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1.
Nat Protoc ; 15(6): 1881-1921, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32341577

RESUMO

Despite decades of research, little is known about the cellular targets and the mode of action of the vast majority of antimalarial drugs. We recently demonstrated that the cellular thermal shift assay (CETSA) protocol in its two variants: the melt curve and the isothermal dose-response, represents a comprehensive strategy for the identification of antimalarial drug targets. CETSA enables proteome-wide target screening for unmodified antimalarial compounds with undetermined mechanisms of action, providing quantitative evidence about direct drug-protein interactions. The experimental workflow involves treatment of P. falciparum-infected erythrocytes with a compound of interest, heat exposure to denature proteins, soluble protein isolation, enzymatic digestion, peptide labeling with tandem mass tags, offline fractionation, and liquid chromatography-tandem mass spectrometry analysis. Methodological optimizations necessary for the analysis of this intracellular parasite are discussed, including enrichment of parasitized cells and hemoglobin depletion strategies to overcome high hemoglobin abundance in the host red blood cells. We outline an effective data processing workflow using the mineCETSA R package, which enables prioritization of drug-target candidates for follow-up studies. The entire protocol can be completed within 2 weeks.


Assuntos
Antimaláricos/farmacologia , Malária Falciparum/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/metabolismo , Descoberta de Drogas/métodos , Eritrócitos/parasitologia , Humanos , Malária Falciparum/metabolismo , Terapia de Alvo Molecular/métodos , Testes de Sensibilidade Parasitária/métodos , Plasmodium falciparum/metabolismo , Proteoma/metabolismo
2.
PLoS Negl Trop Dis ; 14(3): e0008068, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32163414

RESUMO

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.


Assuntos
Antiprotozoários/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Testes de Sensibilidade Parasitária/métodos , Nucleosídeos de Pirimidina/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/crescimento & desenvolvimento , Células 3T3 , Animais , Antiprotozoários/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Células HEK293 , Humanos , Camundongos , Nucleosídeos de Pirimidina/toxicidade
3.
PLoS Negl Trop Dis ; 13(12): e0007885, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31790397

RESUMO

Monitoring the drug susceptibility of Leishmania isolates still largely relies on standard in vitro cell-based susceptibility assays using (patient-isolated) promastigotes for infection. Although this assay is widely used, no fully standardized/harmonized protocol is yet available hence resulting in the application of a wide variety of host cells (primary cells and cell lines), different drug exposure times, detection methods and endpoint criteria. Advocacy for standardization to decrease inter-laboratory variation and improve interpretation of results has already repeatedly been made, unfortunately still with unsatisfactory progress. As a logical next step, it would be useful to reach at least some agreement on the type of host cell and basic experimental design for routine amastigote susceptibility determination. The present laboratory study using different L. infantum strains as a model for visceral leishmaniasis species compared primary cells (mouse peritoneal exudate (PEC), mouse bone marrow derived macrophages and human peripheral blood monocyte derived macrophages) and commercially available cell lines (THP-1, J774, RAW) for either their susceptibility to infection, their role in supporting intracellular amastigote multiplication and overall feasibility/accessibility of experimental assay protocol. The major findings were that primary cells are better than cell lines in supporting infection and intracellular parasite multiplication, with PECs to be preferred for technical reasons. Cell lines require drug exposure of >96h with THP-1 to be preferred but subject to a variable response to PMA stimulation. The fast dividing J774 and RAW cells out-compete parasite-infected cells precluding proper assay read-out. Some findings could possibly also be applicable to cutaneous Leishmania strains, but this still needs cross-checking. Besides inherent limitations in a clinical setting, susceptibility testing of clinical isolates may remain problematic because of the reliance on patient-derived promastigotes which may exhibit variable degrees of metacyclogenesis and infectivity.


