Establishment of an in vitro culture system to study the developmental biology of Onchocerca volvulus with implications for anti-Onchocerca drug discovery and screening.

BACKGROUND: Infections with Onchocerca volvulus nematodes remain a threat in Sub-Saharan Africa after three decades of ivermectin mass drug administration. Despite this effort, there is still an urgent need for understanding the parasite biology especially the mating behaviour and nodule formation as well as the development of more potent drugs that can clear the developmental (L3, L4, L5) and adult stages of the parasite and inhibit parasite reproduction and behaviour. METHODOLOGY/PRINCIPAL FINDINGS: Prior to culture, freshly harvested O. volvulus L3 larvae from dissected Simulium damnosum flies were purified by centrifugation using a 30% Percoll solution to eliminate fly tissue debris and contaminants. Parasites were cultured in both cell-free and cell-based co-culture systems and monitored daily by microscopic visual inspection. Exhausted culture medium was replenished every 2-3 days. The cell-free culture system (DMEM supplemented with 10% NCS) supported the viability and motility of O. volvulus larvae for up to 84 days, while the co-culture system (DMEM supplemented with 10% FBS and seeded on LLC-MK2 feeder cells) extended worm survival for up to 315 days. Co-culture systems alone promoted two consecutive parasite moults (L3 to L4 and L4 to L5) with highest moulting rates (69.2±30%) observed in DMEM supplemented with 10% FBS and seeded on LLC-MK2 feeder cells, while no moult was observed in DMEM supplemented with 10% NCS and seeded on LEC feeder cells. In DMEM supplemented with 10% FBS and seeded on LLC-MK2 feeder cells, O. volvulus adult male worms attached to the vulva region of adult female worms and may have mated in vitro. Apparent early initiation of nodulogenesis was observed in both DMEM supplemented with 10% FBS and seeded on LLC-MK2 and DMEM supplemented with 10% NCS and seeded on LLC-MK2 systems. CONCLUSIONS/SIGNIFICANCE: The present study describes an in vitro system in which O. volvulus L3 larvae can be maintained in culture leading to the development of adult stages. Thus, this in vitro system may provide a platform to investigate mating behaviour and early stage of nodulogenesis of O. volvulus adult worms that can be used as additional targets for macrofilaricidal drug screening.

Investigation of pyrimidine nucleoside analogues as chemical probes to assess compound effects on the proliferation of Trypanosoma cruzi intracellular parasites.

Trypanosoma cruzi parasites utilise de novo pyrimidine biosynthesis to produce DNA and survive within mammalian host cells. This pathway can be hijacked to assess the replication of intracellular parasites with the exogenous addition of a DNA specific probe. To identify suitable probe compounds for this application, a collection of pyrimidine nucleoside analogues was assessed for incorporation into T. cruzi intracellular amastigote DNA using image-based technology and script-based analysis. Associated mammalian cell toxicity of these compounds was also determined against both the parasite host cells (3T3 cells) and HEK293 cells. Incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into parasite DNA was the most effective of the probes tested, with minimal growth inhibition observed following either two or four hours EdU exposure. EdU was subsequently utilised as a DNA probe, followed by visualisation with click chemistry to a fluorescent azide, to assess the impact of drugs and compounds with previously demonstrated activity against T. cruzi parasites, on parasite replication. The inhibitory profiles of these molecules highlight the benefit of this approach for identifying surviving parasites post-treatment in vitro and classifying compounds as either fast or slow-acting. F-ara-EdU resulted in <50% activity observed against T. cruzi amastigotes following 48 hours incubation, at 73 µM. Collectively, this supports the further development of pyrimidine nucleosides as chemical probes to investigate replication of the parasite T. cruzi.

In Vitro Drug Efficacy Testing Against Trypanosoma brucei.

