RESUMEN
Drug combinations and drug repurposing have emerged as promising strategies to develop novel treatments for infectious diseases, including Chagas disease. In this study, we aimed to investigate whether the repurposed drugs chloroquine (CQ) and colchicine (COL), known to inhibit Trypanosoma cruzi infection in host cells, could boost the anti-T. cruzi effect of the trypanocidal drug benznidazole (BZN), increasing its therapeutic efficacy while reducing the dose needed to eradicate the parasite. The combination of BZN and COL exhibited cytotoxicity to infected cells and low antiparasitic activity. Conversely, a combination of BZN and CQ significantly reduced T. cruzi infection in vitro, with no apparent cytotoxicity. This effect seemed to be consistent across different cell lines and against both the partially BZN-resistant Y and the highly BZN-resistant Colombiana strains. In vivo experiments in an acute murine model showed that the BZN+CQ combination was eight times more effective in reducing T. cruzi infection in the acute phase than BZN monotherapy. In summary, our results demonstrate that the concomitant administration of CQ and BZN potentiates the trypanocidal activity of BZN, leading to a reduction in the dose needed to achieve an effective response. In a translational context, it could represent a higher efficacy of treatment while also mitigating the adverse effects of high doses of BZN. Our study also reinforces the relevance of drug combination and repurposing approaches in the field of Chagas disease drug discovery.
Asunto(s)
Enfermedad de Chagas , Nitroimidazoles , Tripanocidas , Trypanosoma cruzi , Ratones , Animales , Reposicionamiento de Medicamentos , Cloroquina/farmacología , Cloroquina/uso terapéutico , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/parasitología , Nitroimidazoles/farmacología , Nitroimidazoles/uso terapéutico , Tripanocidas/farmacología , Tripanocidas/uso terapéuticoRESUMEN
Infection by Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, depends on reactive oxygen species (ROS), which has been described to induce parasite proliferation in mammalian host cells. It is unknown how the parasite manages to increase host ROS levels. Here, we found that intracellular T. cruzi forms release in the host cytosol its major cyclophilin of 19 kDa (TcCyp19). Parasites depleted of TcCyp19 by using CRISPR/Cas9 gene replacement proliferate inefficiently and fail to increase ROS, compared to wild type parasites or parasites with restored TcCyp19 gene expression. Expression of TcCyp19 in L6 rat myoblast increased ROS levels and restored the proliferation of TcCyp19 depleted parasites. These events could also be inhibited by cyclosporin A, (a cyclophilin inhibitor), and by polyethylene glycol-linked to antioxidant enzymes. TcCyp19 was found more concentrated in the membrane leading edges of the host cells in regions that also accumulate phosphorylated p47phox , as observed to the endogenous cyclophilin A, suggesting some mechanisms involved with the translocation process of the regulatory subunit p47phox in the activation of the NADPH oxidase enzymatic complex. We concluded that cyclophilin released in the host cell cytosol by T. cruzi mediates the increase of ROS, required to boost parasite proliferation in mammalian hosts.
Asunto(s)
Ciclofilinas/metabolismo , Citosol/metabolismo , Interacciones Huésped-Parásitos , Especies Reactivas de Oxígeno/metabolismo , Trypanosoma cruzi/crecimiento & desarrollo , Trypanosoma cruzi/metabolismo , Animales , Ciclofilinas/biosíntesis , Ciclofilinas/genética , Citosol/química , Mioblastos/parasitología , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Ratas , Trypanosoma cruzi/genéticaRESUMEN
Chagas disease is an illness caused by the protozoan parasite Trypanosoma cruzi, affecting more than 7 million people in the world. Benznidazole and nifurtimox are the only drugs available for treatment and in addition to causing several side effects, are only satisfactory in the acute phase of the disease. Sirtuins are NAD+-dependent deacetylases involved in several biological processes, which have become drug target candidates in various disease settings. T. cruzi presents two sirtuins, one cytosolic (TcSir2rp1) and the latter mitochondrial (TcSir2rp3). Here, we characterized the effects of human sirtuin inhibitors against T. cruzi sirtuins as an initial approach to develop specific parasite inhibitors. We found that, of 33 compounds tested, two inhibited TcSir2rp1 (15 and 17), while other five inhibited TcSir2rp3 (8, 12, 13, 30, and 32), indicating that specific inhibitors can be devised for each one of the enzymes. Furthermore, all inhibiting compounds prevented parasite proliferation in cultured mammalian cells. When combining the most effective inhibitors with benznidazole at least two compounds, 17 and 32, demonstrated synergistic effects. Altogether, these results support the importance of exploring T. cruzi sirtuins as drug targets and provide key elements to develop specific inhibitors for these enzymes as potential targets for Chagas disease treatment.