Assuntos
Antiprotozoários/farmacologia , Leishmania infantum/efeitos dos fármacos , Testes de Sensibilidade Parasitária/métodos , Animais , Células Cultivadas , Feminino , Humanos , Leishmania infantum/crescimento & desenvolvimento , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/parasitologia , Camundongos , Testes de Sensibilidade Parasitária/normas
4.
Acta Microbiol Immunol Hung ; 67(1): 23-32, 2019 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-31833381

RESUMO

We aimed to compare LDH release assay, trypan blue and fluorescent stainings, and non-nutrient Escherichia coli plate assay in determining treatment efficacy of antiamoebic agents against Acanthamoeba castellanii trophozoites/cysts, in vitro. 1BU trophozoites/cysts were challenged with 0.02% polyhexamethylene biguanid (PHMB), 0.1% propamidine isethionate (PD), and 0.0065% miltefosine (MF). Efficacies of the drugs were determined by LDH release and trypan blue assays, by Hoechst 33343, calcein-AM, and ethidium homodimer-1 fluorescent dyes, and by a non-nutrient agar E. coli plate assay. All three antiamoebic agents induced a significant LDH release from trophozoites, compared to controls (p < 0.0001). Fluorescent-dye staining in untreated 1BU trophozoites/cysts was negligible, but using antiamoebic agents, there was 59.3%-100% trypan blue, 100% Hoechst 33342, 0%-75.3% calcein-AM, and 100% ethidium homodimer-1 positivity. On E. coli plates, in controls and MF-treated 1BU trophozoites/cysts, new trophozoites appeared within 24 h, encystment occurred after 5 weeks. In PHMB- and PD-treated 1BU throphozoites/cysts, irregularly shaped, smaller trophozoites appeared after 72 h, which failed to form new cysts within 5 weeks. None of the enzymatic- and dye-based viability assays tested here generated survival rates for trophozoites/cysts that were comparable with those yielded with the non-nutrient agar E. coli plate assay, suggesting that the culture-based assay is the best method to study the treatment efficacy of drugs against Acanthamoeba.


Assuntos
Acanthamoeba castellanii/efeitos dos fármacos , Antiparasitários/farmacologia , Testes de Sensibilidade Parasitária/métodos , Trofozoítos/efeitos dos fármacos , Escherichia coli , Fluorescência , L-Lactato Desidrogenase/análise , Coloração e Rotulagem
5.
Parasit Vectors ; 12(1): 493, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31640761

RESUMO

BACKGROUND: Over the past five years, as a public service to encourage and accelerate drug discovery for diseases of poverty, the Medicines for Malaria Venture (MMV) has released box sets of 400 compounds named the Malaria, Pathogen and Stasis Boxes. Here, we screened the Pathogen Box against the post-infective larvae (schistosomula) of Schistosoma mansoni using assays particular to the three contributing institutions, namely, the University of California San Diego (UCSD) in the USA, the Swiss Tropical and Public Health Institute (Swiss TPH) in Switzerland, and the Fundação Oswaldo Cruz (FIOCRUZ) in Brazil. With the same set of compounds, the goal was to determine the degree of inter-assay variability and identify a core set of active compounds common to all three assays. New drugs for schistosomiasis would be welcome given that current treatment and control strategies rely on chemotherapy with just one drug, praziquantel. METHODS: Both the UCSD and Swiss TPH assays utilize daily observational scoring methodologies over 72 h, whereas the FIOCRUZ assay employs XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) at 72 h to measure viability as a function of NAD+/NADH redox state. Raw and transformed data arising from each assay were assembled for comparative analysis. RESULTS: For the UCSD and Swiss TPH assays, there was strong concordance of at least 87% in identifying active and inactive compounds on one or more of the three days. When all three assays were compared at 72 h, concordance remained a robust 74%. Further, robust Pearson's correlations (0.48-0.68) were measured between the assays. Of those actives at 72 h, the UCSD, Swiss TPH and FIOCRUZ assays identified 86, 103 and 66 compounds, respectively, of which 35 were common. Assay idiosyncrasies included the identification of unique compounds, the differential ability to identify known antischistosomal compounds and the concept that compounds of interest might include those that increase metabolic activity above baseline. CONCLUSIONS: The inter-assay data generated were in good agreement, including with previously reported data. A common set of antischistosomal molecules for further exploration has been identified .