The recent endorsement of fexinidazole by the European Medicines Agency for the treatment of human African trypanosomiasis has demonstrated the high predictive value of cell-based assays for parasite chemotherapy. Here we describe three in vitro drug susceptibility tests with Trypanosoma brucei that have served as the basis for the identification of fexinidazole as a promising lead: (1) a standard assay with end-point measurement to determine drug efficacy; (2) a wash-out assay to test for reversibility and speed of drug action; (3) isothermal microcalorimetry for real-time measurement of onset of drug action and time to kill. Together, these assays allow to estimate pharmacodynamic parameters in vitro and to devise appropriate treatment regimens for subsequent in vivo experiments.

Phenotypic screening of nonsteroidal anti-inflammatory drugs identified mefenamic acid as a drug for the treatment of schistosomiasis.

BACKGROUND: Treatment and control of schistosomiasis, one of the most insidious and serious parasitic diseases, depend almost entirely on a single drug, praziquantel. Since the funding for drug development for poverty-associated diseases is very limited, drug repurposing is a promising strategy. In this study, 73 nonsteroidal anti-inflammatory drugs (NSAIDs) commonly used in medical and veterinary fields were evaluated for their anti-schistosomal properties. METHODS: The efficacy of NSAIDs was first tested against adult Schistosoma mansoni ex vivo using phenotypic screening strategy, effective drugs were further tested in a murine model of schistosomiasis. The disease parameters measured were worm and egg burden, hepato- and splenomegaly. FINDINGS: From 73 NSAIDs, five (mefenamic acid, tolfenamic acid, meclofenamic acid, celecoxib, and diclofenac) were identified to effectively kill schistosomes. These results were further supported by scanning electron microscopy analysis. In addition, the octanol-water partition coefficient, both for neutral and ionized species, revealed to be a critical property for the ex vivo activity profile. Compounds were then tested in vivo using both patent and a prepatent S. mansoni infection in a mouse model. The most effective NSAID was mefenamic acid, which highly reduced worm burden, egg production, and hepato- and splenomegaly. INTERPRETATION: The treatment regimen used in this study is within the range for which mefenamic acid has been used in clinical practice, thus, it is demonstrated the capacity of mefenamic acid to act as a potent anti-schistosomal agent suitable for clinical repurposing in the treatment of schistosomiasis.

Development of a preliminary in vitro drug screening assay based on a newly established culturing system for pre-adult fifth-stage Onchocerca volvulus worms.

BACKGROUND: The human filarial parasite Onchocerca volvulus is the causative agent of onchocerciasis (river blindness). It causes blindness in 270,000 individuals with an additional 6.5 million suffering from severe skin pathologies. Current international control programs focus on the reduction of microfilaridermia by annually administering ivermectin for more than 20 years with the ultimate goal of blocking of transmission. The adult worms of O. volvulus can live within nodules for over 15 years and actively release microfilariae for the majority of their lifespan. Therefore, protracted treatment courses of ivermectin are required to block transmission and eventually eliminate the disease. To shorten the time to elimination of this disease, drugs that successfully target macrofilariae (adult parasites) are needed. Unfortunately, there is no small animal model for the infection that could be used for discovery and screening of drugs against adult O. volvulus parasites. Here, we present an in vitro culturing system that supports the growth and development of O. volvulus young adult worms from the third-stage (L3) infective stage. METHODOLOGY/PRINCIPAL FINDINGS: In this study we optimized the culturing system by testing several monolayer cell lines to support worm growth and development. We have shown that the optimized culturing system allows for the growth of the L3 worms to L5 and that the L5 mature into young adult worms. Moreover, these young O. volvulus worms were used in preliminary assays to test putative macrofilaricidal drugs and FDA-approved repurposed drugs. CONCLUSION: The culture system we have established for O. volvulus young adult worms offers a promising new platform to advance drug discovery against the human filarial parasite, O. volvulus and thus supports the continuous pursuit for effective macrofilaricidal drugs. However, this in vitro culturing system will have to be further validated for reproducibility before it can be rolled out as a drug screen for decision making in macrofilaricide drug development programs.

Impedance-Based Microfluidic Assay for Automated Antischistosomal Drug Screening.

