International Journal for Parasitology: Drugs and Drug Resistance

Benzimidazole (BZ) anthelmintics are among the most important treatments for parasitic nematode infections in the developing world. Widespread BZ resistance in veterinary parasites and emerging resistance in human parasites raise major concerns for the continued use of BZs. Knowledge of the mechanisms of resistance is necessary to make informed treatment decisions and circumvent resistance. Benzimidazole resistance has traditionally been associated with mutations and natural variants in the C. elegans beta-tubulin gene ben-1 and orthologs in parasitic species. However, variants in ben-1 alone do not explain the differences in BZ responses across parasite populations. Here, we examine the roles of five C. elegans beta-tubulin genes (tbb-1, mec-7, tbb-4, ben-1, and tbb-6) to identify the role each gene plays in BZ response. We generated C. elegans strains with a loss of each beta-tubulin gene, as well as strains with a loss of tbb-1, mec-7, tbb-4, or tbb-6 in a genetic background that also lacks ben-1 to test beta-tubulin redundancy in BZ response. We found that only the individual loss of ben-1 conferred a substantial level of BZ resistance, although the loss of tbb-1 was found to confer a small benefit in the presence of albendazole (ABZ). The loss of ben-1 was found to confer an almost complete rescue of animal development in the presence of 30 {micro}M ABZ, likely explaining why no additive effects caused by the loss of a second beta-tubulin were observed. We demonstrate that ben-1 is the only beta-tubulin gene in C. elegans where loss confers substantial BZ resistance. Highlights- Loss of ben-1 provides almost complete rescue of development in albendazole (ABZ) - Loss of different beta-tubulin genes does not confer ABZ resistance - Loss of ben-1 and a second beta-tubulin does not enhance the ben-1 level of ABZ resistance

Praziquantel (PZQ), an anthelmintic drug discovered in the 1970s, is still used to treat schistosomiasis and various other infections caused by parasitic flatworms. PZQ causes a triad of phenotypic effects on schistosome worms - rapid depolarization, muscle contraction, and damage throughout the worm tegument. The molecular target mediating these effects has been intimated as a Ca2+-permeable ion channel, but native currents evoked by PZQ have not been reported in any schistosome cell type. The properties of the endogenous PZQ activated conductance therefore remain unknown. Here, invasive electrophysiology was used to probe for responses to PZQ from different locales in a living schistosome worm. No direct response was seen in tegument-derived vesicles, or from the sub-tegumental muscle layer despite the presence of voltage-operated currents. However, PZQ rapidly triggered a sustained, non-selective cation current in recordings from neuronal tissue, targeting both the anterior ganglion and the main longitudinal nerve cord. The biophysical signature of this PZQ-evoked current resolved at single channel resolution matched that of a transient receptor potential ion channel named TRPMPZQ, recently proposed as the molecular target of PZQ. The endogenous PZQ-evoked current was also inhibited by a validated TRPMPZQ antagonist. PZQ therefore is a neuroactive anthelmintic, effecting a robust, depolarization through ion channels with the characteristics of TRPMPZQ. Key Findings / Scope StatementO_LIResponses to the anthelmintic drug, praziquantel (PZQ), were examined using invasive electrophysiology in a living schistosome worm. C_LIO_LIPZQ evoked a cation current in recordings from neuronal tissue C_LIO_LIThe biophysical and pharmacological characteristics of the native PZQ current matched the properties of TRPMPZQ. C_LI

Anthelmintic resistant parasitic nematodes present a significant threat to sustainable livestock production worldwide. The ability to detect the emergence of anthelmintic resistance at an early stage, and therefore determine which drugs remain most effective, is crucial for minimising production losses. Despite many years of research into the molecular basis of anthelmintic resistance, no molecular-based tools are commercially available for the diagnosis of resistance as it emerges in field settings. We described a mixed deep amplicon sequencing approach to determine the frequency of the levamisole (LEV) resistant single nucleotide polymorphism (SNP) within arc-8 exon 4 (S168T) in Haemonchus spp., coupled with benzimidazole (BZ) resistance SNPs within {beta}-tubulin isotype-1 and ITS-2 nemabiome. This constitutes the first multi-drug and multi-species molecular diagnostic developed for helminths of veterinary importance. Of the ovine, bovine, caprine and camelid Australian field isolates we tested, S168T was detected in the majority of Haemonchus spp. populations from sheep and goats, but rarely at a frequency greater than 16%; an arbitrary threshold we set based on whole genome sequencing of LEV resistant H. contortus GWBII. Overall, BZ resistance was far more prevalent in Haemonchus spp. than LEV resistance, confirming that LEV is still an important anthelmintic class for small ruminants in New South Wales. The mixed amplicon metabarcoding approach described herein, paves the way towards the use of large scale sequencing as a surveillance technology in the field, the results of which can be translated into evidence-based recommendations for the livestock sector.