Assuntos
Descoberta de Drogas/métodos , Testes de Sensibilidade Parasitária/métodos , Schistosoma mansoni/efeitos dos fármacos , Esquistossomose mansoni/tratamento farmacológico , Esquistossomicidas/farmacologia , Animais , Biomphalaria/parasitologia , Cricetinae , Feminino , Larva/classificação , Larva/efeitos dos fármacos , Estágios do Ciclo de Vida , Mesocricetus , Testes de Sensibilidade Parasitária/normas , Fenótipo , Schistosoma mansoni/classificação , Schistosoma mansoni/crescimento & desenvolvimento , Esquistossomicidas/uso terapêutico
6.
Artigo em Inglês | MEDLINE | ID: mdl-31451503

RESUMO

Trichomoniasis is a sexually transmitted disease with hundreds of millions of annual cases worldwide. Approved treatment options are limited to two related nitro-heterocyclic compounds, yet resistance to these drugs is an increasing concern. New antimicrobials against the causative agent, Trichomonas vaginalis, are urgently needed. We show here that clinically approved anticancer drugs that inhibit the proteasome, a large protease complex with a critical role in degrading intracellular proteins in eukaryotes, have submicromolar activity against the parasite in vitro and on-target activity against the enriched T. vaginalis proteasome in cell-free assays. Proteomic analysis confirmed that the parasite has all seven α and seven ß subunits of the eukaryotic proteasome although they have only modest sequence identities, ranging from 28 to 52%, relative to the respective human proteasome subunits. A screen of proteasome inhibitors derived from a marine natural product, carmaphycin, revealed one derivative, carmaphycin-17, with greater activity against T. vaginalis than the reference drug metronidazole, the ability to overcome metronidazole resistance, and reduced human cytotoxicity compared to that of the anticancer proteasome inhibitors. The increased selectivity of carmaphycin-17 for T. vaginalis was related to its >5-fold greater potency against the ß1 and ß5 catalytic subunits of the T. vaginalis proteasome than against the human proteasome subunits. In a murine model of vaginal trichomonad infection, proteasome inhibitors eliminated or significantly reduced parasite burden upon topical treatment without any apparent adverse effects. Together, these findings validate the proteasome of T. vaginalis as a therapeutic target for development of a novel class of trichomonacidal agents.


Assuntos
Antitricômonas/farmacologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/genética , Vaginite por Trichomonas/tratamento farmacológico , Trichomonas vaginalis/efeitos dos fármacos , Trichomonas vaginalis/genética , Sequência de Aminoácidos , Animais , Anti-Infecciosos/farmacologia , Citoplasma/parasitologia , Resistência a Medicamentos/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Parasitária/métodos , Proteômica/métodos , Doenças Sexualmente Transmissíveis/tratamento farmacológico , Doenças Sexualmente Transmissíveis/parasitologia , Tricomoníase/tratamento farmacológico , Tricomoníase/parasitologia , Vaginite por Trichomonas/parasitologia
7.
BMC Infect Dis ; 19(1): 593, 2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31286895

