Dihydropyrazine-Induced Inactivation of Glyceraldehyde-3-phosphate Dehydrogenase

Abstract

Reactive oxygen species (ROS) can cause oxidative damage to DNA, proteins, and lipids in all living organisms, and the resulting oxidative damage may play roles in several human disorders as well as the aging process.1) Dihydropyrazine (DHP) is produced as a Maillard reaction intermediate by condensation of two molecules of D-glucosamine, 2) and some DHP derivatives generate various radical species and ROS that cause DNA strand cleavage in vitro.3) Since many pyrazine derivatives have been detected in various foods 4) and human urine, 5) we speculated that DHPs, which are presumed to be precursors of pyrazines, would be responsible for certain diseases. However, there are few reports regarding the biological and physiological roles of DHPs.Previously, we reported that methyl-substituted dihydropyrazines (Me-DHPs), such as 2,3-dihydro-5,6-dimethylpyrazine, had DNA strand-breaking activity, inhibited growth and induced mutagenesis in Escherichia coli.6,7) Although phenyl-substituted dihydropyrazines (Ph-DHPs) have much higher DNA strand-breaking activities and produce higher levels of radical species than Me-DHPs, 8,9) DNA repairdeficient bacteria are less sensitive to Ph-DHPs than to Me-DHPs.10,11) Recently, cyclohexyl-DHP, which was designed according to our hypothesis of a relationship between the chemical structures of DHP derivatives and their DNA strand-breaking activities, 12) was shown to possess a stronger DNA strand-breaking activity than Me-DHPs and PhDHPs.13) Moreover, cyclohexyl-DHP can potentially generate superoxide anions, and not only cause breakage of chromosomal DNA leading to mutagenic lesions but also induce damage to cellular proteins. 14) In the present study, we examined enzyme inactivation by DHPs using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a convenient model protein, since GAPDH is known to be highly sensitive to oxidative damage. 15) Our observations reveal that DHPs inactivate GAPDH in vitro and in vivo. MATERIALS AND METHODSSynthesis of DHP Derivatives 2,3-Dihydro-5,6-dimethylpyrazine (Me-DHP-1), 2,3-dihydro-5-methyl-6-phenylpyrazine (Ph-DHP-1), and a mixture of two isomers of cyclohexyl-DHP (Fig. 1), namely 2,3,5,6,7,8-hexahydroquinoxaline (endo-type) and 1,2,3,5,6,7-hexahydroquinoxaline (exo-type), were prepared using a previously described method. 3)Materials Rabbit muscle GAPDH was obtained from MP Biomedicals. DL-glyceraldehyde-3-phosphate was purchased from Sigma. Nicotinamide adenine dinucleotide (NAD) was obtained from Wako. B-PER Bacterial Protein Extraction Reagent was purchased from Pierce.Bacterial Strain The E. coli Dihydropyrazine (DHP), which is produced during the Maillard reaction, generates radicals that not only cause breakage of chromosomal DNA leading to mutagenic lesions but also induce oxidative damage to cellular proteins. In the present study, we show that three DHP derivatives, which generated superoxide anions, caused inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). SH-compounds, such as cysteine, dithiothreitol (DTT), 2-mercap...

Glutathione depression by dihydropyrazine derivative

“…2). Consistent with this, in our previous study, Cyclohexyl-DHP exhibited more potent inhibition of GAPDH than that of methyl derivative of DHP (Takechi et al, 2010). Regarding GSH consumption, however, there were no apparent differences in reactivity with GSH between Cyclohexyl-DHP and Me-DHP-3 (Figs.…”

“…We recently reported that DHP derivatives caused inhibition of GAPDH and that SH-compounds suppressed the inhibition of GAPDH by DHP (Takechi et al, 2010). Therefore, we examined the interaction between DHP and GSH, which is the major intracellular thiol compound in organisms.…”

“…In addition, Cyclohexyl-DHP was shown to possess stronger DNA strand-breaking activity than Me-DHPs and PhDHPs (Maruoka et al, 2005;Yamaguchi et al, 2007). Moreover, we demonstrated that DHPs can potentially generate superoxide anions (Takechi et al, 2009), and not only cause breakage of chromosomal DNA leading to mutagenic lesions but also induce damage to cellular proteins such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Takechi et al, 2010).…”

Generation of Radical Species from Dihydropyrazines Having DNA Strand-Breakage Activity and Other Characteristics

“…However, it is still not known whether a protein or a gene is the target of the radical species. The inhibition effect 20,29) of DHPs on SH-enzymes has only been revealed recently. Further work is needed to elucidate the mechanism of action of DHPs in vitro and in vivo.…”

“…Among DHPs that have been studied, the strength order of both DNA strand-breakage activity and biological effects such as lethality and mutagenesis for Escherichia coli indicate a clear difference based on their structures. 19,20) In this report, free radical species from DHPs were identified and examined in order to clarify the radical's participation in the effects of DHPs.…”