Toxoplasmosis remains a world-wide public health concern, especially for the immunocompromised. Although this population segment is increasing due to therapeutic interventions, organ transplants and infections including HIV, treatment relies almost exclusively on sulfadoxine and pyrimethamine, antifolates developed against malaria but with only moderate efficacy against acute toxoplasmosis and no effect on the chronic stage. Here we explore whether 7-substituted analogues of 7-deazaadenosine (tubercidin) that have shown remarkable efficacy against other protozoan pathogens, might also show anti-toxoplasmic activity. Tubercidin and a series of eleven 7-substituted analogues including 2-deoxy and 3-deoxyribofuranoses was tested against intracellular Toxoplasma gondii tachyzoites. The test compounds yielded EC50 values between 0.012 and 1.72 {micro}M, well below those of the control drug sulfadiazine (11.9 {micro}M) and the previously identified purine analogue adenosine arabinoside (Ara-A; 11.4 {micro}M). The tubercidin analogues displayed at most moderate toxicity to HFF cells, with the most efficacious compound, 7-(3,4-di-Cl-phenyl)-3-deoxytubercidin (FH8513) reaching a selectivity index of >2500. These nucleosides are most likely taken up by T. gondii through one of the four Equilibrative Nucleoside Transporters (ENTs) encoded by the parasites. However, deletion of TgENT2 and/or TgENT3 had no effect on the EC50 values, and deletion of TgAT1 actually sensitised the tachyzoites to most of the tubercidin analogues. We propose that these nucleosides are internalised through the TgENT1 uridine transporter and that the sensitisation in {Delta}TgAT1 cells is the result of reduced uptake of adenosine that competes with the tubercidin analogues for metabolic enzymes such as adenosine kinase.

With resistance to current frontline antimalarials spreading globally, new drug candidates need to be discovered to populate the antimalarial drug development pipeline. We previously screened the Medicines for Malaria Venture Pathogen Box for compounds that prevent Plasmodium falciparum parasites from exiting and invading human erythrocytes, steps essential for the proliferation of parasites in the blood, which causes disease. Compound MMV020512 (M-512) was identified in this screen and live cell imaging here established that it does not specifically inhibit invasion but likely inhibits intraerythrocytic parasite growth. M-512 resistance selection in parasites led to the identification of mutations in the membrane protease PfROM8 and the cation ion channel PfCSC1. PfROM8 was validated as a target of M-512 when a L562R putative resistance mutation was engineered into wildtype parasites reproducing the resistance phenotype. Knockdown of wildtype PfROM8, the L562R mutant and CSC1 reduced parasite growth, indicating the proteins are functionally important. Counterintuitively, the PfROM8 and PfCSC1 knockdown parasites became more resistant to M-512 suggesting that the compound is an agonist of both proteins which may form a functional complex and that dysregulation of this complex is deleterious to parasite growth.

