Development of a Fluorescent Assay to Search New Drugs Using Stable tdTomato-Leishmania, and the Selection of Galangin as a Candidate With Anti-Leishmanial Activity

GARCÍA-BUSTOS, MARÍA FERNANDA; MOYA ÁLVAREZ, AGUSTÍN; PÉREZ BRANDAN, CECILIA; PARODI, CECILIA; SOSA, ANDREA MABEL; BUTTAZZONI ZUÑIGA, VALERIA CAROLINA; PASTRANA, OSCAR MARCELO; MANGHERA, PAULA; PEÑALVA, PABLO ALEJANDRO; MARCO, JORGE DIEGO; BARROSO, PAOLA ANDREA

Frontiers in Cellular and Infection Microbiology

Frontier

Año: 2021 vol. 11

Antimonials continue to be considered the first-line treatment for leishmaniases, but its useentails a wide range of side effects and serious reactions. The search of new drugsrequires the development of methods more sensitive and faster than the conventionalones. We developed and validated a fluorescence assay based in the expression oftdTomato protein by Leishmania, and we applied this method to evaluate the activity invitro of flavonoids and reference drugs. The pIR1SAT/tdTomato was constructed andintegrated into the genome of Leishmania (Leishmania) amazonensis. Parasites wereselected with nourseothricin (NTC). The relation of L. amaz/tc3 fluorescence and thenumber of parasites was determined; then the growth in vitro and infectivity in BALB/cmice was characterized. To validate the fluorescence assay, the efficacy of miltefosine andmeglumine antimoniate was compared with the conventional methods. After that, themethod was used to assess in vitro the activity of flavonoids; and the mechanism of actionof the most active compound was evaluated by transmission electron microscopy andELISA. A linear correlation was observed between the emission of fluorescence of L.amaz/tc3 and the number of parasites (r2 = 0.98), and the fluorescence was stable in theabsence of NTC. No differences were observed in terms of infectivity between L. amaz/tc3and wild strain. The efficacy of miltefosine and meglumine antimoniate determined by thefluorescence assay and the microscopic test showed no differences, however, in vivo thefluorescence assay was more sensitive than limiting dilution assay. Screening assayrevealed that the flavonoid galangin (GAL) was the most active compound with IC50 valuesof 53.09 μM and 20.59 μM in promastigotes and intracellular amastigotes, respectively.Furthermore, GAL induced mitochondrial swelling, lipid inclusion bodies and vacuolizationin promastigotes; and up-modulated the production of IL-12 p70 in infected macrophages. The fluorescence assay is a useful tool to assess the anti-leishmanial activity of new compounds. However, the assay has some limitations in the macrophageamastigote model that might be related with an interfere of flavanol aglycones with the fluorescence readout of tdTomato. Finally, GAL is a promising candidate for the development of new treatment against the leishmaniasis.