Control of aflatoxin biosynthesis by sulfur containing benzimidazole derivatives: In-silico interaction, biological activity, and gene regulation of Aspergillus flavus.

Aspergillus flavus producing aflatoxins is one of the potent contaminants of raw food commodities during pre-and post-harvest crops. Aflatoxins are the group of secondary metabolites a subset of natural polyketides. Our major focus is on the inhibition of the biosynthesis pathway of aflatoxin by targeting the enzymes involved. Benzimidazoles are known antimicrobial compounds. In this study the sulfur containing benzimidazole derivatives were tested for their antifungal and antiaflatoxigenic activity. The fungal growth and aflatoxin production was analysed in culture medium as well as in the rice. Inhibition of specific genes was studied in terms of mRNA expression and the interaction of test compound with polyketide synthases by in-silico molecular docking. Substitution at the 6th position of 2-(2-thienyl) benzimidazole (2-TBD) reduced the antifungal property of benzimidazole but effectively inhibited the aflatoxin synthesis in the culture medium as well as in the rice from the toxigenic strain of A. flavus. Among the derivatives tested, the methyl group containing 2-(2-thienyl)- 6-methylbenzimidazole (6-MTBD) inhibited aflatoxin B1 most effectively followed by carboxylic group containing 2-(2-thienyl) benzimidazole-6-carboxylic acid (6-TBCA) with IC50 value of 12.36 and 18.25 µg/mL respectively. Molecular docking study shows that 2-(2-thienyl) benzimidazole-6-carbonitrile (6-CTBD) and 6-MTBD occupy same pocket on TE domain of PksA with similar range of binding energy, however the experimental data show a different effect on the biosynthesis of AFB1. 6-MTBD effectively inhibited the AFB1 synthesis (97%) while 6-CTBD could not (39.5%). Data obtained from the expression study also supports the experimental observations. These compounds are non-toxic to mammalian cells. These benzimidazole derivatives inhibit toxic secondary metabolites without affecting the growth of the fungi hence can be used during fermentation to avoid mycotoxin contamination.

Inhibition of aflatoxin B1 biosynthesis and down regulation of aflR and aflB genes in presence of benzimidazole derivatives without impairing the growth of Aspergillus flavus.

Aflatoxins are mutagenic secondary metabolites produced by certain ubiquitous saprophytic fungi. These contaminate agricultural crops and pose a serious health threat to humans and livestock all over the world. Benzimidazole and its derivatives are biologically active heterocyclic compounds known for their fungicidal activity. In the present study, second and sixth position substituted benzimidazole derivatives are tested for their antifungal and anti-aflatoxigenic activity. Aflatoxigenic strain of Aspergillus flavus cultured in Yeast extract sucrose (YES) medium as well as in rice in the presence and absence of test compounds. 2-(2-Furyl) benzimidazole (FBD) showed complete inhibition of fungal growth at 50 µg/mL. However, the polar derivatives of FBD viz. 6-NFBD, 6-AFBD, 6-CAFBD, and 6-CFBD did not impair the fungal growth but effectively inhibited aflatoxin B1 biosynthesis. Significant down-regulation of aflR gene involved in regulation and aflB structural gene for aflatoxin B1 biosynthesis was observed in presence of 6-NFBD. These benzimidazole derivatives also showed good anti-aflatoxigenic activity in rice, though the IC50 concentrations in rice were comparatively higher than those in YES medium. This study summarizes the most notable structure-activity relationship (SAR) of 2-(2-Furyl) benzimidazoles for anti-aflatoxigenic and anti-fungal activities. These molecules can be further studied for their applications in industrial fermentation processes vulnerable to mold growth and subsequent aflatoxin B1 synthesis like koji fermentation, cheese production, etc.