Isopropyl Gallate, a Gallic Acid Derivative: In Silico and In Vitro Investigation of Its Effects on Leishmania major

Abstract

Isopropyl gallate (IPG) is a polyphenol obtained from alterations in the gallic acid molecule via acid catalysis with previously reported leishmanicidal and trypanocidal activities. The present study aims to evaluate in silico binding activity towards some targets for antileishmanial chemotherapy against Leishmania major species, and ADMET parameters for IPG, as well as in vitro antileishmanial and cytotoxic effects. Molecular docking was performed using AutoDockVina and BIOVIA Discovery Studio software, whereas in silico analysis used SwissADME, PreADMET and admetSAR software. In vitro antileishmanial activity on promastigotes and amastigotes of Leishmania major, cytotoxicity and macrophages activation were assessed. IPG exhibited affinity for pteridine reductase (PTR1; -8.2 kcal/mol) and oligopeptidase B (OPB; -8.0 kcal/mol) enzymes. ADMET assays demonstrated good lipophilicity, oral bioavailability, and skin permeability, as well as non-mutagenic, non-carcinogenic properties and low risk of cardiac toxicity for IPG. Moreover, IPG inhibited the in vitro growth of promastigotes (IC₅₀ = 90.813 µM), presented significant activity against amastigotes (IC₅₀ = 13.45 μM), promoted low cytotoxicity in macrophages (CC₅₀ = 1260 μM), and increased phagocytic capacity. These results suggest IPG is more selectively toxic to the parasite than to mammalian cells. IPG demonstrated acceptable in silico pharmacokinetics parameters, and reduced infection and infectivity in parasitized macrophages, possibly involving macrophage activation pathways and inhibition of leishmania enzymes.

Keywords: ADMET; antileishmania activity; molecular docking; polyphenols; propyl gallate.

Figure 1

The 2D structure of IPG (isopropyl 3,4,5-trihydroxybenzoate). Molecular weight of 212.20 g/mol. Source: Swiss ADME, 2021.

Figure 2

Interaction affinity between IPG and the molecular targets under study. Nucleoside hydrolase (NH); oligopeptidase B (OPB); leishmanolysin proteinase (gp63); pteridine reductase (PTR1); triparedoxin peroxidase (TxP). Two asterisks (**) Represents the molecular targets (OPB and PTR-1) that showed better binding interaction with IPG.

Figure 3

Molecular docking of the most favorable interaction of IPG with the active site of PTR1 and the active site of OPB. (A) Active site of pteridine reductase—PTR1; (B) 3D interaction of IPG with PTR1; (C) 2D interaction of IPG with PTR1; (D) Active site of oligopeptidase B—OPB; (E) 3D interaction of IPG with OPB; (F) 2D interaction of IPG with OPB.

Figure 4

Murine macrophages experimentally infected by L. major without treatment (A–C) and treated with IPG at concentrations of 19 μM (D–F) and 38 μM (G–I). Amphotericin B was used as a positive control at the concentration of 2.16 μM (J–L). VP—Parasitophorous vacuole. Black arrows indicate internalized amastigote forms. Red arrows indicate smeared macrophages. Magnification 1000×.

Figure 5

Effects of IPG and Anf B as a reference drug to assess infection (infected macrophages (A) and infectivity (B) in the treatment of murine macrophages infected with L. major. The percentage of infection after treatment (A) and the number of amastigotes per macrophage (B) were calculated by counting 100 cells in triplicate. Results are presented as mean ± SEM. of three independent experiments performed in triplicate, * p < 0.05, ** p < 0.01, when compared with control C, Anf B or the concentrations tested.

Figure 6

Influence of IPG on lysosomal volume, phagocytic capacity of murine macrophages and nitric oxide. (A) (Lysosomal volume); (B) (phagocytic capacity), and (C) (nitrite) represents the mean ± SEM of three experiments performed in triplicate, being * p < 0.05; ** p < 0.01; **** p < 0.0001. C—control, LPS—Escherichia coli lipopolysaccharide.