Schistosomiasis is a neglected tropical disease, caused by parasitic worms, which affects almost 200 million people worldwide. For over 40 years, chemotherapeutic treatment has relied on the administration of praziquantel, an efficacious drug against schistosomiasis. However, concerns about developing drug resistance require the discovery of novel drug compounds. Currently, the drug-screening process is mostly based on the visual evaluation of drug effects on worm larvae in vitro by a trained operator. This manual process is extremely labor-intensive, has limited throughput, and may be affected by subjectivity of the operator evaluation. In this paper, we introduce a microfluidic platform with integrated electrodes for the automated detection of worm larvae viability using an impedance-based approach. The microfluidic analysis unit consists of two sets of electrodes and a channel of variable geometry to enable counting and size detection of single parasite larvae and the collective evaluation of the motility of the larvae as an unbiased estimator for their viability. The current platform also allows for multiplexing of the analysis units resulting in increased throughput. We used our platform to record size and motility variations of Schistosoma mansoni larvae exposed to different concentrations of mefloquine, a drug with established in vitro antischistosomal properties. The developed platform demonstrates the potential of integrated microfluidic platforms for high-throughput antischistosomal drug screening.

Experimental models in Chagas disease: a review of the methodologies applied for screening compounds against Trypanosoma cruzi.

One of the main problems of Chagas disease (CD), the parasitic infection caused by Trypanosoma cruzi, is the lack of a completely satisfactory treatment, which is currently based on two old nitroheterocyclic drugs (i.e., nifurtimox and benznidazole) that show important limitations for treating patients. In this context, many laboratories look for alternative therapies potentially applicable to the treatment, and therefore, research in CD chemotherapy works in the design of experimental protocols for detecting molecules with activity against T. cruzi. Phenotypic assays are considered the most valuable strategy for screening these antiparasitic compounds. Among them, in vitro experiments are the first step to test potential anti-T. cruzi drugs directly on the different parasite forms (i.e., epimastigotes, trypomastigotes, and amastigotes) and to detect cytotoxicity. Once the putative trypanocidal drug has been identified in vitro, it must be moved to in vivo models of T. cruzi infection, to explore (i) acute toxicity, (ii) efficacy during the acute infection, and (iii) efficacy in the chronic disease. Moreover, in silico approaches for predicting activity have emerged as a supporting tool for drug screening procedures. Accordingly, this work reviews those in vitro, in vivo, and in silico methods that have been routinely applied during the last decades, aiming to discover trypanocidal compounds that contribute to developing more effective CD treatments.

Phytochemical Study of Salvia leriifolia Roots: Rearranged Abietane Diterpenoids with Antiprotozoal Activity.

Phytochemical investigation of the lipophilic extract of the roots of Salvia leriifolia resulted in the isolation of the new rearranged abietane diterpenoids leriifoliol (1) and leriifolione (2), together with 10 known diterpenoids. Structure elucidations were performed via extensive NMR and HRESIMS data, and the absolute configurations of compounds 1 and 3-5 were established by evaluation of experimental and calculated ECD spectra. The antiplasmodial activity of the new isolates was assayed against Trypanosoma brucei rhodesiense, T. cruzi, Plasmodium falciparum, and Leishmania donovani and also toxicity against rat myoblast (L6) cells. Compound 1 displayed antimalarial and low cytotoxic activity with IC50 values of 0.4 and 33.6 µM, respectively, and a selectivity index of 84. Compound 2 displayed activity against T. brucei, T. cruzi, and L. donovani, with IC50 values of 1.0, 4.6, and 1.0 µM, respectively. Putative biosynthetic pathways toward the formation of 1, 2, and 3 are proposed. Leriifoliol (1) is the first 20- nor-9,10- seco-abietane, while 2 exhibits an uncommon 6-6-5 fused-ring system.

SYBR® Green I-Based Fluorescence Assay to Assess Cell Viability of Malaria Parasites for Routine Use in Compound Screening.

