Proteomic and ultrastructural analysis of the effect of a new nitazoxanide- N -methyl-1 H -benzimidazole hybrid against Giardia intestinalis

In an effort to develop alternative drugs for the treatment of giardiasis our research group has synthesized and evaluated a novel nitazoxanide andN-methyl-1H-benzimidazole hybrid molecule, namedCMC-20. It showed an IC50of 0.010μM onGiardia intestinalis, lower than the IC50values of 0.015, 0.037 and 1.224μM for nitazoxanide, albendazole and metronidazole, respectively. In addition, we report studies carried out on its mechanism of action and effect at the ultrastructural level onG. intestinalis. The proteomic analysis of trophozoites treated withCMC-20revealed significant changes in the expression level of proteins of the cytoskeleton, alpha and beta tubulin, alpha-1, beta giardin and axoneme-associated protein, among other molecules. Ultrastructural studies demonstrated thatCMC-20induces morphological changes on the parasite that loses its characteristic pear shape. Uncommon large bulbous structure at the flagella end, and parasites showing flange membrane bending and a concave depression in the ventral region, resembling an encystation process, were also observed. In addition, some apoptotic and autophagic-like features, such as membrane blebbing, intense vacuolation, chromatin condensation and multilamellar bodies were detected. Phosphatidylserine externalization was determined as an apoptotic marker by flow cytometry and immunofluorescence microscopy; however, a typical ladder-like DNA fragmentation profile was not detected. Although it was found thatCMC-20triggers the encystation process, damage to the cyst wall indicates loss of viability.