Toxoplasmosis is a common protozoan infection that can have severe outcomes in the immunocompromised and during pregnancy, but treatment options are limited. Recently, nucleotide metabolism has received much attention as a target for new antiprotozoal agents and here we focus on pyrimidine salvage by Toxoplasma gondii as a drug target. Whereas uptake of [3H]-cytidine and particularly [3H]-thymidine was at most marginal, [3H]-uracil and [3H]-uridine were readily taken up. Kinetic analysis of uridine uptake was consistent with a single transporter with a Km of 3.3 {+/-} 0.8 {micro}M, which was inhibited by uracil with high affinity (Ki = 1.15 {+/-} 0.07 {micro}M) but not by thymidine or 5-methyluridine, showing that the 5-Me group is incompatible with uptake by T. gondii. Conversely, [3H]-uracil transport displayed a Km of 2.05 {+/-} 0.40 {micro}M, not significantly different from the uracil Ki on uridine transport, and was inhibited by uridine with a Ki 2.44 {+/-} 0.59 {micro}M, also not significantly different from the experimental uridine Km. The reciprocal, complete inhibition, displaying Hill slopes of approximately [~]1, strongly suggest that uridine and uracil share a single transporter with similarly high affinity for both, and we designate it uridine/uracil transporter 1 (TgUUT1). While TgUUT1 excludes 5-methyl substitutions, the smaller 5F substitution was tolerated as 5F-uracil inhibited uptake of [3H]-uracil with a Ki of 6.80 {+/-} 2.12 {micro}M (P > 0.05 compared to uracil Km). Indeed, we found that 5F-Uridine, 5F-uracil and 5F,2-deoxyuridine were all potent antimetabolites against T. gondii with EC50 values well below that of the current first line treatment, sulfadiazine. In vivo evaluation also showed that 5F-uracil and 5F,2-deoxyuridine were similarly effective as sulfadiazine against acute toxoplasmosis. Our preliminary conclusion is that TgUUT1 mediates potential new anti-toxoplasmosis drugs with activity superior to the current treatment.

All the pathology and symptoms associated with malaria are caused by the growth of Plasmodium parasites inside human red blood cells. This process, which in the case of the major human malaria pathogen Plasmodium falciparum takes place over a 48-hour period, involves multiple tightly regulated developmental transitions. Understanding the P. falciparum genes that regulate these key processes could lead to the identification of targets for new drugs. However, while large-scale sequencing efforts have led to a good understanding of the P. falciparum genome and how it evolves over time and space, a disconnect remains between the amount of genome sequence data available and the amount of data describing what exactly the genes contained within it do - the phenotype. We have generated a panel of 66 P. falciparum lines carrying individual gene knockouts tagged with unique DNA barcodes. We then used these lines in a series of assays that combine flow cytometry, cell sorting and DNA barcode quantification using next generation sequencing (Barcode Sequencing or BarSeq) to phenotype key aspects of the parasite life cycle such as growth, replication capacity and cell cycle progression. This approach both yields new data about individual gene function, and outlines a new approach where barcoded P. falciparum lines are used in pooled BarSeq-based assays to generate more precise phenotype data at scale.

Nitroaromatic drugs are of critical importance for the treatment of trypanosome infections in Africa and the Americas. Fexinidazole recently joined benznidazole and nifurtimox in this family when it was approved as the first oral therapy against Human African trypanosomiasis (HAT). Nitroaromatic prodrugs are bioactivated by the trypanosome-specific type I nitroreductase (NTR) enzyme that renders the compounds trypanocidal. A caveat to the specificity of NTR activation is the potential for drug resistance and cross-resistance that can arise if NTR expression or functionality is altered through mutation. The outcomes of NTR bioactivation of nitroaromatic compounds is variable but can include the formation highly reactive open chain nitriles that can damage biomolecules including DNA. A proposed mechanism of action of nitroaromatic compounds is the formation of reactive oxygen species (ROS) resulting in the formation of trypanocidal levels of DNA damage. Fexinidazole made its way to clinical approval without a significant interrogation of its effects on trypanosome biology and a limited understanding of its mechanism of action. Early reports mentioned fexinidazole potentially affects DNA synthesis but without supporting data. In this study, we evaluated and compared the cytotoxic effects of nifurtimox, benznidazole, and fexinidazole on Trypanosoma brucei using in vitro analyses. Specifically, we sought to differentiate between the proposed effects of nitroaromatics on DNA damage and DNA synthesis. Toward this goal we generated a novel {gamma}H2A-based flow cytometry assay that reports DNA damage formation in conjunction with cell cycle progression. Here we report that fexinidazoles cytotoxic outcomes are distinct from the related drugs nifurtimox and benznidazole. Specifically, we show that fexinidazole treatment results in a pronounced defect in DNA synthesis that reduces the population of parasites in S phase. In contrast, treatment with nifurtimox and benznidazole appear accumulate DNA damage early in cell cycle and result in a defective G2 population. The findings presented here bring us closer to understanding the anti-trypanosomatid mechanisms of action of nitroaromatic compounds, which will promote improved drug design and help combat potential drug resistance in the future. Our findings also highlight DNA synthesis inhibition as a powerful anti-parasitic drug target.

