Non-peptidic Cruzain Inhibitors with Trypanocidal Activity Discovered by Virtual Screening and In Vitro Assay

Helton J Wiggers,James H Mckerrow,Juliana Cheleski,William B Fernandes,Zumira A Carneiro,Renata Sesti-Costa,Luiz Juliano,Josmar R Rocha,Carlos A Montanari,João S Silva,Albérico B F Da Silva,Maria H S Cezari

doi:10.1371/journal.pntd.0002370

Abstract

A multi-step cascade strategy using integrated ligand- and target-based virtual screening methods was developed to select a small number of compounds from the ZINC database to be evaluated for trypanocidal activity. Winnowing the database to 23 selected compounds, 12 non-covalent binding cruzain inhibitors with affinity values (K i) in the low micromolar range (3–60 µM) acting through a competitive inhibition mechanism were identified. This mechanism has been confirmed by determining the binding mode of the cruzain inhibitor Nequimed176 through X-ray crystallographic studies. Cruzain, a validated therapeutic target for new chemotherapy for Chagas disease, also shares high similarity with the mammalian homolog cathepsin L. Because increased activity of cathepsin L is related to invasive properties and has been linked to metastatic cancer cells, cruzain inhibitors from the same library were assayed against it. Affinity values were in a similar range (4–80 µM), yielding poor selectivity towards cruzain but raising the possibility of investigating such inhibitors for their effect on cell proliferation. In order to select the most promising enzyme inhibitors retaining trypanocidal activity for structure-activity relationship (SAR) studies, the most potent cruzain inhibitors were assayed against T. cruzi-infected cells. Two compounds were found to have trypanocidal activity. Using compound Nequimed42 as precursor, an SAR was established in which the 2-acetamidothiophene-3-carboxamide group was identified as essential for enzyme and parasite inhibition activities. The IC50 value for compound Nequimed42 acting against the trypomastigote form of the Tulahuen lacZ strain was found to be 10.6±0.1 µM, tenfold lower than that obtained for benznidazole, which was taken as positive control. In addition, by employing the strategy of molecular simplification, a smaller compound derived from Nequimed42 with a ligand efficiency (LE) of 0.33 kcal mol−1 atom−1 (compound Nequimed176) is highlighted as a novel non-peptidic, non-covalent cruzain inhibitor as a trypanocidal agent candidate for optimization.

Highlights

Chagas disease, widespread in Latin America, is caused by the kinetoplastid protozoan parasite Trypanosoma cruzi
Since the sub-library still encompassed a large number of structures, two fast virtual screening methods based on ligand and receptor were employed in order to enrich it
The agreement between ligand- and target-based virtual screening methods was used as the criterion for the selection of an enriched, focused sub-library

Summary

Introduction

Widespread in Latin America, is caused by the kinetoplastid protozoan parasite Trypanosoma cruzi. Despite efforts to reduce the transmission of the parasite by controlling the hematophagous triatomine insect vector, the World Health Organization estimates that 10 million people are infected worldwide, with another 25 million at risk. Most cases are in Latin America, where Chagas disease is endemic, but it is found in Canada, the United States, Europe (mainly in Spain and Portugal), Japan and Australia [1,2,3]. T. cruzi’s complex life cycle involves two replicative forms: the epimastigote, in the gut of the insect vector, and the amastigote, an intracellular form in the infected mammal. Chagas disease has an acute phase and a chronic latent phase. The acute phase, which occurs shortly after infection, lasts for a few weeks or months, whereas the chronic phase develops over many years. The acute phase may not be noticed because it is symptom-free or exhibits only mild symptoms that are not unique

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