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

• Published in: Memórias do Instituto Oswaldo Cruz 2024-01, Vol.119, p.e230223-e230223
• Main Authors: Delvoss, Cleyson Mathias Morais, Inoue, Alexandre Haruo, da Silva, Rosiane Valeriano, Fragoso, Stênio Perdigão, Eger, Iriane
• Format: Article
• Language: English
• Subjects: Animals; Cell Survival - drug effects; Drug Evaluation, Preclinical; Green Fluorescent Proteins - genetics; Inhibitory Concentration 50; Nitroimidazoles - pharmacology; Parasitic Sensitivity Tests; PARASITOLOGY; TROPICAL MEDICINE; Trypanocidal Agents - pharmacology; Trypanosoma cruzi - drug effects; Trypanosoma cruzi - genetics
• ISSN: 0074-0276; 1678-8060; 1678-8060
• DOI: 10.1590/0074-02760230223

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. 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. 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. 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. Our proposed fluorometric assessment is effective and can serve as a viable substitute for the time-consuming microscopic counting of intracellular amastigotes.