Asunto(s)
Enfermedad de Chagas/tratamiento farmacológico , Nitroimidazoles/farmacología , Sirtuinas/antagonistas & inhibidores , Sirtuinas/metabolismo , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Animales , Línea Celular , Sinergismo Farmacológico , Células Epiteliales/efectos de los fármacos , Células Epiteliales/parasitología , Histona Desacetilasas del Grupo III/antagonistas & inhibidores , Concentración 50 Inhibidora , Macaca mulatta , Simulación del Acoplamiento Molecular , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sirtuinas/química , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/genética , Trypanosoma cruzi/patogenicidadRESUMEN
High genetic and phenotypic variability between Leishmania species and strains within species make the development of broad-spectrum antileishmanial drugs challenging. Thus, screening panels consisting of several diverse Leishmania species can be useful in enabling compound prioritization based on their spectrum of activity. In this study, a robust and reproducible high content assay was developed, and 1280 small molecules were simultaneously screened against clinically relevant cutaneous and visceral species: L. amazonensis, L. braziliensis, and L. donovani. The assay is based on THP-1 macrophages infected with stationary phase promastigotes and posterior evaluation of both compound antileishmanial activity and host cell toxicity. The profile of compound activity was species-specific, and out of 51 active compounds, only 14 presented broad-spectrum activity against the three species, with activities ranging from 52% to 100%. Notably, the compounds CB1954, Clomipramine, Maprotiline, Protriptyline, and ML-9 presented pan-leishmanial activity, with efficacy greater than 70%. The results highlight the reduced number of compound classes with pan-leishmanial activity that might be available from diversity libraries, emphasizing the need to screen active compounds against a panel of species and strains. The assay reported here can be adapted to virtually any Leishmania species without the need for genetic modification of parasites, providing the basis for the discovery of broad spectrum anti-leishmanial agents.
Asunto(s)
Leishmaniasis/tratamiento farmacológico , Animales , Antiprotozoarios/uso terapéutico , Evaluación Preclínica de Medicamentos , Humanos , Leishmania/efectos de los fármacos , Leishmania/patogenicidad , Leishmaniasis Visceral/tratamiento farmacológico , Maprotilina/química , Ratones , Protriptilina/química , Especificidad de la Especie , Células THP-1RESUMEN
Leishmaniasis are infectious diseases caused by parasites of genus Leishmania that affect affects 12 million people in 98 countries mainly in Africa, Asia, and Latin America. Effective treatments for this disease are urgently needed. In this study, we present a computer-aided approach to investigate a set of 32 recently synthesized chalcone and chalcone-like compounds to act as antileishmanial agents. As a result, nine most promising compounds and three potentially inactive compounds were experimentally evaluated against Leishmania infantum amastigotes and mammalian cells. Four compounds exhibited EC50 in the range of 6.2-10.98µM. In addition, two compounds, LabMol-65 and LabMol-73, exhibited cytotoxicity in macrophages >50µM that resulted in better selectivity compared to standard drug amphotericin B. These two compounds also demonstrated low cytotoxicity and high selectivity towards Vero cells. The results of target fishing followed by homology modeling and docking studies suggest that these chalcone compounds could act in Leishmania because of their interaction with cysteine proteases, such as procathepsin L. Finally, we have provided structural recommendations for designing new antileishmanial chalcones.