Owing to its fast and reliable assessment of parasite growth, the SYBR® Green I-based fluorescence assay is widely used to monitor drug susceptibility of malaria parasites. Its particular advantages are that it is a simple, one-step procedure and very cost-effective making it especially suited for high through put screening of newly developed drugs and drug combinations. Here we describe a SYBR® Green I-based fluorescence assay protocol to be used for routine screening of compounds and extracts in a research laboratory environment. A variation of the standard protocol is also provided allowing to address stage-specific effects of fast-acting drugs.

The establishment of in vitro culture and drug screening systems for a newly isolated strain of Trypanosoma equiperdum.

Dourine is caused by Trypanosoma equiperdum via coitus with an infected horse. Although dourine is distributed in Equidae worldwide and is listed as an internationally important animal disease by the World Organization for Animal Health (OIE), no effective treatment strategies have been established. In addition, there are no reports on drug discovery, because no drug screening system exists for this parasite. A new T. equiperdum strain was recently isolated from the genital organ of a stallion that showed typical symptoms of dourine. In the present study, we adapted T. equiperdum IVM-t1 from soft agarose media to HMI-9 liquid media to develop a drug screening assay for T. equiperdum. An intracellular ATP-based luciferase assay using CellTiter-Glo reagent and an intracellular dehydrogenase activity-based colorimetric assay using WTS-8 tetrazolium salt (CCK-8 reagent) were used in order to examine the trypanocidal effects of each compound. In addition, the IC50 values of 4 reference trypanocidal compounds (pentamidine, diminazene, suramin and melarsomine) were evaluated and compared using established assays. The IC50 values of these reference compounds corresponded well to previous studies involving other strains of T. equiperdum. The luciferase assay would be suitable for the mass screening of chemical libraries against T. equiperdum because it allows for the simple and rapid-evaluation of the trypanocidal activities of test compounds, while a simple, inexpensive colorimetric assay will be applicable in developing countries for the evaluation of the drug sensitivity of epidemic trypanosome strains.

Perfil químico y actividad antibacterial de los extractos de Peltigera laciniata (Merrill ex Riddle) Gyeln / Chemical profile and antibacterial activity of extracts from Peltigera laciniata (Merrill ex Riddle) Gyeln

Introducción: los líquenes, al presentar metabolitos secundarios como xantonas, antraquinonas y alcaloides, se han postulado como material con alto potencial biológico (e. g. antibiótico y antiviral), siendo el antibacterial muy promisorio, el cual se determina por medio de antibiogramas por difusión, punto central de esta investigación. Objetivo: evaluar la actividad antibacterial de los extractos de Peltigera laciniata (Merrill ex Riddle) Gyeln. Olmo de hoja cortada. Métodos: el material liquénico se sometió a percolación con etanol 96 por ciento. Al extracto crudo etanólico se le realizó el aislamiento de alcaloides totales y flavonoides totales con adición de HCL 3 por ciento y metanol, respectivamente. Ambas fracciones, fueron monitoreados por cromatografía de capa fina y fraccionados utilizando cromatografía de columna. Los extractos y fracciones se sometieron a bioensayos sobre Escherichia coli y Staphylococcus aureus para la valoración de los halos de inhibición, utilizando como control Sultamicilina. Los ensayos fueron realizados tres veces con 2 réplicas. Resultados: al realizar la separación cromatográfica de los alcaloides, se observó aumento de la inhibición en comparación con la mezcla alcaloidal. La fracción A1 presenta valores de inhibición cercanos al control y presentó los menores valores de inhibición con respecto a los demás tratamientos evaluados. El efecto de la fracción de los flavonoides totales tuvo menor impacto sobre E. coli y S. aureus, sin embargo, es importante destacar la acción antibacterial de los compuestos nitrogenados de tipo alcaloidal sobre microoganismos Gram positivos. Conclusiones: en el perfil químico realizado a partir de los extractos de la especie de estudio se visualizó la presencia de metabolitos secundarios de tipo alcaloide y flavonoide, evidenciando el efecto antimicrobiano de los alcaloides presentes en el extracto y la fracción, lo cual ratifica el potencial farmacológico de tipo antibacterial, atribuido al núcleo Protoberberínico(AU)

