Reactivity of Biliatresone, a Natural Biliary Toxin, with Glutathione, Histamine, and Amino Acids

Abstract

In our previous work, we identified a natural toxin, biliatresone, from Dysphania glomulifera and D. littoralis, endemic plants associated with outbreaks of biliary atresia in Australian neonatal livestock. Biliatresone is a very rare isoflavonoid with an α-methylene ketone between two phenyls, 1,2-diaryl-2-propenone, along with methylenedioxy, dimethoxyl, and hydroxyl functional groups, that causes extrahepatic biliary toxicity in zebrafish. The toxic core of biliatresone is a methylene in the α-position relative to the ketone of 1,2-diaryl-2-propenone that serves as an electrophilic Michael acceptor. The α-methylene of biliatresone spontaneously conjugated with water and methanol (MeOH), respectively, via Michael addition in a reverse phase high-performance liquid chromatography (RP-HPLC) analysis. We here report the reactivity of biliatresone toward glutathione (GSH), several amino acids, and other thiol- or imidazole-containing biomolecules. LC-MS and HPLC analysis of the conjugation reaction showed the reactivity of biliatresone to be in the order histidine > N-acetyl-d-cysteine (D-NAC) = N-acetyl-l-cysteine (L-NAC) > histamine > glutathione ≥ cysteine ≫ glycine > glutamate > phenylalanine, while serine and adenine had no reactivity due to intramolecular hydrogen bonding in the protic solvents. The reactivity of ethyl vinyl ketone (EVK, 1-penten-3-one), an example of a highly reactive α,ß-unsaturated ketone, toward GSH gave a 6.7-fold lower reaction rate constant than that of biliatresone. The reaction rate constant of synthetic 1,2-diaryl-2-propen-1-one (DP), a core structure of the toxic molecule, was 10-fold and 1.5-fold weaker in potency compared to the reaction rate constants of biliatresone and EVK, respectively. These results demostrated that the methylenedioxy, dimethoxyl, and hydroxyl functional groups of biliatresone contribute to the stronger reactivity of the Michael acceptor α-methylene ketone toward nucleophiles compared to that of DP and EVK.

The effect of rosemary (Rosmarinus officinalis L.) extract on the oxidative stability of lipids in cow and soy milk enriched with fish oil

“…2B). 1-penten-3-one is a highly reactive α,ß-unsaturated ketone that could react further with other compounds like protein, which caused its reduction during the later stage of the storage period (Koo, Waisbourd-Zinman, Wells, Pack, & Porter, 2016). This was in agreement with our previous studies on the lipid oxidation progress of fish oil enriched cow milk (Alemán et al, 2015;Sørensen et al, 2015).…”

Hsp72 Is an Intracellular Target of the α,β-Unsaturated Sesquiterpene Lactone, Parthenolide

“… 2 , 33 Furthermore, electrophilic modification of histidine by electrophiles 4-HNE and biliatresone (an α,β-unsaturated ketone) suggests that modification is possible with a secondary amine as nucleophile, such as an imidazole group. 32 , 34 , 35 Hsp72 has 50 lysine residues, 7 of which we found to be modified. To try to understand why those seven were targeted as well as the other cysteines and histidine, we used the PDB2PQR server to run a PROPKA3 calculation to find p K a and surface exposure for all of the ionizable amino acids in Hsp72.…”

“…Modification of lysine and histidine residues by electrophilic compounds containing α,β-unsaturated carbonyls have been reported in the literature. 4 , 31 , 32 Although parthenolide modification of such residues on proteins under aqueous conditions has not been reported previously, the reaction of primary and secondary amines with parthenolide in methanol is a synthetic route for the development of new chemotherapeutic drugs. 2 , 33 Furthermore, electrophilic modification of histidine by electrophiles 4-HNE and biliatresone (an α,β-unsaturated ketone) suggests that modification is possible with a secondary amine as nucleophile, such as an imidazole group.…”

The toxin biliatresone causes mouse extrahepatic cholangiocyte damage and fibrosis through decreased glutathione and SOX17

“…After demonstrating the effects of biliatresone on cholangiocytes in spheroid and duct explant culture, we studied its mechanism of action. We have shown that biliatresone contains an α‐methylene‐ketone reactive group and binds GSH strongly and rapidly . Given that decreases in GSH in some cell types have been reported to result in diminished cellular tubulin staining, as we observed in cholangiocytes after biliatresone treatment, we hypothesized that biliatresone acts through GSH.…”