classes of peptidases in host invasion, both are capable of cleaving components of the epidermis and dermal extracellular matrix, as well as proteins involved in the host immune response against the migrating parasite.

A model of cutaneous extracellular matrix was used to determine if live Dirofilaria immitis larvae secrete proteases which are active at physiological pH and capable of degrading macromolecules found in cutaneous tissue. After 72 hr, 100 third-stage larvae (L3) degraded 24% of the total matrix, while fourth-stage larvae (L4) degraded 10%. A sharp increase in the amount of matrix degraded by L3 corresponded with the onset of the molting process. L3 and L4 degraded comparable amounts of the glycoprotein and elastin components of the matrix, but molting L3 degraded nearly twice the amount of the collagen component (62% vs 35%). Characterization of proteases present in larval-soluble extracts and excretory-secretory products using synthetic substrates and protease inhibitors demonstrated cysteine-protease and metalloprotease activity. Cysteine protease activity was found in whole worm extracts of both L3 and L4. Metalloprotease was secreted at higher levels by molting L3, but was also secreted by L4. Partial separation of the metalloprotease by size-exclusion chromatography indicated that the molecular weight of the native enzyme was in the 49-54 kDa range. The cysteine protease activity was demonstrated in fractions corresponding to 34-39 kDa. The biological function of the D. immitis larval proteases remains to be conclusively determined; however, these data suggest that they are involved in degradation of components of cutaneous tissue and in the molting process.

Cysteine protease gene fragments from three protozoan parasites Trypanosoma cruzi, Trypanosoma brucei, and Entamoeba histolytica were amplified by the polymerase chain reaction (PCR) from genomic DNA using degenerate oligonucleotide primers. The primers used for the amplification were designed based upon amino acid sequences flanking the active site cysteine and asparagine residues that are conserved in the eukaryotic cysteine proteases analyzed to date. The amplified DNA fragments, representing approximately 70% of the coding regions of the cysteine protease genes, were subcloned and sequenced. Sequence analysis and alignment showed significant sequence similarity to other members of the eukaryotic cysteine protease family (45% identical to chicken cathepsin L) and conservation of the cysteine, histidine, and asparagine residues which form the catalytic triad. These gene fragments provide molecular probes for further analysis of the structure and function of these important metabolic enzymes.