Introduction: Due to their content of secondary metabolites such as xanthones, anthraquinones and alkaloids, lichens have been suggested to be a material of high biological potential (e.g. antibiotic and antiviral). Their very promising antibacterial potential may be determined by diffusion antibiograms, the main concern of the present study. Objective: Evaluate the antibacterial activity of extracts obtained from Peltigera laciniata (Merrill ex Riddle) Gyeln, cutleaf elm. Methods: The lichenic material was percolated with 96 percent ethanol. Total alkaloids and total flavonoids were isolated from the crude ethanolic extract by adding 3 percent HCL and methanol, respectively. Both fractions were monitored by thin-layer chromatography and fractioned by column chromatography. Extracts and fractions were subjected to bioassays against Escherichia coli and Staphylococcus aureus for inhibition haloes, using sultamicillin as control. The assays were conducted 3 times with 2 replications. Results: Upon chromatographic separation of the alkaloids, an increase was observed in inhibition when compared with the alkaloidal mixture. Fraction A1 displayed inhibition values close to the control. Fraction FT showed lower inhibition values than the other treatments evaluated. The fraction of total flavonoids had a lesser impact on E. coli and S. aureus, but alkaloidal nitrogenated compounds had significant antibacterial activity against Gram-positive microorganisms. Conclusions: The chemical profile of extracts from the study species revealed the presence of alkaloidal and flavonoidal secondary metabolites, as well as the antimicrobial effect of the alkaloids contained in the extract and the fraction. This confirms the antibacterial pharmacological potential attributed to the protoberberine core(AU)

Perfil químico y actividad antibacterial de los extractos de Peltigera laciniata (Merrill ex Riddle) Gyeln / Chemical profile and antibacterial activity of extracts from Peltigera laciniata (Merrill ex Riddle) Gyeln

Introducción: los líquenes, al presentar metabolitos secundarios como xantonas, antraquinonas y alcaloides, se han postulado como material con alto potencial biológico (e. g. antibiótico y antiviral), siendo el antibacterial muy promisorio, el cual se determina por medio de antibiogramas por difusión, punto central de esta investigación. Objetivo: evaluar la actividad antibacterial de los extractos de Peltigera laciniata (Merrill ex Riddle) Gyeln. Olmo de hoja cortada. Métodos: el material liquénico se sometió a percolación con etanol 96 por ciento. Al extracto crudo etanólico se le realizó el aislamiento de alcaloides totales y flavonoides totales con adición de HCL 3 por ciento y metanol, respectivamente. Ambas fracciones, fueron monitoreados por cromatografía de capa fina y fraccionados utilizando cromatografía de columna. Los extractos y fracciones se sometieron a bioensayos sobre Escherichia coli y Staphylococcus aureus para la valoración de los halos de inhibición, utilizando como control Sultamicilina. Los ensayos fueron realizados tres veces con 2 réplicas. Resultados: al realizar la separación cromatográfica de los alcaloides, se observó aumento de la inhibición en comparación con la mezcla alcaloidal. La fracción A1 presenta valores de inhibición cercanos al control y presentó los menores valores de inhibición con respecto a los demás tratamientos evaluados. El efecto de la fracción de los flavonoides totales tuvo menor impacto sobre E. coli y S. aureus, sin embargo, es importante destacar la acción antibacterial de los compuestos nitrogenados de tipo alcaloidal sobre microoganismos Gram positivos. Conclusiones: en el perfil químico realizado a partir de los extractos de la especie de estudio se visualizó la presencia de metabolitos secundarios de tipo alcaloide y flavonoide...(AU)

