Toward New Therapeutics for Visceral Leishmaniasis: Efficacy and Mechanism of Action of Amides Inspired by Gibbilimbol B

The problems with current strategies to control canine visceral Leishmaniasis (CVL), which include the euthanasia of infected animals, and also the toxicity of the drugs currently used in human treatments for CVL, add urgency to the search for new therapeutic agents. This study aimed to evaluate the activity against Leishmania (L.) infantum of 12 amides that are chemically inspired by gibbilimbol B, a bioactive natural product that was initially obtained from Piper malacophyllum. Three of these compounds-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 studies predicted that these compounds would show high levels of gastrointestinal absorption, would be able to penetrate the blood-brain barrier, would show moderate solubility, and would not show unwanted molecular interactions. Due to their promising pharmacological 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 9 induced strong alkalinization of acidocalcisomes, which resulted in a significant and rapid increase in intracellular Ca²⁺ levels, thereby causing 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, but it caused neither acidocalcisome alkalinization nor mitochondrial membrane potential depolarization. Finally, the MALDI-TOF/MS assessment of protein alterations in L. (L.) infantum treated separately with compounds 9 and 10 revealed changes in mass spectral profiles from both treatments. These results highlight the anti-L. (L.) infantum potential of these amides-especially for compounds 9 and 10-and they suggest that these compounds could be promising candidates for future in vivo studies in VL-models.