RESUMO

BACKGROUND: Current drug regimens for cutaneous leishmaniasis (CL) include toxic systemic therapies such as amphotericin B (AB) and pentavalent antimonials. Fluconazole (FZ) is a well-tolerated potential oral alternative for the management CL. To date, few objective data exist to guide clinical decision-making when selecting a therapeutic agent a priori, and standardized, clinically-approved drug susceptibility testing platforms for Leishmania spp. have yet to be established. The Sensititre™ YeastOne™ YO9 plate is a commercialized drug susceptibility plate including AB and FZ used for routine testing of non-fastidious yeast. Our objective was to adapt the readily available Sensititre™ YeastOne™ YO9 plate, to determine drug susceptibility profiles of AB and FZ in cultured isolates of Old World and New World Leishmania spp. for the treatment of CL. METHODS: Promastigotes were cultured in Tobie's medium with Locke's overlay until log phase growth was achieved, inoculated into the Sensititre™ system, and incubated over 96 H. minimum inhibitory concentrations (MICs) were determined colorimetrically, and promastigote death was assessed by conventional microscopy out to 96- h. Colour change correlated to MIC values. RESULTS: All strains tested exhibited MIC values for FZ that were ≥ 256 µg/mL. New World strains demonstrated reduced susceptibility to AB (0.25 µg/mL - 0.50 µg/mL AB) compared to Old World strains at 0.12 µg/mL AB (p = 0.02). Seventeen (61%) of 28 Viannia isolates versus 82% (27/33) of non-Viannia isolates were resistant at 0.12 µg/mL AB (p = 0.09). For L. V. braziliensis isolates, mean MIC for AB was 0.375 ± 0.14 µg/mL (range 0.25-0.50 µg/mL), while for isolates of L. V. panamensis it was 0.314 ± 0.26 µg/mL (range 0.12-1.0 µg/mL). CONCLUSIONS: We adapted the Sensititre™ YeastOne™ YO9 plate for testing of Leishmania spp. susceptibility profiles for commonly used antifungals in the treatment of CL, including AB and FZ. Given its current utility in mycology, optimization of the system for potential clinical implementation in parasitology should be pursued. However evaluation of clinically relevant amastigote-stage stages, and higher concentrations of FZ beyond the upper limit concentration of the Sensititre™ YeastOne™ Y09 plate would be required.


Assuntos
Anfotericina B/farmacologia , Fluconazol/farmacologia , Leishmania/efeitos dos fármacos , Testes de Sensibilidade Parasitária/métodos , Humanos , Leishmaniose/parasitologia , Testes de Sensibilidade Microbiana
8.
Vet Parasitol ; 271: 7-13, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31303208

RESUMO

In the current study, the egg hatch test (EHT) has been evaluated as an in vitro technique to detect albendazole (ABZ) resistance in Fasciola hepatica. The intra- and inter-assay variations of the EHT were measured by means of the coefficient of variation in different fluke isolates and over time; then, the results of the EHT were compared with the "gold standard" controlled efficacy test, which assesses the in vivo anthelmintic efficacy. The EHT was used later to evaluate the intra-herd variability regarding the level of ABZ resistance in calves infected by the same fluke isolate. Finally, several factors of the initial protocol were modified to improve the simplicity of the assay, including the incubation time of eggs with the drug and the use of eggs collected from faeces. The greatest uniformity between results within the assay and over time until 8 weeks after gallbladder collection (the deadline proposed for egg analysis) was obtained with an ABZ concentration of 0.5 µM. The length of exposure to ABZ was shown to be critical, as prolonged incubation (15 days) led to a change of ovicidal activity. The ABZ concentration of 0.5 µM is suggested as a possible discriminating dose to predict ABZ resistance, due to the close agreement between the results of the EHT at an ABZ concentration of 0.5 µM and those of the in vivo assays.


Assuntos
Albendazol/farmacologia , Doenças dos Bovinos/parasitologia , Fasciola hepatica/efeitos dos fármacos , Fasciolíase/veterinária , Testes de Sensibilidade Parasitária/métodos , Animais , Anti-Helmínticos/farmacologia , Bovinos , Doenças dos Bovinos/diagnóstico , Resistência a Medicamentos , Fasciolíase/diagnóstico , Fasciolíase/parasitologia , Fezes/parasitologia , Óvulo/efeitos dos fármacos , Fatores de Tempo
9.
SLAS Discov ; 24(7): 755-765, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31180789

