Evaluation of the Antileishmanial Activity of Some Benzimidazole Derivatives Using In Vitro and In Silico Techniques

Abstract

Benzimidazole derivatives are well known for their anthelmintic activity. Investigating the potential efficacy of new derivatives of this class against various parasites is essential to identify novel drug candidates. For this purpose, an in-house molecular database was screened, and four benzimidazole-based molecules were chosen to evaluate antiprotozoal activity. The compounds (K1-K4) had been previously synthesized through a four-step procedure. The potential in vitro cytotoxic properties of the compounds were assessed against the Leishmania (L.) major strain and L929 mouse fibroblast cells. The tests indicated that K1 (3-Cl phenyl) demonstrated an antileishmanial effect (IC₅₀ = 0.6787 µg/mL) and cytotoxicity at elevated concentrations (CC₅₀ = 250 µg/mL) in healthy cells. These findings were comparable to those of AmpB. The antileishmanial activity values were determined as follows: K2; 8.89 µg/mL, K3; 45.11 µg/mL, K4; and 69.19 µg/mL. The CC₅₀ values were determined as follows: K2, 63 µg/mL; K3; 0.56 µg/mL; and K4, 292 µg/mL. Molecular docking and dynamic simulations were conducted to elucidate the potential mechanisms of action of the test substances. In silico investigations indicated interactions between the compounds and the active site of pteridine reductase 1 (PTR1), which is a biosynthetic enzyme essential for parasite proliferation. N-alkyl benzimidazole-based compounds exhibit potential inhibitory activity against L. (L.) major promastigotes. Therefore, these findings suggest that in vivo evaluation is warranted, and structural modifications may lead to the identification of more effective antileishmanial agents.

Keywords: Leishmania major; antileismanial activity; benzimidazole derivates; in silico; in vitro; pteridine reductase 1.

Scheme 1

Compounds evaluated for antileishmanial activity.

Figure 1

The efficacy of K2, K3, and K4 compounds against L. major promastigotes.

Figure 2

The efficacy of the AmpB and K1 compound against L. major promastigotes.

Figure 3

Control (a) and serial dilutions of the K1 compound (ranging from 0.23 µg/mL to 30 µg/mL) (b–l) in wells containing L. (L.) major promastigotes (100× magnification).

Figure 4

The 3D (A) and 2D (B) docking poses of the compound PTR1-K1 enzyme complex (PDBID: 5L4N). Blue dots represents π–π interactions, light blue dots represents aromatic hydrogen bonds, and yellow dots represents hydrogen bonds.

Figure 5

The 3D (A) and 2D (B) docking poses of the compound PTR1-K2 enzyme complex (PDBID: 5L4N). Blue dots represents π–π interactions, light blue dots represents aromatic hydrogen bonds, and yellow dots represents hydrogen bonds.

Figure 6

Interaction plots of the MDS results for the enzyme complexes of K1–PTR1. (A) A diagram of the ligand properties; (B) the RMSD plot of the ligand and of the protein throughout the simulation; (C) RMSF-plots of amino acids and their interactions; (D) the interaction between the fraction and residue diagram; (E) the plot of the number of interactions and interaction types over time, purple: hydrophobic interaction, green: hydrogen bond, blue: water-mediated hydrogen bonds; and (F) the plot of the total interaction residues over time.