Parasitic nematodes infect billions of people and are mainly controlled by anthelmintic mass drug administration (MDA). While there are growing efforts to better understand mechanisms of anthelmintic resistance in human and animal populations, it is unclear how resistance mechanisms that alter susceptibility to one drug affect the interactions and efficacy of drugs used in combination. Mutations that alter drug permeability across primary nematode barriers have been identified as potential resistance mechanisms using the model nematode Caenorhabditis elegans. We leveraged high-throughput assays in this model system to measure altered anthelmintic susceptibility in response to genetic perturbations of potential cuticular, amphidial, and alimentary routes of drug entry. Mutations in genes associated with these tissue barriers differentially altered susceptibility to the major anthelmintic classes (macrocyclic lactones, benzimidazoles, and nicotinic acetylcholine receptor agonists) as measured by animal development. We investigated two-way anthelmintic interactions across C. elegans genetic backgrounds that confer resistance or hypersensitivity to one or more drugs. We observe that genetic perturbations that alter susceptibility to a single drug can shift the drug interaction landscape and lead to the appearance of novel synergistic and antagonistic interactions. This work establishes a framework for investigating combinatorial therapies in model nematodes that can potentially be translated to amenable parasite species.

Schistosoma mansoni is a parasitic helminth that is vectored through freshwater snails. While the anti-schistosome defense of the South American snail, Biomphalaria glabrata, is well studied, little is known about the immune response of the African snail, Biomphalaria sudanica. We measured expression of five candidate immune genes in B. sudanica 8, 24, and 72 hours post-exposure to S. mansoni using reverse transcription quantitative PCR. Expression patterns of resistant snails were compared to susceptible snails and those sham exposed. We also assessed how diet (lettuce vs. pellet) affected expression of three genes, given prior findings that pellet-fed snails were more susceptible to S. mansoni. Results indicated that resistant snails constitutively expressed higher levels of superoxide dismutase 1 (SOD1) than susceptible snails, consistent with expression patterns of resistant B. glabrata. Parasite-induced expression occurred at 8 hours in SOD1, biomphalysin, thioester protein 1 (TEP1), and granulin (GRN); however, for biomphalysin and TEP1, induced expression was only detected for susceptible snails. At 24 hours, biomphalysin expression increased in exposed resistant snails, and at 72 hours, all exposed snails decreased biomphalysin expression compared to controls. Parasite-induced expression of SOD1, biomphalysin, TEP1, and GRN supports the hypothesis that these genes play a role in B. sudanica anti-schistosome defense, however increased expression does not necessarily yield clearance of S. mansoni. SOD1 expression was higher in lettuce-fed snails at 8 and 24 hours, consistent with their greater resistance. Together, these results demonstrate the conserved and unique aspects of the B. sudanica anti-schistosome response.

With emerging resistance to frontline treatments, it is vital that new antimalarial drugs are identified to target Plasmodium falciparum. We have recently described a compound, MMV020291, as a specific inhibitor of red blood cell invasion, and have generated analogues with improved potency. Here, we identify actin and profilin as putative targets of the MMV020291 series through resistance selection and whole genome sequencing of three MMV020291 resistant populations. This revealed three non-synonymous single nucleotide polymorphisms in two genes; two in profilin (N154Y, K124N) and a third one in actin-1 (M356L). Using CRISPR-Cas9, we engineered these mutations into wildtype parasites which rendered them resistant to MMV020291. We demonstrate that MMV020291 reduces actin polymerisation that is required by the merozoite stage parasites to invade red blood cells. Additionally, the series inhibits the actin-1 dependent process of apicoplast segregation, leading to a delayed death phenotype. In vitro co-sedimentation experiments using recombinant P. falciparum actin-1 and profilin proteins indicate that potent MMV020291 analogues amplify the actin-monomer sequestering effect of profilin, thereby reducing the formation of filamentous actin. Altogether, this study identifies the first compound series targeting the actin-1/profilin interaction in P. falciparum and paves the way for future antimalarial development against the highly dynamic process of actin polymerisation.

