Safranal and its analogs inhibit Escherichia coli ATP synthase and cell growth

Abstract

Safranal, a dominant component of saffron, is known to have antitumor, cytotoxic, and antibacterial properties. In this study, we examined safranal and its structural analogs— thymol, carvacrol, damascenone, cuminol, 2,6,6-Trimethyl-2-cyclohexene-1,4-dione (TMCHD), 4-Isopropylbenzyl bromide (IPBB), and 4-tert-Butylphenol (TBP) induced inhibition of Escherichia coli membrane bound F1Fo ATP synthase. Safranal and its analogs inhibited wild-type enzyme to variable degrees. While safranal caused 100% inhibition of wild-type F1Fo ATP synthase only, about 50% inhibition occurred for αR283D mutant ATP synthase. Moreover, safranal, thymol, carvacrol, damascenone, cuminol, TMCHD, IPBB, and TBP all fully abrogated the growth of wild-type E. coli cells and had partial or no effect on the growth of null and mutant E. coli strains. Therefore, the antimicrobial properties of safranal, thymol, carvacrol, damascenone, cuminol, TMCHD, IPBB, and TBP can be linked to their binding and inhibition of ATP synthase. Total loss of growth in wild-type and partial or no growth loss in null or mutant E. coli strains demonstrates that ATP synthase is a molecular target for safranal and its structural analogs. Partial inhibition of the αArg-283 mutant enzyme establishes that αArg-283 residue is required in the polyphenol binding pocket of ATP synthase for the binding of safranal. Furthermore, partial growth loss for the null and mutant strains in the presence of inhibitors also suggests the role of other targets and residues in the process of inhibition.

Understanding the link between antimicrobial properties of dietary olive phenolics and bacterial ATP synthase

“…The inhibition of microbial cell growth in the presence of phytochemicals from previous studies [11, 25, 49, 50, 56, 76] and the olive phenolics—tyrosol, hydroxytyrosol, DHPG, and oleuropein—from the current study suggest that ATP synthase can be used as a potential molecular drug target to combat microbial infections. In conclusion, the tyrosol induced inhibition of ATPase activity and E. coli cell growth in the current study indicate that the antimicrobial properties of tyrosol can be linked to its inhibitory effects on ATP synthase.…”

“…The maximal volume of DMSO in ATPase assays was less than 1%. In the current study and previous studies, we noted that up to 40% DMSO by itself had no effect on membrane-bound F 1 F o of E. coli ATP synthase [25, 49, 50]. All other chemicals and reagents used in the current study were ultrapure, analytical grade and purchased from Sigma-Aldrich Chemical Company or Fisher Scientific Company.…”

“…Other dietary polyphenolic compounds have also been shown to bind to the polyphenol binding pocket [25, 49, 50, 56, 57]. Recently, site-directed mutagenic studies confirmed that safranal, a phenolic compound, binds at the polyphenol binding site and that the αArg-283 residue is essential for that binding [25]. Therefore, in the current study, we mutated αArg-283 to αAsp-283, αGlu-284 to αArg-284, βVal-265 to βGln-265, and γThr-273 to γAla-273 to confirm the role of these residues in the binding of the olive phenolics tyrosol, hydroxytyrosol, DHPG, and oleuropein at the polyphenol binding site.…”

“…Tyrosol is mainly glucuronidated after intake and is one of the main metabolite forms of tyrosol in humans [65]. Recently, another phenolic phytochemical, safranal, was shown to cause inhibition of ATP synthase at high concentrations [25]. It was also shown that high concentrations of safranal were essential to cause the observed pharmacological effects, and these concentrations are non-toxic in animals and humans [66].…”

“…A large number of dietary phenolic compounds from a variety of sources have been shown to have potential antitumor or antimicrobial properties [21–25]. Olives and its constituent phenolic compounds have nutraceutical properties and have been assessed for their strong antimicrobial properties at low concentrations against human intestinal and respiratory tract infections, such as those caused by Gram positive bacteria ( Bacillus cereus , B. subtilis , and Staphylococcus aureus ), Gram negative bacteria ( Pseudomonas aeruginosa , E. coli , and Klebsiella pneumoniae ), and fungi ( Candida albicans and Cryptococcus neoformans ) [26].…”