Introduction: Due to their content of secondary metabolites such as xanthones, anthraquinones and alkaloids, lichens have been suggested to be a material of high biological potential (e.g. antibiotic and antiviral). Their very promising antibacterial potential may be determined by diffusion antibiograms, the main concern of the present study. Objective: Evaluate the antibacterial activity of extracts obtained from Peltigera laciniata (Merrill ex Riddle) Gyeln, cutleaf elm. Methods: The lichenic material was percolated with 96 percent ethanol. Total alkaloids and total flavonoids were isolated from the crude ethanolic extract by adding 3 percent HCL and methanol, respectively. Both fractions were monitored by thin-layer chromatography and fractioned by column chromatography. Extracts and fractions were subjected to bioassays against Escherichia coli and Staphylococcus aureus for inhibition haloes, using sultamicillin as control. The assays were conducted 3 times with 2 replications. Results: Upon chromatographic separation of the alkaloids, an increase was observed in inhibition when compared with the alkaloidal mixture. Fraction A1 displayed inhibition values close to the control. Fraction FT showed lower inhibition values than the other treatments evaluated. The fraction of total flavonoids had a lesser impact on E. coli and S. aureus, but alkaloidal nitrogenated compounds had significant antibacterial activity against Gram-positive microorganisms. Conclusions: The chemical profile of extracts from the study species revealed the presence of alkaloidal and flavonoidal secondary metabolites, as well as the antimicrobial effect of the alkaloids contained in the extract and the fraction. This confirms the antibacterial pharmacological potential attributed to the protoberberine core(AU)

Fluorescence/luminescence-based markers for the assessment of Schistosoma mansoni schistosomula drug assays.

BACKGROUND: Schistosomiasis is responsible for a tremendous public health burden, yet only a single drug, praziquantel, is available. New antischistosomal treatments should therefore be developed. The accuracy, speed and objectivity of in vitro drug screening depend on the assay read-out. Microscopy is still the current gold standard and is in need of updating to an automated format. The aim of the present study was to investigate a panel of fluorescence/luminescence dyes for their applicability as viability markers in drug sensitivity assays for Schistosoma mansoni schistosomula. METHODS: A search for available viability and cytotoxicity marker assays and dyes was carried out and a short-list of the most interesting candidates was created. The selected kits and dyes were tested on S. mansoni Newly Transformed Schistosomula (NTS), first to assess whether they correlate with parasite viability, with comparatively low background noise, and to optimise assay conditions. Markers fulfilling these criteria were then tested in a dose-response drug assay using standard and experimental drugs and those for which an IC50 value could be accurately and reproducibly calculated were also tested on a subset of a compound library to determine their hit-identification accuracy. RESULTS: Of the 11 markers selected for testing, resazurin, Vybrant® and CellTiter-Glo® correlated best with NTS viability, produced signals ≥ 3-fold stronger than background noise and revealed a significant signal-to-NTS concentration relationship. Of these, CellTiter-Glo® could be used to accurately determine IC50 values for antischistosomals. Use of CellTiter-Glo® in a compound subset screen identified 100% of hits that were identified using standard microscopic evaluation. CONCLUSION: This study presents a comprehensive overview of the utility of colorimetric markers in drug screening. Our study demonstrates that it is difficult to develop a simple, cheap "just add" colorimetric marker-based drug assay for the larval stage of S. mansoni. CellTiter-Glo® can likely be used for endpoint go/no go screens and potentially for drug dose-response studies.

Activity of Herbal Medicines on Plasmodium falciparum Gametocytes: Implications for Malaria Transmission in Ghana.

