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.

Molecular Basis of the Leishmanicidal Activity of the Antidepressant Sertraline as a Drug Repurposing Candidate.

Drug repurposing affords the implementation of new treatments at a moderate cost and under a faster time-scale. Most of the clinical drugs against Leishmania share this origin. The antidepressant sertraline has been successfully assayed in a murine model of visceral leishmaniasis. Nevertheless, sertraline targets in Leishmania were poorly defined. In order to get a detailed insight into the leishmanicidal mechanism of sertraline on Leishmania infantum, unbiased multiplatform metabolomics and transmission electron microscopy were combined with a focused insight into the sertraline effects on the bioenergetics metabolism of the parasite. Sertraline induced respiration uncoupling, a significant decrease of intracellular ATP level, and oxidative stress in L. infantum promastigotes. Metabolomics evidenced an extended metabolic disarray caused by sertraline. This encompasses a remarkable variation of the levels of thiol-redox and polyamine biosynthetic intermediates, as well as a shortage of intracellular amino acids used as metabolic fuel by Leishmania Sertraline killed Leishmania through a multitarget mechanism of action, tackling essential metabolic pathways of the parasite. As such, sertraline is a valuable candidate for visceral leishmaniasis treatment under a drug repurposing strategy.

Design of Bactericidal Peptides Against Escherichia coli O157:H7, Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus.

BACKGROUND: Antimicrobial peptides are on the first line of defense against pathogenic microorganisms of many living beings. These compounds are considered natural antibiotics that can overcome bacterial resistance to conventional antibiotics. Due to this characteristic, new peptides with improved properties are quite appealing for designing new strategies for fighting pathogenic bacteria. METHODS: Sixteen designed peptides were synthesized using Fmoc chemistry; five of them are new cationic antimicrobial peptides (CAMPs) designed using a genetic algorithm that optimizes the antibacterial activity based on selected physicochemical descriptors and 11 analog peptides derived from these five peptides were designed and constructed by single amino acid substitutions. These 16 peptides were structurally characterized and their biological activity was determined against Escherichia coli O157:H7 (E. coli O157:H7), and methicillin-resistant strains of Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (P. aeruginosa) were determined. RESULTS: These 16 peptides were folded into an α-helix structure in membrane-mimicking environment. Among these 16 peptides, GIBIM-P5S9K (ATKKCGLFKILKGVGKI) showed the highest antimicrobial activity against E. coli O157:H7 (MIC=10µM), methicillin-resistant Staphylococcus aureus (MRSA) (MIC=25µM) and Pseudomonas aeruginosa (MIC=10 µM). Peptide GIBIM-P5S9K caused permeabilization of the bacterial membrane at 25 µM as determined by the Sytox Green uptake assay and the labelling of these bacteria by using the fluoresceinated peptide. GIBIM-P5S9K seems to be specific for these bacteria because at 50 µM, it provoked lower than 40% of erythrocyte hemolysis. CONCLUSION: New CAMPs have been designed using a genetic algorithm based on selected physicochemical descriptors and single amino acid substitution. These CAMPs interacted quite specifically with the bacterial cell membrane, GIBIM-P5S9K exhibiting high antibacterial activity on Escherichia coli O157:H7, methicillin-resistant strains of Staphylococcus aureus and P. aeruginosa.

A Synthetic Strategy for Conjugation of Paromomycin to Cell-Penetrating Tat(48-60) for Delivery and Visualization into Leishmania Parasites.

A successful approach to deliver paromomycin, a poorly absorbed aminoglycoside antibiotic, to parasite cells is reported, based on selective protection of amino and hydroxyl groups followed by conjugation to a fluorolabeled, PEG-functionalized cell-penetrating Tat(48-60) peptide. The resulting construct is efficiently internalized into Leishmania cells, evidencing the fitness of cell-penetrating peptides as vectors for efficiently transporting low-bioavailability drugs into cells.

Cyclobenzaprine Raises ROS Levels in Leishmania infantum and Reduces Parasite Burden in Infected Mice.

BACKGROUND: The leishmanicidal action of tricyclic antidepressants has been studied and evidences have pointed that their action is linked to inhibition of trypanothione reductase, a key enzyme in the redox metabolism of pathogenic trypanosomes. Cyclobenzaprine (CBP) is a tricyclic structurally related to the antidepressant amitriptyline, differing only by the presence of a double bond in the central ring. This paper describes the effect of CBP in experimental visceral leishmaniasis, its inhibitory effect in trypanothione reductase and the potential immunomodulatory activity. METHODOLOGY/PRINCIPAL FINDINGS: In vitro antileishmanial activity was determined in promastigotes and in L. infantum-infected macrophages. For in vivo studies, L. infantum-infected BALB/c mice were treated with CBP by oral gavage for five days and the parasite load was estimated. Trypanothione reductase activity was assessed in the soluble fraction of promastigotes of L. infantum. For evaluation of cytokines, L. infantum-infected macrophages were co-cultured with BALB/c splenocytes and treated with CBP for 48 h. The supernatant was analyzed for IL-6, IL-10, MCP-1, IFN-γ and TNF-α. CBP demonstrated an IC50 of 14.5±1.1µM and an IC90 of 74.5±1.2 µM in promastigotes and an IC50 of 12.6±1.05 µM and an IC90 of 28.7±1.3 µM in intracellular amastigotes. CBP also reduced the parasite load in L. infantum-infected mice by 40.4±10.3% and 66.7±10.5% in spleen at 24.64 and 49.28 mg/kg, respectively and by 85.6±5.0 and 89.3±4.8% in liver at 24.64 and 49.28mg/kg, after a short-term treatment. CBP inhibited the trypanothione reductase activity with a Ki of 86 ± 7.7 µM and increased the ROS production in promastigotes. CBP inhibited in 53% the production of IL-6 in infected macrophages co-culture. CONCLUSION/SIGNIFICANCE: To the best of our knowledge, this study is the first report of the in vivo antileishmanial activity of the FDA-approved drug CBP. Modulation of immune response and induction of oxidative stress in parasite seem to contribute to this efficacy.