Leishmania mexicana, like other trypanosomatids, possesses a unique kinetochore--the protein complex crucial for chromosome segregation during mitosis. To investigate the functional significance of specific phosphorylation sites on essential kinetochore proteins, we adapted a selection-free precision editing strategy using CRISPR-Cas9 in Leishmania mexicana promastigotes. Our method targeted genomic DNA with 160-bp double-stranded DNA repair templates and guide RNAs to introduce targeted modifications. We focused on six phosphosites within the kinetochore proteins KKT2, KKT4, and KKT7, generating phosphodeficient, phosphomimetic, and synonymous mutants for each site. Across 18 independent transfections, we achieved a successful editing rate of 27.5% as determined by PCR screening, with 30.4% of clones confirmed as edited by Sanger sequencing. A significant portion of these edited clones (22.1%) were homozygous. Despite these precise genomic modifications, none of the phosphosite mutant clones exhibited any apparent growth defects or cell cycle dysregulation, suggesting these phosphorylation sites individually may not be critical for these processes under standard culture conditions. To facilitate higher-throughput precision editing, we developed a Python script that automates the design of the 160-bp repair templates. This script uses a FASTA file, a codon usage table, and a simple configuration file to design templates with a single nonsynonymous mutation and additional synonymous mutations for screening purposes. It also generates a corresponding synonymous-only repair template and primers for both screening and repair template generation, offering a "ready-to-go" approach. While designed for Leishmania, this powerful tool is adaptable for use with other kinetoplastids.

Schistosomiasis is a neglected tropical disease caused by human-infective schistosomes (Trematoda: Schistosoma). Intestinal schistosomiasis in sub-Saharan Africa and the Neotropics is caused primarily by Schistosoma mansoni and is transmitted by several Biomphalaria planorbid snail species. Adult male and female parasites in the definitive mammalian host pair and reside in the mesenteric vasculature; females lay eggs that traverse the intestinal wall to be excreted, but a significant proportion become trapped in host tissues, especially the liver, eliciting granulomatous immune responses that underlie most disease pathology. S. mansoni is the primary lab model for research and, due to the abundance and ease of harvesting, liver-derived eggs are almost exclusively used to maintain the life cycle and to study miracidia and subsequent larval stages. However, recent evidence shows that eggs from the liver or intestine have key morphometric, transcriptomic, and antigenic differences, which can profoundly affect experimental outcomes. To determine whether these differences extend to the miracidia stage, we compared miracidia hatched from mouse liver and intestine-derived eggs, sequencing their transcriptomes and assessing their unstimulated behaviors over time in an arena allowing for high-resolution tracking of miracidia behavior at a large spatiotemporal scale. We found that while transcriptomic profiles of miracidia are distinguishable based on egg tissue origin, only a small subset of genes is differentially expressed. Further, basic, unstimulated behavior of miracidia that developed in different niches of the definitive host was significantly different. These different behavioral programs may reflect intrinsic developmental programming or differential viability and hardiness related to tissue origin. These findings underscore the importance of egg source in experimental design and interpretation, with significant implications for the maintenance of laboratory life cycles and the use of miracidia in schistosomiasis research.

Helminth infections in grazing ruminants are of major concern for animal welfare and cause substantial economic losses, prompting the widespread use of ivermectin (IVM). The emergence of IVM resistance, driven by complex and poorly understood mechanisms, increasingly compromises treatment efficacy. Drug efflux transporters, particularly P-glycoproteins (PGPs), are suspected to contribute to resistance. Yet, the study of their individual and functional role is hindered by their diversity in nematodes. This study aimed to dissect the role of specific PGPs in mediating IVM resistance. Thus, Caenorhabditis elegans strain IVR10, selected for IVM resistance and reported to overexpress pgps, was used as a model. We generated different IVR10 strains each lacking one of six key pgps, and assessed changes in IVM tolerance. Remarkably, only the deletion of pgp-9 significantly increased IVM susceptibility. Furthermore, transgenic expression of Haemonchus contortus pgp-9.1 rescued the resistant phenotype, demonstrating a conserved function across species. To explore drug dynamics, we developed a fluorescent IVM analog, which revealed reduced drug accumulation in IVR10, a phenotype reversed by pgp-9 deletion. Altogether, these findings show that nematode PGP-9 modulates IVM tolerance by controlling drug efflux and highlight it as a potential therapeutic target.

