Quest for new antimicrobial membrane-active peptide leads, either from de novo design or from natural sources, including genome mining and drug repurposing.
Optimization by SAR studies. Elucidation of their mechanism of action and targets involved; if feasible further validation by a variety of biophysical and biochemical techniques. Recombinant production. To date we have successfully defined the leishmanicidal activity of cecropin-melittin peptides gramicidin S analogues , and of AS-48, histatin 5, and eosinophils cationic protein as paradigms of antimicrobial polypeptide with intracellular targets.
Electron micrograph of Leishmania donovani promastigotes after 4h incubation with 5µM of LF chimera, a membrane –active peptide. To note, an all-or-none effect , with parasites apparently unaffected, preserving a normal intracellular morphology with those fully permeabilized , whose intracellular content was lost
Application of cell penetrating peptide technology (CPP)in Trypanosomatids.
We have assayed a variety of CPP on these cells, quantified their uptake and defined their pathways. Definition of cargo molecules capable to be transported by CPPs and their application into reversal of resistance due to a faulty accumulation of drug, as miltefosine in Leishmania. We improved CPP specificity by incorporation of peptide sequences with higher Leishmania recognition, from biopanning of phage peptide expression libraries, and targeting intracellular organelles, either by modification of the physicochemical characteristics of CPPs on Leishmania or by incorporation into their sequence of organelle import sequences.
Confocal microscopy of Leishmania donovani promastigotes incubated for 4h with 36 µM Tat-GFP, the green fluorescent protein fused with the cell penetrating peptide Tat at its N-terminal end ( green fluorescence) in the presence of 1 mg/ml rhodaminated dextran as fluid phase marker.
Definition of new drugs
Definition of new drugs targeting the bioenergetic metabolism of Leishmania, with a particular emphasis on those causing mitochondrial dysfunction, as oxidative phosphorylation contributed most to the bioenergetic requirements of the parasite. Drug modification to improve its accumulation in mitochondria. Assessment of inhibition of mitochondrial functionality and definition of their site of action by functional and enzymatic assays, molecular docking and lately, metabolomics. Mitochondrial targets for musalanone, sitamaquine, miltefosine, 14-hydroxylunularin and styrylquinolines have been achieved .
Docking of 14-hydroxilunularin (2,5,4’-trihydroxybibenzyl)(blue) and VP55 (E-2,5,4’-trihydroxystilbene) (yellow) on its binding site of mitochondrial F1-ATP synthase of Leishmania donovani, modeled on the crystallographic structure of bovine F1-ATP synthase (Prof. A. Romero)