Improving in vitro screening compounds anti-Trypanosoma cruzi by GFP-expressing parasites

BACKGROUND Conventional microscopic counting is a widely utilised method for evaluating the trypanocidal effects of drugs on intracellular amastigotes. This is a low-cost approach, but it is time-consuming and reliant on the expertise of the microscopist. So, there is a pressing need for developing technologies to enhance the efficiency of low-cost anti-Trypanosoma cruzi drug screening. OBJECTIVES In our laboratory, we aimed to expedite the screening of anti-T. cruzi drugs by implementing a fluorescent method that correlates emitted fluorescence from green fluorescent protein (GFP)-expressing T. cruzi (Tc-GFP) with cellular viability. METHODS Epimastigotes (Y strain) were transfected with the pROCKGFPNeo plasmid, resulting in robust and sustained GFP expression across epimastigotes, trypomastigotes, and intracellular amastigotes. Tc-GFP epimastigotes and intracellular amastigotes were exposed to a serial dilution of benznidazole (Bz). Cell viability was assessed through a combination of microscopic counting, MTT, and fluorimetry. FINDINGS The fluorescence data indicated an underestimation of the activity of Bz against epimastigotes (IC50 75 µM x 14 µM). Conversely, for intracellular GFP-amastigotes, both fluorimetry and microscopy yielded identical IC50 values. Factors influencing the fluorimetry approach are discussed. MAIN CONCLUSIONS Our proposed fluorometric assessment is effective and can serve as a viable substitute for the time-consuming microscopic counting of intracellular amastigotes.

Subcellular localisation of FLAG tagged enzymes of the dynamic protein S-palmitoylation cycle of Trypanosoma cruzi epimastigotes

Dynamic S-palmitoylation of proteins is the addition of palmitic acid by zDHHC palmitoyl transferases (PATs) and depalmitoylation by palmitoyl protein thioesterases (PPTs). A putative PAT (TcPAT1) has been previously identified in Trypanosoma cruzi, the etiological agent of Chagas disease. Here we analyse other 14 putative TcPATs and 2 PPTs in the parasite genome. T. cruzi cell lines expressing TcPATs and TcPPTs plus a FLAG tag at the C terminus were produced for most enzymes, with positive detection by indirect immunofluorescence. Overexpressed TcPATs were mostly found as single spots at the parasite anterior end, while the TcPPTs were dispersed throughout the parasite body.

Trypanocidal activity of coumarins and styryl-2-pyrones from Polygala sabulosa A.W. Bennett (Polygalaceae) / Atividade tripanocida de cumarinas e estiril-2-pironas de Polygala sabulosa A.W. Bennett (Polygalaceae)

A bioatividade das frações e compostos obtidos de Polygala sabulosa contra as formas epimastigota, tripomastigota sanguínea e amastigota de Trypanosoma cruzi foram avaliadas in vitro. Frações diclorometano e acetato de etila mostraram potente atividade tripanocida sob as formas epimastigotas (IC50 < 10,4 µg/mL). Análises por cromatografia em camada delgada destas frações confirmaram a presença de compostos previamente descritos (dihidroestiril-2-pironas, estiril-2-pironas e 6-metoxi-7-preniloxicumarina). Após o fracionamento da fração diclorometano por cromatografia em coluna, obteve-se o composto α-espinasterol e da fração acetato de etila obtiveram-se os compostos apigenina, quercetina e uma quercetina-3- O-glicosídeo, todos descritos pela primeira vez para o gênero Polygala. 4-metoxi-6-(11,12-metilenodioxi-14-metoxidihidroestiril)-2-pirona, 4-metoxi-6-(11,12-dimetoxi-14-metoxiestiril)-2-pirona, 6-metoxi-7-preniloxicumarina e quercetina-3- O-glicosídeo mostraram fraca atividade contra a forma tripomastigota sanguínea (IC50 < 1008,6 µg/mL). A cumarina prenilada foi o composto mais ativo contra ambas as formas epimastigota e tripomastigota, com IC50 10,5 e 88,2 µg/mL, respectivamente. A atividade hemolítica e a toxicidade celular de cada composto foram também avaliadas. Além disso, 4-metoxi-6-(11,12-metilenodioxi-14-metoxidihidroestiril)-2-pirona e 6-metoxi-7-preniloxicumarina reduziram em quatro vezes a infecção em ratos por Vero Cells nas concentrações de 100 e 50 µg/mL respectivamente. Esses resultados mostram, pela primeira vez, a atividade de compostos de P. sabulosa contra T. cruzi.

Bioactivity of fractions and compounds obtained from Polygala sabulosa against Trypanosoma cruzi epimastigote, blood trypomastigote and amastigote forms were evaluated in vitro. Dichloromethane and ethyl acetate fractions showed a strong trypanocidal activity on epimastigotes (IC50 < 10.4 µg/mL). Chromatographic analysis by TLC of these fractions confirmed the presence of previously described compounds (dihydrostyryl-2-pyrones, styryl-2-pyrones and 6-methoxy-7-prenyloxycoumarin). The dichloromethane fraction was fractioned by silica gel column chromatography to afford the compound α-spinasterol and the ethyl acetate fraction yielded apigenin, quercetin and a quercetin-3-O-glucoside, being the first description for the Polygala genus. 4-Methoxy-6-(11,12-methylenedioxy-14-methoxydihydrostyryl)-2-pyrone, 4-methoxy-6-(11,12-dimethoxystyryl)-2-pyrone, 6-methoxy-7-prenyloxycoumarin and quercetin-3-O-glucoside showed a weak activity against blood trypomastigotes (IC50 < 1008.6 µg/mL). The prenylated coumarin was the most active compound against both epimastigote and trypomastigote forms, IC50 10.5 and 88.2 µg/mL, respectively. The hemolytic activity and cell toxicity of each active compound was also assessed. Furthermore, 4-methoxy-6-(11,12-methylenedioxy-14-methoxydihydrostyryl)-2-pyrone and 6-methoxy-7-prenyloxycoumarin reduced 4 times the T. cruzi infection rate for Vero cells at 100 and 50 µg/mL, respectively. These results show for the first time active compounds against T. cruzi in P. sabulosa.