Asunto(s)
Antiprotozoarios/farmacología , Chalconas/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Leishmania infantum/efectos de los fármacos , Nitrofuranos/farmacología , Piperazinas/farmacología , Piperidinas/farmacología , Anfotericina B/farmacología , Animales , Antiprotozoarios/síntesis química , Antiprotozoarios/química , Chalconas/síntesis química , Chalconas/química , Chlorocebus aethiops , Simulación por Computador , Inhibidores de Cisteína Proteinasa/síntesis química , Inhibidores de Cisteína Proteinasa/química , Bases de Datos Factuales , Descubrimiento de Drogas , Humanos , Simulación del Acoplamiento Molecular , Nitrofuranos/síntesis química , Nitrofuranos/química , Piperazinas/síntesis química , Piperazinas/química , Piperidinas/síntesis química , Piperidinas/química , Relación Estructura-Actividad , Células VeroRESUMEN
Flavonoids have previously been identified as antiparasitic agents and pteridine reductase 1 (PTR1) inhibitors. Herein, we focus our attention on the chroman-4-one scaffold. Three chroman-4-one analogues (1-3) of previously published chromen-4-one derivatives were synthesized and biologically evaluated against parasitic enzymes (Trypanosoma brucei PTR1-TbPTR1 and Leishmania major-LmPTR1) and parasites (Trypanosoma brucei and Leishmania infantum). A crystal structure of TbPTR1 in complex with compound 1 and the first crystal structures of LmPTR1-flavanone complexes (compounds 1 and 3) were solved. The inhibitory activity of the chroman-4-one and chromen-4-one derivatives was explained by comparison of observed and predicted binding modes of the compounds. Compound 1 showed activity both against the targeted enzymes and the parasites with a selectivity index greater than 7 and a low toxicity. Our results provide a basis for further scaffold optimization and structure-based drug design aimed at the identification of potent anti-trypanosomatidic compounds targeting multiple PTR1 variants.
Asunto(s)
Antiparasitarios/química , Antiparasitarios/farmacología , Cromanos/química , Cromanos/farmacología , Oxidorreductasas/antagonistas & inhibidores , Antiparasitarios/síntesis química , Sitios de Unión , Cromanos/síntesis química , Activación Enzimática/efectos de los fármacos , Concentración 50 Inhibidora , Leishmania major/efectos de los fármacos , Leishmania major/enzimología , Conformación Molecular , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Estructura Molecular , Oxidorreductasas/química , Unión Proteica , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/enzimologíaRESUMEN
Members of the ATP-binding cassette (ABC)-type transporter superfamily have been implicated in multidrug resistance in malaria, and various mechanistic models have been postulated to explain their interaction with diverse antimalarial drugs. To gain insight into the pharmacological benefits of inhibiting ABC-type transporters in malaria chemotherapy, we investigated the in vitro chemosensitization potential of various P-glycoprotein inhibitors. A fluorescent chloroquine derivative was synthesized and used to assess the efflux dynamics of chloroquine in MDR and wild type Plasmodium falciparum parasites. This novel BODIPY-based probe accumulated in the digestive vacuole (DV) of CQ-sensitive parasites but less so in MDR cells. Pre-exposure of the MDR parasites to non-cytocidal concentrations of unlabeled chloroquine resulted in a diffused cytoplasmic retention of the probe whereas a similar treatment with the CQR-reversing agent, chlorpheniramine, resulted in DV accumulation. A diffused cytoplasmic distribution of the probe was also obtained following treatment with the P-gp specific inhibitors zosuquidar and tariquidar, whereas treatments with the tyrosine kinase inhibitors gefitinib or imatinib produced a partial accumulation within the DV. Isobologram analyses of the interactions between these inhibitors and the antimalarial drugs chloroquine, mefloquine, and artemisinin revealed distinct patterns of drug synergism, additivity and antagonism. Taken together, the data indicate that competitive tyrosine kinase and noncompetitive P-glycoprotein ATPase-specific inhibitors represent two new classes of chemosensitizing agents in malaria parasites, but caution against the indiscriminate use of these agents in antimalarial drug combinations.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Antimaláricos/farmacología , Plasmodium falciparum/efectos de los fármacos , Artemisininas/farmacología , Benzamidas/farmacología , Compuestos de Boro/química , Cloroquina/farmacología , Clorfeniramina/farmacología , Dibenzocicloheptenos/farmacología , Interacciones Farmacológicas , Resistencia a Múltiples Medicamentos , Eritrocitos/parasitología , Colorantes Fluorescentes/química , Gefitinib , Humanos , Mesilato de Imatinib , Mefloquina/farmacología , Piperazinas/farmacología , Plasmodium falciparum/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Quinazolinas/farmacología , Quinolinas/farmacologíaRESUMEN