Ascarid parasites, such as Ascaridia galli and Heterakis gallinarum, are nearly ubiquitous in poultry and can cause serious production losses. H. gallinarum is of particular concern because of its role as a vector for the protozoan Histomonas meleagridis, the cause of blackhead disease. Currently, only the benzimidazole anthelmintic, fenbendazole (FBZ), is approved for use in poultry, and recently, FBZ resistance has been discovered and validated in populations of the turkey ascarid Ascaridia dissimilis and in ascarid of gallinaceous birds H. gallinarum. Here, we further explore the prevalence of resistance in poultry ascarids by testing FBZ efficacy against thirteen isolates of A. galli and eight isolates of H. gallinarum. Isolates were used to infect day-old naive chickens. Four weeks after infection, animals to be treated received the label-recommended dosage of FBZ (SafeGuard Aquasol) for five days, per the manufacturers directions. One week after the fifth day of treatment, animals were euthanized and parasite burdens were counted to determine treatment efficacy between the untreated and treated groups. Resistance was identified and validated in a single isolate of A. galli, marking the first confirmed case in the species. All isolates of H. gallinarum were found to be resistant. The emergence of resistance in A. galli and the high prevalence of resistance in H. gallinarum highlight the growing concern of resistance in parasites of poultry. Without approved alternative treatments, the detrimental effects of infections cannot be mitigated in resistant populations, significantly impacting profit margins. Diagnostics that enable broader surveys are necessary to understand the full scope of the problem. However, we show that resistance is present across production species and should act as an impetus for the discovery of new treatments and the adoption of new management strategies.

We report the synergistic interaction between the complex III inhibitor atovaquone and an ATP synthase inhibitor, venturicidin A, against Plasmodium falciparum. Our results suggest the simultaneous inhibition of the parasites primary and alternative pathways for generating the mitochondrial membrane potential to be responsible for this phenomenon, whereby the alternative pathway relies on ATP synthase running in reverse mode. We hypothesize that the synergistic interaction between atovaquone and proguanil could follow a similar mechanism.

Animal African trypanosomosis (AAT), caused by protist parasites of the genus Trypanosoma, puts upward of a million head of livestock at risk across 37 countries in Africa. The economic impact of AAT and the presence of human-infectious trypanosomes in animals place a clear importance on improving diagnostics for animal trypanosomes to map the distribution of the veterinary parasites and identify reservoirs of human-infectious trypanosomes. We have adapted the CRISPR-based detection toolkit SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) for trypanosomatid parasites responsible for AAT (SHERLOCK4AAT) including Pan- trypanosomatid, Trypanozoon, T. vivax, T. congolense, T. theileri, T. simiae and T. suis assays. To test the applicability of this technique in the field, we analysed dried blood spots collected from 200 farm and 224 free-ranging pigs in endemic and historical human African trypanosomiasis foci in Guinea and Cote dIvoire, respectively. The results revealed that SHERLOCK4AAT can detect and discriminate between trypanosome species involved in multiple infections with a high sensitivity. 62.7 % [58.1, 67.3] of pigs were found infected with at least one trypanosome species. T. brucei gambiense, a human-infectious trypanosome, was found in one animal at both sites, highlighting the risk that these animals may act as persistent reservoirs. These data suggest that, due to their proximity to humans and their attractiveness to tsetse flies, pigs could act as sentinels to monitor T. b. gambiense circulation using the SHERLOCK4AAT toolbox.

