Inhibition of Leishmania infantum Trypanothione Reductase by Azole‐Based Compounds: a Comparative Analysis with Its Physiological Substrate by X‐ray Crystallography

Abstract

Herein we report a study aimed at discovering a new class of compounds that are able to inhibit Leishmania donovani cell growth. Evaluation of an in-house library of compounds in a whole-cell screening assay highlighted 4-((1-(4-ethylphenyl)-2-methyl-5-(4-(methylthio)phenyl)-1H-pyrrol-3-yl)methyl)thiomorpholine (compound 1) as the most active. Enzymatic assays on Leishmania infantum trypanothione reductase (LiTR, belonging to the Leishmania donovani complex) shed light on both the interaction with, and the nature of inhibition by, compound 1. A molecular modeling approach based on docking studies and on the estimation of the binding free energy aided our rationalization of the biological data. Moreover, X-ray crystal structure determination of LiTR in complex with compound 1 confirmed all our results: compound 1 binds to the T(SH)2 binding site, lined by hydrophobic residues such as Trp21 and Met113, as well as residues Glu18 and Tyr110. Analysis of the structure of LiTR in complex with trypanothione shows that Glu18 and Tyr110 are also involved in substrate binding, according to a competitive inhibition mechanism.

Novel Heteroaryl Selenocyanates and Diselenides as Potent Antileishmanial Agents

“…On the other hand, nitrofuran compounds (34,35), the most relevant registered as nifurtimox, and derivatives of the benzodioxol core (36) have been selected. In addition, substituted five-membered heterocyclic rings such as isoxazol (37) and thiophenyl (38) or pirrol (39) have been tested as leishmanicidal agents. Furthermore, related to heterocycles derivatives, some fused aryl azo and triazo molecules have been described (34,40).…”

Dibenzosuberyl substituted polyamines and analogs of clomipramine as effective inhibitors of trypanothione reductase; molecular docking, and assessment of trypanocidal activities

“…Several crystal structures show aromatic moieties of inhibitors bound at the "hydrophobic wall" of TR, and interacting with Trp22 and Met114 (corresponding to BR 4), and often the inhibitor molecule extends into the region near Tyr111 (BR 3). For example, crystal structures locate in the region of Trp21/22 and Met113/114: the acridine rings of mepacrine (quinacrine) 42 and quinacrine mustard; 43 the dihydroquinazoline ring of T. brucei TR inhibitors (with groups also being close to Tyr110 and also in a conformationally induced sub-pocket); 34 also biaryl inhibitors 96 and a diarylpyrrole 32 (both of which also extended into region near Tyr110). Representative ligand poses compiled from all protonated versions and DBS/DBA configurations of compounds (1) to (7) …”

“…65 There is previous kinetic evidence for the binding of more than one inhibitor molecule to the large active site of TR. 58 Additionally, crystal structures of TR with bound quinacrine mustard, 43 and TR with a diarylpyrrole 32 show two molecules located in the active site. Our enzyme studies did not show evidence of the binding of more than one inhibitor molecule.…”

“…The active site has several locations where ligands can bind 40,96 and for some inhibitors is large enough to accommodate two molecules. 32 The residues directly involved in catalysis (including the redox active Cys53 and Cys58, along with His461') are positioned at the deepest part of the cleft, and close to the dimer interface (note, in one subunit the residues are labeled with primed numbers). At the interface of the subunits there is a tunnel that connects both active sites, which has a wider volume at the center of the enzyme, i.e., an interface pocket.…”

“…7 Also, several genetic studies on Leishmania and Trypanosoma have shown the importance of TR, including studies in which trypanosomatids modified to produce decreased levels of TR were found to be acutely sensitive to oxidative stress with severely compromised abilities to infect cells. [28][29][30][31] Additionally, the structure and properties of TR from different genera of trypanosomatids are very similar, 16,[32][33][34] indicating that an inhibitor of TR may be active against a range of trypanosomatids.…”