Staphylococcus aureus is increasingly recognized as a facultative intracellular pathogen, although the significance and pervasiveness of its intracellular lifestyle remain controversial. Here, we applied fluorescence microscopy-based infection assays and automated image analysis to profile the interaction of 191 S. aureus isolates from patients with bone/joint infections, bacteremia, and infective endocarditis, with four host cell types, at five times post-infection. This multiparametric analysis revealed that almost all isolates are internalized and that a large fraction replicate and persist within host cells, presenting distinct infection profiles in non-professional vs. professional phagocytes. Phenotypic clustering highlighted interesting sub-groups, including one comprising isolates exhibiting high intracellular replication and inducing delayed host death in vitro and in vivo. These isolates are deficient for the cysteine protease staphopain A. This study establishes S. aureus intracellular lifestyle as a prevalent feature of infection, with potential implications for the effective treatment of staphylococcal infections.
Asunto(s)
Bacteriemia , Infecciones Estafilocócicas , Humanos , Staphylococcus aureus , Microscopía , Estilo de VidaRESUMEN
Cutinases are enzymes produced by phytopathogenic fungi like Moniliophthora roreri. The three genome-located cutinase genes of M. roreri were amplified from cDNA of fungi growing in different induction culture media for cutinase production. The mrcut1 gene was expressed in the presence of a cacao cuticle, while the mrcut2 and mrcut3 genes were expressed when an apple cuticle was used as the inducer. The sequences of all genes were obtained and analyzed by bioinformatics tools to determine the presence of signal peptides, introns, glycosylation, and regulatory sequences. Also, the theoretical molecular weight and pI were obtained and experimentally confirmed. Finally, cutinase 1 from M. roreri (MRCUT1) was selected for heterologous expression in Escherichia coli. Successful overexpression of MRCUT1 was observed with the highest enzyme activity of 34,036 U/mg under the assay conditions at 40°C and pH 8. Furthermore, the degradation of different synthetic polyesters was evaluated; after 21 days, 59% of polyethylene succinate (PES), 43% of polycaprolactone (PCL), and 31% of polyethylene terephthalate (PET) from plastic residues were degraded. IMPORTANCE Plastic pollution is exponentially increasing; even the G20 has recognized an urgent need to implement actions to reduce it. In recent years, searching for enzymes that can degrade plastics, especially those based on polyesters such as PET, has been increasing as they can be a green alternative to the actual plastic degradation process. A promising option in recent years refers to biological tools such as enzymes involved in stages of partial and even total degradation of some plastics. In this context, the MRCUT1 enzyme can degrade polyesters contained in plastic residues in a short time. Besides, there is limited knowledge about the biochemical properties of cutinases from M. roreri. Commonly, fungal enzymes are expressed as inclusion bodies in E. coli with reduced activity. Interestingly, the successful expression of one cutinase of M. roreri in E. coli with enhanced activity is described.
Asunto(s)
Agaricales/metabolismo , Biodegradación Ambiental , Hidrolasas de Éster Carboxílico/metabolismo , Poliésteres/metabolismo , Tereftalatos Polietilenos/metabolismo , Polietilenos/metabolismo , Succinatos/metabolismo , Agaricales/enzimología , Agaricales/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cacao/genética , Hidrolasas de Éster Carboxílico/genética , Contaminantes Ambientales/metabolismo , Contaminación Ambiental/análisis , Escherichia coli/genética , Escherichia coli/metabolismo , Amplificación de Genes/genética , Expresión Génica/genética , Técnicas de Amplificación de Ácido Nucleico , Plásticos/metabolismoRESUMEN
High content analysis enables automated, robust, and unbiased evaluation of in vitro Leishmania infection. Here, we describe a protocol based on the infection of THP-1 macrophages with Leishmania promastigotes and the quantification of parasite load by high content analysis. The technique is capable of detecting and quantifying intracellular amastigotes, providing a multiparametric readout of the total number of cells, ratio of infected cells, total number of parasites, and number of parasites per infected cells. The technique can be used to quantitate infection of any Leishmania species in virtually all types of permissive host cells and can be applied to quantification of drug activity and studies of the Leishmania intracellular life cycle stage.