The apicomplexan parasite Toxoplasma gondii infects 25-30% of the global human population and can cause life-threatening diseases in immunocompromised patients. The chronically infectious forms of the parasite, bradyzoites, persist within cysts in brain and muscle tissue and are responsible for its transmission and remission of the disease. Currently available treatment options are very limited and are only effective against the fast-replicating tachyzoites but fail to eradicate the chronic stages of T. gondii. The cause of these treatment failures remains unclear. Here, we utilized our recently developed human myotube-based culture model to screen compounds from the MMV Pathogen Box against pan-resistant in vitro bradyzoites and identified multiple compounds with simultaneous activity against tachyzoites and bradyzoites. Stable isotope-resolved metabolic profiling on tachyzoites and bradyzoites identified the mitochondrial bc1-complex as a target of bradyzocidal compounds and defines their metabolic impacts on both parasite forms. Our data suggest that mature bradyzoites rely on mitochondrial ATP production.

Inhibitors for Plasmodium falciparum dihydrofolate reductase (DHFR) form an important class of antimalarial drugs widely used for malaria treatment, but have been compromised by development of resistance to the drugs. Mutations in DHFR are the main contributing factors to the resistance. Although new, rationally designed antifolates active against resistant P. falciparum, such as P218, have been developed, the activity against the quadruple mutant P. falciparum (V1/S) has only been demonstrated in vitro, and in vivo activity has only been shown in SCID mice. A convenient in vivo model for antifolate testing is desirable. In this study, the endogenous P. berghei dihydrofolate reductase-thymidylate synthase (Pbdhfr-ts) gene was successfully replaced by quadruple dhfr-ts mutant gene from P. falciparum (N51I+C59R+S108N+I164L). The transgenic parasite gained resistance to pyrimethamine but not to other class of antimalarial drugs. While 30 mg/kg of pyrimethamine could not inhibit the transgenic parasite, P218 could inhibit the transgenic parasite with the ED50 of 0.11{+/-}0.02 mg/kg, a level similar to the P. falciparum in SCID mice model. These results demonstrated the validity of our model and showed that P218 was very potent against quadruple Pfdhfr-ts mutant parasite, in vivo.

Anthelmintic drugs are used to control soil-transmitted helminths that infect a third of the worlds human population. There is increasing concern about the development of resistance to anthelmintic drugs because of the limited number of compounds available and there is an unmet need for new resistance-busting drugs. Here we describe the presence of a previously unrecognized endogenous acetylcholine analogue, {beta}-alanine betaine, which may serve as an endogenous ligand for an alternate subfamily of nicotinic receptors (DEG-3/DES-2) that could be developed as novel drug targets because their analogues are not present in their human or animal hosts. We collected peri-enteric fluid from female Ascaris suum (a model for the human parasite, Ascaris lumbricoides) and subjected it to chromatography and MS/MS to reveal signals consistent with acetylcholine, choline, and {beta}-alanine betaine but we did not recover betaine. We injected betaine into female Ascaris suum which produced no effect. However, injection of {beta}-alanine betaine, produced characteristic pretzel coiling and injection of levamisole produced a rod-like spastic paralysis. The differences between {beta}-alanine betaine and levamisole suggested that they activate different nAChRs subfamilies. PCR showed that messages of the DEG-3 subfamily of nAChR channels, which are betaine targets and were present in the intestine and body wall of A. suum. Calcium signaling experiments showed that {beta}-alanine betaine increased intracellular calcium of the intestine enterocytes and electrophysiology of the body muscle cells demonstrated that {beta}-alanine betaine produced membrane potential depolarization. In N2 elegans, application of {beta}-alanine betaine produced gradual inhibition of motility, which was reduced in acr-20, acr-23, des-2, deg-3 and lgc-41 null-mutants. These observations suggest that, in addition to acetylcholine, {beta}-alanine betaine - an anaerobic analog of betaine - may function as an endogenous ligand in anaerobic nematodes such as A. suum. An expanded repertoire of nicotinic acetylcholine receptor subfamilies in nematodes relative to mammals may reflect a corresponding need for diversification of cholinergic endogenous ligands in these organisms. This repertoire could allow their simpler neuronal system to perform more complex controls and be exploited for development of different and novel subfamily selective cholinergic anthelmintics.