RESUMO

The current methodologies used to identify promising new anthelmintic compounds rely on subjective microscopic examination of worm motility or involve genetic modified organisms. We describe a new methodology to detect worm viability that takes advantage of the differential incorporation of the fluorescent molecular marker propidium iodide and the 2,1,3-benzothiadiazole core, which has been widely applied in light technology. The new assay developed could be validated using the "Pathogen Box" library. By use of this bioassay, it was possible to identify three molecules with activity against Caenorhabditis elegans that were previously described as effective in in vitro assays against other pathogens, such as Schistosoma mansoni, Mycobacterium tuberculosis, and Plasmodium falciparum, accelerating the identification of molecules with anthelmintic potential. The current fluorescence-based bioassay may be used for assessing C. elegans viability. The described methodology replaces the subjectivity of previous assays and provides an enabling technology that is useful for rapid in vitro screens of both natural and synthetic compound libraries. It is expected that the results obtained from these robust in vitro screens would select the most effective compounds for follow-up in vivo experimentation with pathogenic helminths.


Assuntos
Anti-Helmínticos/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Descoberta de Drogas/métodos , Corantes Fluorescentes/química , Testes de Sensibilidade Parasitária/métodos , Tiadiazóis/química , Animais , Cinética , Estrutura Molecular , Imagem Óptica/métodos
10.
Biomaterials ; 216: 119221, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31195301

RESUMO

Hypnozoites are the liver stage non-dividing form of the malaria parasite that are responsible for relapse and acts as a natural reservoir for human malaria Plasmodium vivax and P. ovale as well as a phylogenetically related simian malaria P. cynomolgi. Our understanding of hypnozoite biology remains limited due to the technical challenge of requiring the use of primary hepatocytes and the lack of robust and predictive in vitro models. In this study, we developed a malaria liver stage model using 3D spheroid-cultured primary hepatocytes. The infection of primary hepatocytes in suspension led to increased infectivity of both P. cynomolgi and P. vivax infections. We demonstrated that this hepatic spheroid model was capable of maintaining long term viability, hepatocyte specific functions and cell polarity which enhanced permissiveness and thus, permitting for the complete development of both P. cynomolgi and P. vivax liver stage parasites in the infected spheroids. The model described here was able to capture the full liver stage cycle starting with sporozoites and ending in the release of hepatic merozoites capable of invading simian erythrocytes in vitro. Finally, we showed that this system can be used for compound screening to discriminate between causal prophylactic and cidal antimalarials activity in vitro for relapsing malaria.


Assuntos
Antimaláricos/farmacologia , Hepatócitos/parasitologia , Malária/tratamento farmacológico , Plasmodium/efeitos dos fármacos , Animais , Técnicas de Cultura de Células/métodos , Linhagem Celular , Células Cultivadas , Hepatócitos/citologia , Humanos , Fígado/citologia , Fígado/parasitologia , Macaca fascicularis , Macaca mulatta , Testes de Sensibilidade Parasitária/métodos , Recidiva , Prevenção Secundária , Esferoides Celulares/citologia , Esferoides Celulares/parasitologia , Esporozoítos/efeitos dos fármacos
11.
EBioMedicine ; 43: 370-379, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31027918

RESUMO

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.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Ácido Mefenâmico/farmacologia , Testes de Sensibilidade Parasitária , Schistosoma/efeitos dos fármacos , Esquistossomicidas/farmacologia , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Feminino , Humanos , Ácido Mefenâmico/administração & dosagem , Camundongos , Testes de Sensibilidade Parasitária/métodos , Schistosoma mansoni/efeitos dos fármacos , Esquistossomose/tratamento farmacológico , Esquistossomose/parasitologia , Esquistossomicidas/administração & dosagem
12.
PLoS Negl Trop Dis ; 13(1): e0007108, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30653499

