Enzymatic and Molecular Characterization of Anti- Leishmania Molecules That Differently Target Leishmania and Mammalian eIF4A Proteins, LieIF4A and eIF4AMus

Abstract

Previous investigations of the Leishmania infantum eIF4A-like protein (LieIF4A) as a potential drug target delivered cholestanol derivatives inhibitors. Here, we investigated the mode of action of cholesterol derivatives as a novel scaffold structure of LieIF4A inhibitors on the RNA-dependent ATPase activity of LieIF4A and its mammalian ortholog (eIF4AI). We compared their biochemical effects on RNA-dependent ATPase activities of both proteins and investigated if rocaglamide, a known inhibitor of eIF4A, could affect LieIF4A as well. Kinetic measurements were conducted at different concentrations of ATP, of the compound and in the presence of saturating whole yeast RNA concentrations. Kinetic analyses showed different ATP binding affinities for the two enzymes as well as different sensitivities to 7-α-aminocholesterol and rocaglamide. The 7-α-aminocholesterol inhibited LieIF4A with a higher binding affinity relative to cholestanol analogs. Cholesterol, another tested sterol, had no effect on the ATPase activity of LieIF4A or eIF4AI. The 7-α-aminocholesterol demonstrated an anti-Leishmania activity on L. infantum promastigotes. Additionally, docking simulations explained the importance of the double bond between C5 and C6 in 7-α-aminocholesterol and the amino group in the C7 position. In conclusion, Leishmania and mammalian eIF4A proteins appeared to interact differently with effectors, thus making LieIF4A a potential drug against leishmaniases.

Keywords: 7-α-aminocholesterol; Leishmania infantum; drug design; inhibitor; translation-initiation factor.

Figure 1

Chemical structures of the compounds used in this study. Chemical structures tested on the LieIF4A ATPase activity (a) cholestanol derivatives (6-α-aminocholestanol and 6-ketocholestanol), (b) cholesterol derivatives (7-α-aminocholesterol and cholesterol), (c) and rocaglate derivative (rocaglamide). The 6-ketocholestanol presents a keto group at position 6 (in red). The sterols present double bond between carbons 5 and 6 (in blue).

Figure 2

ATPase reaction velocities of eIF4A_Mus with 340 ng/μL yRNA and different concentrations of ATP. The means and standard deviations are shown for three independent experiments. The values were fit to the nonlinear Michaelis-Menten equation using GraphPad Prism software. All the reactions were performed in the presence of 10% DMSO and 820 nM LieIF4A.

Figure 3

Relative ATPase reaction rates of the two proteins in the presence of increasing concentrations of 7-α-aminocholesterol and 340 ng/μL RNA. Mammalian eIF4AI was used at 1200 nM and LieIF4A at 820 nM. The error bars represent the mean and standard deviations of three independent measurements made. The relative reaction velocities were normalized to one in the absence of the inhibitor compound to facilitate comparisons. Data were fit to an exponential decay for visualization purposes and IC₅₀ calculation. The relative reaction rate of LieIF4A (◌) and eIF4A_Mus (□) were plotted as a function of compound concentrations. The 7-α-aminocholesterol had different kinetic effects on the two proteins and clearly showed stronger inhibition on the Leishmania protein.

Figure 4

MTT cell viability assay shows a promising anti-leishmanial activity of the cholestanol and sterol derivatives in a dose-dependent manner and a little cellular toxicity at the active concentrations. (a) Effects on L. infantum promastigotes. (b) Effects on uninfected THP-1-derived macrophages. The cholesterol was not active at the tested concentrations.

Figure 5

Docking poses of 7-α-aminocholesterol and its analogs on LieIF4A protein. Best scored docking pose of (A) 7-α-aminocholesterol, (B) cholesterol, (C) 6-α-aminocholestanol and (D) 6-ketocholestanol. Compounds 7-aminocholesterol and 6-α-aminocholestanol established a stable H-bond with Asp332 from motif V, shown in dashed lines with their estimated length in Å.

Figure 6

Protein-inhibitor interaction diagrams as determined with LigPlot+. Residues involved in hydrophobic interactions are shown as red half-circles, and H-bonds are shown in green dotted lines along with their lengths in Angstroms. (A) Diagram of interaction between LieIF4A and 7-α-aminocholesterol. (B) Diagram of interaction between LieIF4A and cholesterol. (C) Diagram of interaction between LieIF4A and 6-α-aminocholestanol. (D) Diagram of interaction between LieIF4A and 6-ketocholestanol.