Asunto(s)
Procesamiento de Imagen Asistido por Computador , Leishmania/crecimiento & desarrollo , Leishmaniasis/patología , Estadios del Ciclo de Vida , Macrófagos/parasitología , Humanos , Leishmania/citología , Leishmaniasis/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Carga de Parásitos/métodos , Células THP-1RESUMEN
Cell-based screening has become the major compound interrogation strategy in Chagas disease drug discovery. Several different cell lines have been deployed as host cells in screening assays. However, host cell characteristics and host-parasite interactions may play an important role when assessing anti-T. cruzi compound activity, ultimately impacting on hit discovery. To verify this hypothesis, four distinct mammalian cell lines (U2OS, THP-1, Vero and L6) were used as T. cruzi host cells in High Content Screening assays. Rates of infection varied greatly between different host cells. Susceptibility to benznidazole also varied, depending on the host cell and parasite strain. A library of 1,280 compounds was screened against the four different cell lines infected with T. cruzi, resulting in the selection of a total of 82 distinct compounds as hits. From these, only two hits were common to all four cell lines assays (2.4%) and 51 were exclusively selected from a single assay (62.2%). Infected U2OS cells were the most sensitive assay, as 55 compounds in total were identified as hits; infected THP-1 yielded the lowest hit rates, with only 16 hit compounds. Of the selected hits, compound FPL64176 presented selective anti-T. cruzi activity and could serve as a starting point for the discovery of new anti-chagasic drugs.
RESUMEN
Tritryps diseases are devastating parasitic neglected infections caused by Leishmania spp., Trypanosoma cruzi and Trypanosoma brucei subspecies. Together, these parasites affect more than 30 million people worldwide and cause high mortality and morbidity. Leishmaniasis comprises a complex group of diseases with clinical manifestation ranging from cutaneous lesions to systemic visceral damage. Antimonials, the first-choice drugs used to treat leishmaniasis, lead to high toxicity and carry significant contraindications limiting its use. Drug-resistant parasite strains are also a matter for increasing concern, especially in areas with very limited resources. The current scenario calls for novel and/or improvement of existing therapeutics as key research priorities in the field. Although several studies have shown advances in drug discovery towards leishmaniasis in recent years, key knowledge gaps in drug discovery pipelines still need to be addressed. In this review we discuss not only scientific and non-scientific bottlenecks in drug development, but also the central role of public-private partnerships for a successful campaign for novel treatment options against this devastating disease.