RESUMO

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.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Filaricidas/farmacologia , Onchocerca volvulus/efeitos dos fármacos , Onchocerca volvulus/crescimento & desenvolvimento , Testes de Sensibilidade Parasitária/métodos , Animais , Feminino , Masculino
13.
PLoS Negl Trop Dis ; 13(1): e0007088, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30640901

RESUMO

Trypanosoma cruzi has three distinct life cycle stages; epimastigote, trypomastigote, and amastigote. Amastigote is the replication stage in host mammalian cells, hence this stage of parasite has clinical significance in drug development research. Presence of extracellular amastigotes (EA) and their infection capability have been known for some decades. Here, we demonstrate that EA can be utilized as an axenic culture to aid in stage-specific study of T. cruzi. Amastigote-like property of axenic amastigote can be sustained in LIT medium at 37°C at least for 1 week, judging from their morphology, amastigote-specific UTR-regulated GFP expression, and stage-specific expression of selected endogenous genes. Inhibitory effect of benznidazole and nifurtimox on axenic amastigotes was comparable to that on intracellular amastigotes. Exogenous nucleic acids can be transfected into EA via conventional electroporation, and selective marker could be utilized for enrichment of transfectants. We also demonstrate that CRISPR/Cas9-mediated gene knockout can be performed in EA. Essentiality of the target gene can be evaluated by the growth capability of the knockout EA, either by continuation of axenic culturing or by host infection and following replication as intracellular amastigotes. By taking advantage of the accessibility and sturdiness of EA, we can potentially expand our experimental freedom in studying amastigote stage of T. cruzi.


Assuntos
Expressão Gênica , Técnicas de Inativação de Genes/métodos , Genética Microbiana/métodos , Biologia Molecular/métodos , Testes de Sensibilidade Parasitária/métodos , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/genética , Antiprotozoários/farmacologia , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Eletroporação , Nifurtimox/farmacologia , Nitroimidazóis/farmacologia , Trypanosoma cruzi/crescimento & desenvolvimento
14.
Biotechniques ; 66(4): 179-185, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30543114

RESUMO

Automated cell counters that utilize still images of sample cells are widely used. However, they are not well suited to counting slender, aggregate-prone microorganisms such as Trypanosoma cruzi. Here, we developed a motion-based cell-counting system, using an image-recognition method based on a cubic higher-order local auto-correlation feature. The software successfully estimated the cell density of dispersed, aggregated, as well as fluorescent parasites by motion pattern recognition. Loss of parasites activeness due to drug treatment could also be detected as a reduction in apparent cell count, which potentially increases the sensitivity of drug screening assays. Moreover, the motion-based approach enabled estimation of the number of parasites in a co-culture with host mammalian cells, by disregarding the presence of the host cells as a static background.


Assuntos
Contagem de Células/métodos , Processamento de Imagem Assistida por Computador/métodos , Imagem Óptica/métodos , Reconhecimento Automatizado de Padrão/métodos , Trypanosoma cruzi/isolamento & purificação , Doença de Chagas/parasitologia , Humanos , Aprendizado de Máquina , Microscopia de Fluorescência/métodos , Movimento (Física) , Testes de Sensibilidade Parasitária/métodos , Software , Trypanosoma cruzi/citologia
15.
Mem Inst Oswaldo Cruz ; 113(12): e180279, 2018 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-30540020

