Appraisal of Cinnamaldehyde Analogs as Dual-Acting Antibiofilm and Anthelmintic Agents

Abstract

Cinnamaldehyde has a broad range of biological activities, which include antibiofilm and anthelmintic activities. The ever-growing problem of drug resistance and limited treatment options have created an urgent demand for natural molecules with antibiofilm and anthelmintic properties. Hence, we hypothesized that molecules with a scaffold structurally similar to that of cinnamaldehyde might act as dual inhibitors against fungal biofilms and helminths. In this regard, eleven cinnamaldehyde analogs were tested to determine their effects on fungal Candida albicans biofilm and nematode Caenorhabditis elegans. α-Methyl and trans-4-methyl cinnamaldehydes efficiently inhibited C. albicans biofilm formation (>90% inhibition at 50 μg/mL) with minimum inhibitory concentrations (MICs) of ≥ 200 μg/mL and 4-bromo and 4-chloro cinnamaldehydes exhibited anthelmintic property at 20 μg/mL against C. elegans. α-Methyl and trans-4-methyl cinnamaldehydes inhibited hyphal growth and cell aggregation. Scanning electron microscopy was employed to determine the surface architecture of C. albicans biofilm and cuticle of C. elegans, and confocal laser scanning microscopy was used to determine biofilm characteristics. The perturbation in gene expression of C. albicans was investigated using qRT-PCR analysis and α-methyl and trans-4-methyl cinnamaldehydes exhibited down-regulation of ECE1, IFD6, RBT5, UCF1, and UME6 and up-regulation of CHT4 and YWP1. Additionally, molecular interaction of these two molecules with UCF1 and YWP1 were revealed by molecular docking simulation. Our observations collectively suggest α-methyl and trans-4-methyl cinnamaldehydes are potent biofilm inhibitors and that 4-bromo and 4-chloro cinnamaldehydes are anthelmintic agents. Efforts are required to determine the range of potential therapeutic applications of cinnamaldehyde analogs.

Keywords: Candida albicans; anthelmintic; antibiofilm; protein interaction; trans-4-methyl cinnamaldehyde; α-methyl cinnamaldehyde.

SCHEME 1

Schematic representation of the strategies used in the present study.

FIGURE 1

In vitro antibiofilm activities of cinnamaldehyde analogs against C. albicans. Influences of eleven cinnamaldehyde analogs and trans-cinnamaldehyde (tC) on biofilm formation by the C. albicans DAY185 at 100 μg/mL (A), C. albicans DAY185 cell growth was investigated in the presence of the eight antibiofilm cinnamaldehyde analogs and the trans-cinnamaldehyde control at 100 μg/mL (B), Chemical structures and MIC (C). The antibiofilm activities of α-methyl cinnamaldehyde (α-MeC) and trans-4-methyl cinnamaldehyde (t-4MeC) at 20 and 50 μg/mL, and trans-cinnamaldehyde (tC) at 20–200 μg/mL against C. albicans DAY185 (D) and C. albicans ATCC 10231 (E). Error bars indicate standard deviations. *P < 0.05 vs. non-treated controls (None).

FIGURE 2

Inhibition of hyphal filamentation and aggregation by α-methyl cinnamaldehyde (α-MeC) and trans-4-methyl cinnamaldehyde (t-4MeC). C. albicans DAY185 was streaked onto PDA solid plates in the absence or presence of α-methyl cinnamaldehyde (α-MeC; 50 μg/mL), trans-4-methyl cinnamaldehyde (t-4MeC; 50 μg/mL), or trans-cinnamaldehyde (tC; 50 and 150 μg/mL). Colony morphologies were observed periodically over 6 days at 37°C (A), C. albicans DAY185 yeast-hyphae transition was assessed in PDB in the presence of α-methyl cinnamaldehyde (α-MeC; 50 μg/mL), trans-4-methyl cinnamaldehyde (t-4MeC; 50 μg/mL), or trans-cinnamaldehyde (tC; 50 and 150 μg/mL) after incubation for 24 h (B). The scale bars in panels (A,B) represent 100 μm. None indicates the non-treated control.

FIGURE 3

Effect of α-methyl (α-MeC) and trans-4-methyl (t-4MeC) cinnamaldehydes on C. albicans DAY185 biofilms. C. albicans DAY185 biofilm formation in the presence of α-methyl cinnamaldehyde (α-MeC), trans-4-methyl cinnamaldehyde (t-4MeC), or trans-cinnamaldehyde (tC) at 50 μg/mL was examined by confocal laser microscopy (A), C. albicans DAY185 biofilm formations were quantified by COMSTAT analysis (B), C. albicans DAY185 biofilm formations on nylon membranes grown in the presence of α-methyl cinnamaldehyde (α-MeC), trans-4-methyl cinnamaldehyde (t-4MeC), or trans-cinnamaldehyde (tC) at 50 μg/mL were observed by SEM (C). The scale bars in panel (A) represent 100 μm and in panel (B) represent 50 and 10 μm. Error bars indicate standard deviations. *P < 0.05 vs. non-treated controls (None). None indicates biofilm formation without treatment after 24 h incubation.

FIGURE 4

The relative transcriptional profile of ten biofilm- and hypha-related genes. C. albicans DAY185 cells incubated with or without α-methyl cinnamaldehyde (α-MeC) or trans-4-methyl cinnamaldehyde (t-4MeC) at 50 μg/mL for 6 h and relative transcriptional expressions profiles were obtained by qRT-PCR. Fold changes represent alteration in the transcription of treated vs. untreated C. albicans DAY185. RDN18 was used for endogenous normalization of expression levels and the experiment was performed in duplicate (six qRT-PCR reactions were performed per gene) (A). A plausible mechanism of C. albicans biofilm inhibition by α-methyl cinnamaldehyde (α-MeC) and trans-4-methyl cinnamaldehyde (t-4MeC) illustrating all the phenotypic and gene expressional changes (B). Error bars indicate standard deviations. *P < 0.05 vs. non-treated controls (None).

FIGURE 5

Amino acid residue interactions between UCF1 receptor protein and α-methyl cinnamaldehyde (α-MeC) (A), trans-4-methyl cinnamaldehyde (t-4MeC) (B), and trans-cinnamaldehyde (tC) (C), Amino acid residue interactions between YWP1 receptor protein and α-methyl cinnamaldehyde (α-MeC) (D), trans-4-methyl cinnamaldehyde (t-4MeC) (E), and trans-cinnamaldehyde (tC) (F).

FIGURE 6

Anthelmintic activities of cinnamaldehyde analogs were evaluated against C. elegans for 5 days. Error bars indicate standard deviations. *P < 0.05 vs. non-treated controls (None).