Asunto(s)
Descubrimiento de Drogas/métodos , Leishmania/efectos de los fármacos , Leishmaniasis/tratamiento farmacológico , Animales , Antiprotozoarios/efectos adversos , Antiprotozoarios/uso terapéutico , Antiprotozoarios/toxicidad , Enfermedad de Chagas/tratamiento farmacológico , Sistemas de Liberación de Medicamentos/métodos , Sistemas de Liberación de Medicamentos/estadística & datos numéricos , Descubrimiento de Drogas/legislación & jurisprudencia , Descubrimiento de Drogas/estadística & datos numéricos , Descubrimiento de Drogas/tendencias , Humanos , Enfermedades Desatendidas/tratamiento farmacológico , Enfermedades Desatendidas/parasitología , Asociación entre el Sector Público-Privado , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma cruzi/efectos de los fármacos , Trypanosomatina/efectos de los fármacosRESUMEN
Basing on a library of thiadiazole derivatives showing anti-trypanosomatidic activity, we have considered the thiadiazoles opened forms and reaction intermediates, thiosemicarbazones, as compounds of interest for phenotypic screening against Trypanosoma brucei (Tb), intracellular amastigote form of Leishmania infantum (Li) and Trypanosoma cruzi (Tc). Similar compounds have already shown interesting activity against the same organisms. The compounds were particularly effective against T. brucei and T. cruzi. Among the 28 synthesized compounds, the best one was (E)-2-(4-((3.4-dichlorobenzyl)oxy)benzylidene) hydrazinecarbothioamide (A14) yielding a comparable anti-parasitic activity against the three parasitic species (TbEC50â¯=â¯2.31⯵M, LiEC50â¯=â¯6.14⯵M, TcEC50â¯=â¯1.31⯵M) and a Selectivity Index higher than 10 with respect to human macrophages, therefore showing a pan-anti-trypanosomatidic activity. (E)-2-((3'.4'-dimethoxy-[1.1'-biphenyl]-3-yl)methyle ne) hydrazinecarbothioamide (A12) and (E)-2-(4-((3.4-dichlorobenzyl)oxy)benzylidene)hydrazine carbothioamide (A14) were able to potentiate the anti-parasitic activity of methotrexate (MTX) when evaluated in combination against T. brucei, yielding a 6-fold and 4-fold respectively Dose Reduction Index for MTX. The toxicity profile against four human cell lines and a panel of in vitro early-toxicity assays (comprising hERG, Aurora B, five cytochrome P450 isoforms and mitochondrial toxicity) demonstrated the low toxicity for the thosemicarbazones class in comparison with known drugs. The results confirmed thiosemicarbazones as a suitable chemical scaffold with potential for the development of properly decorated new anti-parasitic drugs.
Asunto(s)
Antiprotozoarios/farmacología , Enfermedad de Chagas/tratamiento farmacológico , Tiosemicarbazonas/farmacología , Trypanosoma/efectos de los fármacos , Antiprotozoarios/síntesis química , Antiprotozoarios/química , Relación Dosis-Respuesta a Droga , Humanos , Macrófagos/efectos de los fármacos , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Relación Estructura-Actividad , Tiosemicarbazonas/síntesis química , Tiosemicarbazonas/químicaRESUMEN
Chalcones display a broad spectrum of pharmacological activities. Herein, a series of 2'-hydroxy methoxylated chalcones was synthesized and evaluated towards Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum. Among the synthesized library, compounds 1, 3, 4, 7 and 8 were the most potent and selective anti-T. brucei compounds (EC50 = 1.3-4.2 µM, selectivity index >10-fold). Compound 4 showed the best early-tox and antiparasitic profile. The pharmacokinetic studies of compound 4 in BALB/c mice using hydroxypropil-ß-cyclodextrins formulation showed a 7.5 times increase in oral bioavailability.
Asunto(s)
Antiparasitarios/química , Antiparasitarios/farmacología , Chalconas/química , Chalconas/farmacología , Animales , Antiparasitarios/farmacocinética , Antiparasitarios/toxicidad , Línea Celular Tumoral , Chalconas/farmacocinética , Chalconas/toxicidad , Ciclodextrinas/química , Portadores de Fármacos/química , Ratones , Solubilidad , Trypanosomatina/efectos de los fármacosRESUMEN
Pteridine reductase-1 (PTR1) is a promising drug target for the treatment of trypanosomiasis. We investigated the potential of a previously identified class of thiadiazole inhibitors of Leishmania major PTR1 for activity against Trypanosoma brucei (Tb). We solved crystal structures of several TbPTR1-inhibitor complexes to guide the structure-based design of new thiadiazole derivatives. Subsequent synthesis and enzyme- and cell-based assays confirm new, mid-micromolar inhibitors of TbPTR1 with low toxicity. In particular, compound 4m, a biphenyl-thiadiazole-2,5-diamine with IC50 = 16 µM, was able to potentiate the antitrypanosomal activity of the dihydrofolate reductase inhibitor methotrexate (MTX) with a 4.1-fold decrease of the EC50 value. In addition, the antiparasitic activity of the combination of 4m and MTX was reversed by addition of folic acid. By adopting an efficient hit discovery platform, we demonstrate, using the 2-amino-1,3,4-thiadiazole scaffold, how a promising tool for the development of anti-T. brucei agents can be obtained.