RESUMO

BACKGROUND The main strategy to control human malaria still relies on specific drug treatment, limited now by Plasmodium falciparum-resistant parasites, including that against artemisinin derivatives. Despite the large number of active compounds described in the literature, few of them reached full development against human malaria. Drug repositioning is a fast and less expensive strategy for antimalarial drug discovery, because these compounds are already approved for human use. OBJECTIVES To identify new antimalarial drugs from compounds commercially available and used for other indications. METHODS Accuvit®, Ginkgo® and Soyfit®, rich in flavonoids, and also the standard flavonoids, hesperidin, quercetin, and genistein were tested against blood cultures of chloroquine-resistant P. falciparum, as well as chloroquine, a reference antimalarial. Inhibition of parasite growth was measured in immunoenzymatic assay with monoclonal anti-P. falciparum antibodies, specific to the histidine-rich protein II. Tests in mice with P. berghei malaria were based on percent of parasitaemia reduction. These compounds were also evaluated for in vitro cytotoxicity. FINDINGS The inhibition of parasite growth in vitro showed that Accuvit® was the most active drug (IC50 5 ± 3.9 µg/mL). Soyfit® was partially active (IC50 13.6 ± 7.7 µg/mL), and Ginkgo® (IC50 38.4 ± 14 µg/mL) was inactive. All such compounds were active in vivo at a dose of 50 mg/kg body weight. Accuvit® and quercetin induced the highest reduction of P. berghei parasitaemia (63% and 53%, respectively) on day 5 after parasite inoculation. As expected, the compounds tested were not toxic. MAIN CONCLUSIONS The antimalarial activity of Accuvit® was not related to flavonoids only, and it possibly results from synergisms with other compounds present in this drug product, such as multivitamins. Multivitamins in Accuvit® may explain its effect against the malaria parasites. This work demonstrated for the first time the activity of these drugs, which are already marketed.


Assuntos
Antimaláricos/química , Antimaláricos/farmacologia , Genisteína/farmacologia , Hesperidina/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Quercetina/farmacologia , Animais , Modelos Animais de Doenças , Resistência a Medicamentos , Feminino , Dose Letal Mediana , Malária/tratamento farmacológico , Camundongos , Parasitemia/tratamento farmacológico , Testes de Sensibilidade Parasitária/métodos
16.
Future Med Chem ; 10(22): 2619-2639, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30499742

RESUMO

In the absence of clinically proven vaccines and emerging resistance to common antimalarials and insecticides, the onus of interrupting the life cycle of Plasmodium falciparum, is upon the transmission-blocking drugs. Current transmission-blocking drug primaquine finds its use restricted because of associated hemolytic toxicity issues in Glucose-6-Phosphate-Dehydrogenase deficient individuals. This article provides an extensive review of the assays used by the investigators to evaluate the transmission-blocking activity of drugs. Furthermore, limitations in existing transmission-blocking assessment approaches/studies are also covered in detail. This review is expected to help in the identification of lacunae in current understanding of transmission-blocking strategies, which are hindering our efforts to develop sustainable and effective transmission-blocking interventions.


Assuntos
Antimaláricos/farmacologia , Descoberta de Drogas/métodos , Malária/tratamento farmacológico , Malária/transmissão , Plasmodium/efeitos dos fármacos , Animais , Antimaláricos/uso terapêutico , Gametogênese/efeitos dos fármacos , Humanos , Estágios do Ciclo de Vida/efeitos dos fármacos , Malária/parasitologia , Terapia de Alvo Molecular/métodos , Testes de Sensibilidade Parasitária/métodos , Plasmodium/crescimento & desenvolvimento
17.
J Biol Chem ; 293(52): 19974-19981, 2018 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-30463941

RESUMO

Human babesiosis is an emerging tick-borne disease caused by apicomplexan parasites of the genus Babesia Clinical cases caused by Babesia duncani have been associated with high parasite burden, severe pathology, and death. In both mice and hamsters, the parasite causes uncontrolled fulminant infections, which ultimately lead to death. Resolving these infections requires knowledge of B. duncani biology, virulence, and susceptibility to anti-infectives, but little is known and further research is hindered by a lack of relevant model systems. Here, we report the first continuous in vitro culture of B. duncani in human red blood cells. We show that during its asexual cycle within human erythrocytes, B. duncani develops and divides to form four daughter parasites with parasitemia doubling every ∼22 h. Using this in vitro culture assay, we found that B. duncani has low susceptibility to the four drugs recommended for treatment of human babesiosis, atovaquone, azithromycin, clindamycin, and quinine, with IC50 values ranging between 500 nm and 20 µm These data suggest that current practices are of limited effect in treating the disease. We anticipate this new disease model will set the stage for a better understanding of the biology of this parasite and will help guide better therapeutic strategies to treat B. duncani-associated babesiosis.