BACKGROUND: Malaria still remains a major health issue in Ghana despite the introduction of Artemisinin-based combination therapy (ACT) coupled with other preventative measures such as the use of insecticide treated nets (ITNs). The global quest for eradication of malaria has heightened the interest of identifying drugs that target the sexual stage of the parasite, referred to as transmission-blocking drugs. This study aimed at assessing the efficacy and gametocydal effects of some commonly used herbal malaria products in Ghana. METHODOLOGY/PRINCIPAL FINDINGS: After identifying herbal anti-malarial products frequently purchased on the Ghanaian market, ten of them were selected and lyophilized. In vitro drug sensitivity testing of different concentrations of the herbal products was carried out on asexual and in vitro generated gametocytes of the 3D7 strain of Plasmodium falciparum. The efficacies of the products were assessed by microscopy. Cultures containing low dose of RT also produced the least number of late stage gametocytes. Two of the herbal products CM and RT inhibited the growth of late stage gametocytes by > 80% at 100 µg/ml whilst KG was the most inhibitory to early stage gametocytes at that same concentration. However at 1 µg/ml, only YF significantly inhibited the survival of late stage gametocytes although at that same concentration YF barely inhibited the survival of early stage gametocytes. CONCLUSIONS/SIGNIFICANCE: Herbal product RT (Aloe schweinfurthii, Khaya senegalensis, Piliostigma thonningii and Cassia siamea) demonstrated properties of a highly efficacious gametocydal product. Low dose of herbal product RT exhibited the highest gametocydal activity and at 100 µg/ml, RT exhibited >80% inhibition of late stage gametocytes. However inhibition of asexual stage parasite by RT was not optimal. Improving the asexual inhibition of RT could convert RT into an ideal antimalarial herbal product. We also found that generally C. sanguinolenta containing herbal products exhibited gametocydal activity in addition to high asexual efficacy. Herbal products with high gametocydal activity can help in the fight to reduce malaria transmission.

New compound sets identified from high throughput phenotypic screening against three kinetoplastid parasites: an open resource.

Using whole-cell phenotypic assays, the GlaxoSmithKline high-throughput screening (HTS) diversity set of 1.8 million compounds was screened against the three kinetoplastids most relevant to human disease, i.e. Leishmania donovani, Trypanosoma cruzi and Trypanosoma brucei. Secondary confirmatory and orthogonal intracellular anti-parasiticidal assays were conducted, and the potential for non-specific cytotoxicity determined. Hit compounds were chemically clustered and triaged for desirable physicochemical properties. The hypothetical biological target space covered by these diversity sets was investigated through bioinformatics methodologies. Consequently, three anti-kinetoplastid chemical boxes of ~200 compounds each were assembled. Functional analyses of these compounds suggest a wide array of potential modes of action against kinetoplastid kinases, proteases and cytochromes as well as potential host-pathogen targets. This is the first published parallel high throughput screening of a pharma compound collection against kinetoplastids. The compound sets are provided as an open resource for future lead discovery programs, and to address important research questions.

Protocols for the routine screening of drug sensitivity in the human parasite Trichomonas vaginalis.

Trichomonas vaginalis is a sexually transmitted protozoan parasite of humans. Treatment of trichomoniasis is almost completely dependent on the old drug metronidazole and is hampered by resistance. New drug development, like routine screening for drug resistance, has however been hampered by the lack of reliable screening protocols with sufficient throughput. Here we report on two separate in vitro protocols that use fluorescent dyes and allow for standardized drug sensitivity testing on the required scale.

Fragment-based screening in tandem with phenotypic screening provides novel antiparasitic hits.

Methods to discover biologically active small molecules include target-based and phenotypic screening approaches. One of the main difficulties in drug discovery is elucidating and exploiting the relationship between drug activity at the protein target and disease modification, a phenotypic endpoint. Fragment-based drug discovery is a target-based approach that typically involves the screening of a relatively small number of fragment-like (molecular weight <300) molecules that efficiently cover chemical space. Here, we report a fragment screening on TbrPDEB1, an essential cyclic nucleotide phosphodiesterase (PDE) from Trypanosoma brucei, and human PDE4D, an off-target, in a workflow in which fragment hits and a series of close analogs are subsequently screened for antiparasitic activity in a phenotypic panel. The phenotypic panel contained T. brucei, Trypanosoma cruzi, Leishmania infantum, and Plasmodium falciparum, the causative agents of human African trypanosomiasis (sleeping sickness), Chagas disease, leishmaniasis, and malaria, respectively, as well as MRC-5 human lung cells. This hybrid screening workflow has resulted in the discovery of various benzhydryl ethers with antiprotozoal activity and low toxicity, representing interesting starting points for further antiparasitic optimization.

