Research Article: Dissecting a novel allosteric mechanism of cruzain: A computer-aided approach

Date Published: January 25, 2019

Publisher: Public Library of Science

Author(s): Lilian Hernández Alvarez, Diego Enry Barreto Gomes, Jorge Enrique Hernández González, Pedro Geraldo Pascutti, Freddie Salsbury.


Trypanosoma cruzi is the causative agent of Chagas disease, a neglected infection affecting millions of people in tropical regions. There are several chemotherapeutic agents for the treatment of this disease, but most of them are highly toxic and generate resistance. Currently, the development of allosteric inhibitors constitutes a promising research field, since it can improve the accessibility to more selective and less toxic medicines. To date, the allosteric drugs prediction is a state-of-the-art topic in rational structure-based computational design. In this work, a simulation strategy was developed for computational discovery of allosteric inhibitors, and it was applied to cruzain, a promising target and the major cysteine protease of T. cruzi. Molecular dynamics simulations, binding free energy calculations and network-based modelling of residue interactions were combined to characterize and compare molecular distinctive features of the apo form and the cruzain-allosteric inhibitor complexes. By using geometry-based criteria on trajectory snapshots, we predicted two main allosteric sites suitable for drug targeting. The results suggest dissimilar mechanisms exerted by the same allosteric site when binding different potential allosteric inhibitors. Finally, we identified the residues involved in suboptimal paths linking the identified site and the orthosteric site. The present study constitutes the first approximation to the design of cruzain allosteric inhibitors and may serve for future pharmacological intervention. Here, no major effects on active site structure were observed due to compound binding (modification of distance and angles between catalytic residues), which indicates that allosteric regulation in cruzain might be mediated via alterations of its dynamical properties similarly to allosteric inhibition of human cathepsin K (HCatK). The current findings are particularly relevant for the design of allosteric modulators of papain-like cysteine proteases.

Partial Text

Cruzain is the major papain-like cysteine protease of Trypanosoma cruzi, the protozoan responsible for Chagas disease. This enzyme is indispensable for the survival and propagation of the parasite and, therefore, is considered a potential drug target for the disease control [1–3]. Toxicity and inefficiency of the available chemotherapy [4–6], fueled the pursuit for alternative drugs, which in turn, has led to the discovery of many cruzain inhibitors. A tangible evidence of the latter, is the occurrence of twenty-five crystal structures of cruzain in complex with different competitive inhibitors in the Protein Data Bank (PDB) [1, 7–9]. On the other hand, numerous experimental studies and computational predictions have been performed to characterize the cruzain binding site and specificity, facilitating the design of active-site directed drugs [8–12]. However, the lack of interest of pharmaceutical industry in low profit products (which is the case of tropical diseases) [13], and the failure to find a “real drug” that reaches the production and distribution scales, have encouraged the search for new scaffolds of cruzain inhibitors, as well as different strategies of enzyme inhibition and, ultimately, novel therapeutic targets.

Notwithstanding cruzain being an attractive therapeutic target for Chagas disease, few of the reported inhibitors of this protease have been selected for clinical trials in humans to date. This is probably due to their toxicity, low pharmacologic profiles and/or off-target effects. There are some aspects of this enzyme requiring further study that may foster the discovery of new drugs, such as the search for potential druggable sites to design non-competitive inhibitors by using conformational-sampling technics. So far, there are no reported studies on the identification of allosteric inhibitors of cruzain through structure-based virtual screening approaches. In this sense, our results constitute an in silico strategy for designing novel allosteric inhibitors of this enzyme. In the current work, several approaches of structure-based drug design were employed in order to rank the best hits that could contribute to a more specific inhibition of this protease.




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