Assuntos
Antiparasitários/farmacologia , Babesia/efeitos dos fármacos , Babesiose/tratamento farmacológico , Babesiose/parasitologia , Eritrócitos/parasitologia , Testes de Sensibilidade Parasitária/métodos , Atovaquona/farmacologia , Azitromicina/farmacologia , Babesia/crescimento & desenvolvimento , Técnicas de Cultura de Células/métodos , Clindamicina/farmacologia , Humanos , Quinina/farmacologia
18.
ACS Sens ; 3(12): 2613-2620, 2018 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-30426744

RESUMO

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.


Assuntos
Impedância Elétrica , Técnicas Eletroquímicas/métodos , Mefloquina/farmacologia , Técnicas Analíticas Microfluídicas/métodos , Esquistossomicidas/farmacologia , Animais , Dimetil Sulfóxido/química , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Técnicas Eletroquímicas/instrumentação , Eletrodos , Desenho de Equipamento , Técnicas Analíticas Microfluídicas/instrumentação , Testes de Sensibilidade Parasitária/instrumentação , Testes de Sensibilidade Parasitária/métodos , Schistosoma mansoni/efeitos dos fármacos
19.
Parasitol Res ; 117(11): 3367-3380, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30232605

RESUMO

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.


Assuntos
Doença de Chagas/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos/métodos , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Doença de Chagas/parasitologia , Humanos , Estágios do Ciclo de Vida/efeitos dos fármacos , Camundongos , Modelos Teóricos , Nitroimidazóis/farmacologia , Testes de Sensibilidade Parasitária/métodos
20.
Artigo em Inglês | MEDLINE | ID: mdl-29987140

RESUMO

Statins are inhibitors of cholesterol synthesis, but other biological properties, such as antimicrobial effects, have also been assigned to them, leading to their designation as pleiotropic agents. Our goal was to investigate the activity and selectivity of atorvastatin (AVA) against Trypanosoma cruzi by using in vitro models, aiming for more effective and safer therapeutic options through drug repurposing proposals for monotherapy and therapy in combination with benznidazole (BZ). Phenotypic screening was performed with different strains (Tulahuen [discrete typing unit {DTU} VI] and Y [DTU II]) and forms (intracellular forms, bloodstream trypomastigotes, and tissue-derived trypomastigotes) of the parasite. On assay of the Tulahuen strain, AVA was more active against intracellular amastigotes (selectivity index [SI] = 3). Also, against a parasite of another DTU (Y strain), this statin was more active (2.1-fold) and selective (2.4-fold) against bloodstream trypomastigotes (SI = 51) than against the intracellular forms (SI = 20). A cytomorphological approach using phalloidin-rhodamine permitted us to verify that AVA did not induced cell density reduction and that cardiac cells (CC) maintained their typical cytoarchitecture. Combinatory approaches using fixed-ratio methods showed that AVA and BZ gave synergistic interactions against both trypomastigotes and intracellular forms (mean sums of fractional inhibitory concentration indexes [∑FICIs] of 0.46 ± 0.12 and 0.48 ± 0.03, respectively). Thus, the repurposing strategy for AVA, especially in combination with BZ, which leads to a synergistic effect, is encouraging for future studies to identify novel therapeutic protocols for Chagas disease treatment.


Assuntos
Atorvastatina/farmacologia , Doença de Chagas/tratamento farmacológico , Nitroimidazóis/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Linhagem Celular , Doença de Chagas/parasitologia , Reposicionamento de Medicamentos/métodos , Sinergismo Farmacológico , Quimioterapia Combinada/métodos , Coração/parasitologia , Camundongos , Testes de Sensibilidade Parasitária/métodos
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