Antiparasitic activity of furanyl N-acylhydrazone derivatives against Trichomonas vaginalis: in vitro and in silico analyses

Abstract

Background: Trichomonas vaginalis is the causative agent of trichomoniasis, which is one of the most common sexually transmitted diseases worldwide. Trichomoniasis has a high incidence and prevalence and is associated with serious complications such as HIV transmission and acquisition, pelvic inflammatory disease and preterm birth. Although trichomoniasis is treated with oral metronidazole (MTZ), the number of strains resistant to this drug is increasing (2.5-9.6%), leading to treatment failure. Therefore, there is an urgent need to find alternative drugs to combat this disease.

Methods: Herein, we report the in vitro and in silico analysis of 12 furanyl N-acylhydrazone derivatives (PFUR 4, a-k) against Trichomonas vaginalis. Trichomonas vaginalis ATCC 30236 isolate was treated with seven concentrations of these compounds to determine the minimum inhibitory concentration (MIC) and 50% inhibitory concentration (IC₅₀). In addition, compounds that displayed anti-T. vaginalis activity were analyzed using thiobarbituric acid reactive substances (TBARS) assay and molecular docking. Cytotoxicity analysis was also performed in CHO-K1 cells.

Results: The compounds PFUR 4a and 4b, at 6.25 µM, induced complete parasite death after 24 h of exposure with IC₅₀ of 1.69 µM and 1.98 µM, respectively. The results showed that lipid peroxidation is not involved in parasite death. Molecular docking studies predicted strong interactions of PFUR 4a and 4b with T. vaginalis enzymes, purine nucleoside phosphorylase, and lactate dehydrogenase, while only PFUR 4b interacted in silico with thioredoxin reductase and methionine gamma-lyase. PFUR 4a and 4b led to a growth inhibition (< 20%) in CHO-K1 cells that was comparable to the drug of choice, with a promising selectivity index (> 7.4).

Conclusions: Our results showed that PFUR 4a and 4b are promising molecules that can be used for the development of new trichomonacidal agents for T. vaginalis.

Keywords: Antiparasitic; Lipid peroxidation; Molecular docking; Trichomonacidal; Trichomoniasis.

Fig. 1

Chemical structures of PFUR 4 and 4a-k synthesized by the reaction of 2-(H₂NNHCO-furan) and aryl aldehydes (EtOH, RCHO, r.t., 1–72 h, 40–97%)

Fig. 2

In vitro trichomonacidal activity of PFUR 4 and 4a-k at 100 µM against Trichomonas vaginalis ATCC 30236 isolate, confirmed by the trypan blue (0.4%) assay after 24 h of exposure: Control (untreated trophozoites), 0.6% DMSO (vehicle for solubilization), MTZ (metronidazole at 100 µM). Viability of 100% in control corresponds to 2.6 × 10⁵ trophozoites/ml. Data are presented as the mean ± standard deviation of at least three independent experiments. Different letters show a significant difference at P < 0.05

Fig. 3

Anti-Trichomonas vaginalis assay. MIC and IC₅₀ for the antiparasitic activity of PFUR 4a (a) and PFUR 4b (b) against Trichomonas vaginalis ATCC 30236 after exposure to 1.5625, 3.125, 6.25, 12.5, 25, 50 and 100 µM concentrations for 24 h. Kinetic growth curves of Trichomonas vaginalis ATCC 30236 after 1, 6, 12, 24, 48, 72 and 96 h of treatment with PFUR 4a (c) and PFUR 4b (d) at 6.25 µM. Growth was completely inhibited after 24 h. Control (untreated trophozoites), 0.6% DMSO (vehicle for solubilization), MTZ (metronidazole at 100 µM). Viability of 100% in control corresponds to 2.6 × 10⁵ trophozoites/ml. Data are presented as the mean ± standard deviation of at least three independent experiments. Different letters show a significant difference at P < 0.05

Fig. 4

Lipid peroxidation levels measured through thiobarbituric acid reactive substances assay with malondialdehyde (MDA) as a biomarker, after 24 h of exposure to PFUR 4a and 4b, both at 6.25 µM. Control (untreated trophozoites), DMSO (vehicle for solubilization), MTZ (metronidazole at 100 µM). Data are presented as the mean ± standard deviation of at least three independent experiments

Fig. 5

Representation of 2D projection and predicted binding mode of PFUR 4a with the T. vaginalis enzymes TvPNP (a, b) and TvLDH (c, d). The distance (Å) of the hydrogen bonds between specific residues and PFUR 4a is shown in green

Fig. 6

Representation of 2D projection and predicted binding mode of PFUR 4b with the T. vaginalis enzymes TvPNP (a, b), TvTrxR (c, d), TvLDH (e, f) and TvMGL (g, h). The distance (Å) of the hydrogen bonds between specific residues and PFUR 4b is shown in green

Fig. 7

Representation of 2D projection and predicted binding mode of metronidazole (MTZ) with the T. vaginalis enzymes TvPNP (a, b), TvLDH (c, d) and TvMGL (e, f). The distance (Å) of the hydrogen bonds between specific residues and MTZ is shown in green

Fig. 8

Cytotoxicity effect of PFUR 4a (a) and PFUR 4b (b) at 1.5625, 3.125, 6.25 and 12.5 µM on CHO-K1 cells through MTT assay after 24 h of exposure; 0.6% DMSO (vehicle for solubilization) and MTZ (metronidazole at 100 µM). Data are presented as the mean ± standard deviation of at least three independent experiments. Different letters show a significant difference between treatments at P < 0.05