Energy metabolism as a target for cyclobenzaprine: A drug candidate against Visceral Leishmaniasis

Leishmaniases have a broad spectrum of clinical manifestations, ranging from a cutaneous to a progressive and fatal visceral disease. Chemotherapy is nowadays the almost exclusive way to fight the disease but limited by its scarce therapeutic arsenal, on its own compromised by adverse side effects and clinical resistance. Cyclobenzaprine (CBP), an FDA-approved oral muscle relaxant drug has previously demonstrated in vitro and in vivo activity against Leishmania sp., but its targets were not fully unveiled. This study aimed to define the role of energy metabolism as a target for the leishmanicidal mechanisms of CBP. Methodology to assess CBP leishmanicidal mechanism variation of intracellular ATP levels using living Leishmania transfected with a cytoplasmic luciferase. Induction of plasma membrane permeability by assessing depolarization with DiSBAC(2)3 and entrance of the vital dye SYTOX® Green. Mitochondrial depolarization by rhodamine 123 accumulation. Mapping target site within the respiratory chain by oxygen consumption rate. Reactive oxygen species (ROS) production using MitoSOX. Morphological changes by transmission electron microscopy. CBP caused on L. infantum promastigotes a decrease of intracellular ATP levels, with irreversible depolarization of plasma membrane, the collapse of the mitochondrial electrochemical potential, mild uncoupling of the respiratory chain, and ROS production, with ensuing intracellular Ca2+ imbalance and DNA fragmentation. Electron microscopy supported autophagic features but not a massive plasma membrane disruption. The severe and irreversible mitochondrial damage induced by CBP endorsed the bioenergetics metabolism as a relevant target within the lethal programme induced by CBP in Leishmania. This, together with the mild-side effects of this oral drug, endorses CBP as an appealing novel candidate as a leishmanicidal drug under a drug repurposing strategy.
This work was supported by grants of Sao Paulo State Research Foundation (FAPESP 2019/10434-4 to S.E.T.B. and 2021/04464-8 and 2017/50333-7 to A.G.T.); Subdirección General de Redes y Centros de Investigación Cooperativa - FEDER (RICET RD16/0027/0010, and Ministerio de Ciencia e Innovación España. Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020-FEDER PID2019- 108166GB-100 /AEI/10.13039/501100011033 to L. R.), EADS-CASA/Brazilian Air Force (FAB) mobility program to M.L.L., and Coordenaçao de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) to M. L.L.