BACKGROUND: Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and leads to ~10,000 deaths each year. Nifurtimox and benznidazole are the only two drugs available but have significant adverse effects and limited efficacy. New chemotherapeutic agents are urgently required. Here we identified inhibitors of the acidic M17 leucyl-aminopeptidase from T. cruzi (LAPTc) that show promise as novel starting points for Chagas disease drug discovery. METHODOLOGY/PRINCIPAL FINDINGS: A RapidFire-MS screen with a protease-focused compound library identified novel LAPTc inhibitors. Twenty-eight hits were progressed to the dose-response studies, from which 12 molecules inhibited LAPTc with IC50 < 34 μM. Of these, compound 4 was the most potent hit and mode of inhibition studies indicate that compound 4 is a competitive LAPTc inhibitor, with Ki 0.27 μM. Compound 4 is selective with respect to human LAP3, showing a selectivity index of >500. Compound 4 exhibited sub-micromolar activity against intracellular T. cruzi amastigotes, and while the selectivity-window against the host cells was narrow, no toxicity was observed for un-infected HepG2 cells. In silico modelling of the LAPTc-compound 4 interaction is consistent with the competitive mode of inhibition. Molecular dynamics simulations reproduce the experimental binding strength (-8.95 kcal/mol), and indicate a binding mode based mainly on hydrophobic interactions with active site residues without metal cation coordination. CONCLUSIONS/SIGNIFICANCE: Our data indicates that these new LAPTc inhibitors should be considered for further development as antiparasitic agents for the treatment of Chagas disease.

Biology Centre Czech Academy of Sciences Institute of Parasitology České Budějovice Czech Republic

Centre for Pharmaceuticals Research and Development La Habana Cuba

Centre for Protein Studies Faculty of Biology University of Havana La Habana Cuba

Department of Biochemistry Faculty of Biology University of Havana La Habana Cuba

Department of Parasitology Faculty of Science Charles University Prague Biocev Vestec Czech Republic

Drug Discovery Unit Wellcome Centre for Anti Infectives Research University of Dundee Dundee United Kingdom

School of Life Sciences University of Dundee Dundee United Kingdom

Wellcome Centre for Anti Infectives Research Division of Biological Chemistry and Drug Discovery School of Life Sciences University of Dundee Dundee United Kingdom

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