Toward new therapeutics for visceral Leishmaniasis: efficacy and mechanism of action of amides inspired by Gibbilimbol B

Abstract

The problems with current strategies to control canine visceral Leishmaniasis (CVL), which include the euthanasia ofinfected animals, and also the toxicity of the drugs currently used in human treatments for CVL, add urgency to the search for newtherapeutic agents. This study aimed to evaluate the activity against Leishmania (L.) infantum of 12 amides that are chemicallyinspired by gibbilimbol B, a bioactive natural product that was initially obtained from Piper malacophyllum. Three of thesecompounds N-(2-ethylhexyl)-4-chlorobenzamide (9), N-(2-ethylhexyl)-4-nitrobenzamide (10), and N-(2-ethylhexyl)-4-(tert-butyl)benzamide (12) demonstrated activity against the intracellular amastigotes without toxicity to mammalian host cells (CC50> 200 μM); compounds 9, 10, and 12 resulted in EC50 values of 12.7, 12.2, and 5.1 μM, respectively. In silico drug-likeness studiespredicted that these compounds would show high levels of gastrointestinal absorption, would be able to penetrate the blood-brainbarrier, would show moderate solubility, and would not show unwanted molecular interactions. Due to their promisingpharmacological profiles, compounds 9 and 10 were selected for mechanism of action studies (MoA). The MoA studies in L. (L.)infantum revealed that neither of the compounds affected the permeabilization of the plasma membrane. Nevertheless, compound 9induced strong alkalinization of acidocalcisomes, which resulted in a significant and rapid increase in intracellular Ca2+ levels, therebycausing the depolarization of the mitochondrial membrane potential and a reduction in the levels of reactive oxygen species (ROS).In contrast, compound 10 induced a gradual increase in intracellular Ca2+ levels and a similarly gradual reduction in ROS levels, butit caused neither acidocalcisome alkalinization nor mitochondrial membrane potential depolarization. Finally, the MALDI-TOF/MSassessment of protein alterations in L. (L.) infantum treated separately with compounds 9 and 10 revealed changes in mass spectralprofiles from both treatments. These results highlight the anti-L. (L.) infantum potential of these amides especially for compounds9 and 10 and they suggest that these compounds could be promising candidates for future in vivo studies in VL-models.