Gene expression, molecular docking, and molecular dynamics studies to identify potential antifungal compounds targeting virulence proteins/genes VelB and THR as possible drug targets against Curvularia lunata

Abstract

Curvuluria lunata is a melanized fungus pathogenic to both plants and animals including humans, causing from mild, febrile to life-threatening illness if not well treated. In humans, it is an etiological agent of keratomycosis, sinusitis, and onychomycosis in immunocompromised and immunocompetent patients. The development of multiple-drug-resistant strains poses a critical treatment issue as well as public health problem. Natural products are attractive prototypes for drug discovery due to their broad-spectrum efficacy and lower side effects. The present study explores possible targets of natural antifungal compounds (α-pinene, eugenol, berberine, and curcumin) against C. lunata via gene expression analysis, molecular docking interaction, and molecular dynamics (MD) studies. Curcumin, berberine, eugenol, and α-pinene exhibited in vitro antifungal activity at 78 μg/ml, 156 μg/ml, 156 μg/ml, and 1250 μg/ml, respectively. In addition, treatment by these compounds led to the complete inhibition of conidial germination and hindered the adherence when observed on onion epidermis. Several pathogenic factors of fungi are crucial for their survival inside the host including those involved in melanin biosynthesis, hyphal growth, sporulation, and mitogen-activated protein kinase (MAPK) signalling. Relative gene expression of velB, brn1, clm1, and pks18 responsible for conidiation, melanin, and cell wall integrity was down-regulated significantly. Results of molecular docking possessed good binding affinity of compounds and have confirmed their potential targets as THR and VelB proteins. The docked structures, having good binding affinity among all, were further refined, and rescored from their docked poses through 100-ns long MD simulations. The MDS study revealed that curcumin formed a stable and energetically stabilized complex with the target protein. Therefore, the study concludes that the antifungal compounds possess significant efficacy to inhibit C. lunata growth targeting virulence proteins/genes involved in spore formation and melanin biosynthesis.

Keywords: Curvularia lunata; bioactive molecules; molecular docking; molecular dynamics; virulence proteins.

FIGURE 1

(A) C. lunata infection on the leaves of the rice plant; (B) C. lunata colony morphology on potato dextrose agar; and (C) C. lunata conidia.

FIGURE 2

Microscopic images depicting conidia of C.lunata Control (A,B); α-pinene (C,D); curcumin (E,F); berberine (G,H); and eugenol (I,J). Scale bar = 10 µm.

FIGURE 3

Relative quantification of brn1, velB, pks18, and clm1 gene expression in C. lunata (normalised to the house-keeping gene GAPDH). Data were reported as mean of fold changes with standard deviation from two independent experiments amplified in triplicate. p ≤ 0.05 was considered statistically significant.

FIGURE 4

Predicted secondary structure validation of THR (A) and VelB (B) proteins of C. lunata using SOPMA. The blue line represents α-helices, red colour represents the extended strand, green colour represents β-turn, and magenta colour represents the random coil in graphical representation. The X-axis represents position of the amino acid; the Y-axis shows the score for each predicted state.

FIGURE 5

Ramachandran plot of predicted 3D structures of THR (A) and VelB (B) proteins of C. lunata using PROCHECK software.

FIGURE 6

Binding of (A) α-pinene, (B) curcumin, (C) berberine, and (D) eugenol with VelB. Ribbon and 2D representation of VelB protein showing various interactions and docking fit of compounds.

FIGURE 7

Binding of (A) α-pinene, (B) curcumin, (C) berberine, and (D) eugenol with THR. Ribbon and 2D representation of THR protein showing various interactions and docking fit of compounds.

FIGURE 8

Structural order parameter analysis of the THR complex (curcumin) with respect to the THR receptor. (A) Root mean square deviation (RMSD), (B) solvent-accessible surface area (SASA), (C) root mean square fluctuation (RMSF), and (D) radius of gyration (RoG) analysis.

FIGURE 9

Structural order parameter analysis of the VelB complex (curcumin) with respect to the VelB receptor. (A) Root mean square deviation (RMSD), (B) solvent-accessible surface area (SASA), (C) root mean square fluctuation (RMSF), and (D) radius of gyration (RoG) analysis.

FIGURE 10

Distribution of the hydrogen bonds formed by curcumin with THR and VelB receptors at the binding site.