Fast in vitro methods to determine the speed of action and the stage-specificity of anti-malarials in Plasmodium falciparum.

BACKGROUND: Recent whole cell in vitro screening campaigns identified thousands of compounds that are active against asexual blood stages of Plasmodium falciparum at submicromolar concentrations. These hits have been made available to the public, providing many novel chemical starting points for anti-malarial drug discovery programmes. Knowing which of these hits are fast-acting compounds is of great interest. Firstly, a fast action will ensure rapid relief of symptoms for the patient. Secondly, by rapidly reducing the parasitaemia, this could minimize the occurrence of mutations leading to new drug resistance mechanisms.An in vitro assay that provides information about the speed of action of test compounds has been developed by researchers at GlaxoSmithKline (GSK) in Spain. This assay also provides an in vitro measure for the ratio between parasitaemia at the onset of drug treatment and after one intra-erythrocytic cycle (parasite reduction ratio, PRR). Both parameters are needed to determine in vitro killing rates of anti-malarial compounds. A drawback of the killing rate assay is that it takes a month to obtain first results. METHODS: The approach described in the present study is focused only on the speed of action of anti-malarials. This has the advantage that initial results can be achieved within 4-7 working days, which helps to distinguish between fast and slow-acting compounds relatively quickly. It is expected that this new assay can be used as a filter in the early drug discovery phase, which will reduce the number of compounds progressing to secondary, more time-consuming assays like the killing rate assay. RESULTS: The speed of action of a selection of seven anti-malarial compounds was measured with two independent experimental procedures using modifications of the standard [3H]hypoxanthine incorporation assay. Depending on the outcome of both assays, the tested compounds were classified as either fast or non-fast-acting. CONCLUSION: The results obtained for the anti-malarials chloroquine, artesunate, atovaquone, and pyrimethamine are consistent with previous observations, suggesting the methodology is a valid way to rapidly identify fast-acting anti-malarial compounds. Another advantage of the approach is its ability to discriminate between static or cidal compound effects.

Non-peptidic cruzain inhibitors with trypanocidal activity discovered by virtual screening and in vitro assay.

A multi-step cascade strategy using integrated ligand- and target-based virtual screening methods was developed to select a small number of compounds from the ZINC database to be evaluated for trypanocidal activity. Winnowing the database to 23 selected compounds, 12 non-covalent binding cruzain inhibitors with affinity values (K i) in the low micromolar range (3-60 µM) acting through a competitive inhibition mechanism were identified. This mechanism has been confirmed by determining the binding mode of the cruzain inhibitor Nequimed176 through X-ray crystallographic studies. Cruzain, a validated therapeutic target for new chemotherapy for Chagas disease, also shares high similarity with the mammalian homolog cathepsin L. Because increased activity of cathepsin L is related to invasive properties and has been linked to metastatic cancer cells, cruzain inhibitors from the same library were assayed against it. Affinity values were in a similar range (4-80 µM), yielding poor selectivity towards cruzain but raising the possibility of investigating such inhibitors for their effect on cell proliferation. In order to select the most promising enzyme inhibitors retaining trypanocidal activity for structure-activity relationship (SAR) studies, the most potent cruzain inhibitors were assayed against T. cruzi-infected cells. Two compounds were found to have trypanocidal activity. Using compound Nequimed42 as precursor, an SAR was established in which the 2-acetamidothiophene-3-carboxamide group was identified as essential for enzyme and parasite inhibition activities. The IC50 value for compound Nequimed42 acting against the trypomastigote form of the Tulahuen lacZ strain was found to be 10.6±0.1 µM, tenfold lower than that obtained for benznidazole, which was taken as positive control. In addition, by employing the strategy of molecular simplification, a smaller compound derived from Nequimed42 with a ligand efficiency (LE) of 0.33 kcal mol(-1) atom(-1) (compound Nequimed176) is highlighted as a novel non-peptidic, non-covalent cruzain inhibitor as a trypanocidal agent candidate for optimization.