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.