Identification of new leishmanicidal peptide lead structures by automated real-time monitoring of changes in intracellular ATP

Abstract

Leishmanicidal drugs interacting stoichiometrically with parasite plasma membrane lipids, thus promoting permeability, have raised significant expectations for Leishmania chemotherapy due to their nil or very low induction of resistance. Inherent in this process is a decrease in intracellular ATP, either wasted by ionic pumps to restore membrane potential or directly leaked through larger membrane lesions caused by the drug. We have adapted a luminescence method for fast automated real-time monitoring of this process, using Leishmania donovani promastigotes transfected with a cytoplasmic luciferase form, previously tested for anti-mitochondrial drugs. The system was first assayed against a set of well-known membrane-active drugs [amphotericin B, nystatin, cecropin A–melittin peptide CA(1-8)M(1-18)], plus two ionophoric polyethers (narasin and salinomycin) not previously tested on Leishmania, then used to screen seven new cecropin A–melittin hybrid peptides. All membrane-active compounds showed a good correlation between inhibition of luminescence and leishmanicidal activity. Induction of membrane permeability was demonstrated by dissipation of membrane potential, SYTOXTM Green influx and membrane damage assessed by electron microscopy, except for the polyethers, where ATP decrease was due to inhibition of its mitochondrial synthesis. Five of the test peptides showed an ED50 around 1 μM on promastigotes. These peptides, with equal or better activity than 26-residue-long CA(1-8)M(1-18), are the shortest leishmanicidal peptides described so far, and validate our luminescence assay as a fast and cheap screening tool for membrane-active compounds.