Synthesis, molecular modeling and NAD(P)H:quinone oxidoreductase 1 inducer activity of novel cyanoenone and enone benzenesulfonamides
Mostafa M. GhorabMaureen HigginsMansour S. AlsaidReem K. ArafaAbdelaaty A. ShahatAlbena T. Dinkova‐Kostova
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In biological systems, the Keap1/Nrf2/antioxidant response element pathway determines the ability of mammalian cells to adapt and survive conditions of oxidative, electrophilic and inflammatory stress by regulating the production of cytoprotective enzymes NAD(P)H:quinone oxidoreductase 1 (NQO1, EC 1.6.99.2) being one of them. Novel biologically active benzenesulfonamides 2, 3, 5–7, penta-2,4-dienamide 4 and chromene-2-carboxamide 8 structurally augmented with an electron-deficient Michael acceptor enone or cyanoenone functionalities were prepared. A new biological activity was conferred to these molecules, that of induction of NQO1. The potency of induction was increased by incorporation of a nitrile group adjacent to the enone and the dinitrophenyl derivative 3 was the most promising inducer. Also, molecular docking of the new compounds in the Nrf2-binding site of Keap1 was performed to assess their ability to inhibit Keap1 which biologically leads to a consequent Nrf2 accumulation and enhanced gene expression of NQO1. Docking results showed considerable interactions between the new molecules and essential binding site amino acids.Keywords:
Enone
Docking (animal)
KEAP1
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Owing to its unique fragrance, 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF) is widely used as a food flavoring agent and has high demand. Enone oxidoreductase is a vital enzyme involved in HEMF production. In this study, an enone oxidoreductase from Naumovozyma dairenensis CBS 421 (NDEO) was used for HEMF production for the first time. The mutant NDEOT183W,K290W was obtained through semirational protein engineering, which increased the HEMF yield by 75.2%. Finally, the engineered strain BM4 produced the highest HEMF yield, 194.42 mg L-1 in 132 h. Our study revealed that HEMF production can be improved in Saccharomyces cerevisiae and that this is an efficient method to improve the activity of enone oxidoreductase, which is important for the industrial synthesis of furanone.
Enone
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Questing the extremes of the cellular NAD(H) level using an Escherichia coli NAD+ auxotrophic mutant
NAD(H) is an essential cofactor in cell activity participating in over 300 redox reactions in vivo. However, it is difficult to determine the extremes of the cellular NAD(H) level in live cells because the NAD+ is tightly controlled with the biosynthesis regulation mechanism. Here, we developed a directed manipulation strategy to determine the extreme NAD(H) levels in Escherichia coli cells by providing exogenous NAD+ using the NAD(H) transporter NTT4, and blocking the NAD+ biosynthesis pathways. Firstly, we validated the function of NTT4 expressed in an E. coli mutant lacking the de novo NAD+ biosynthesis pathway. We then constructed the NAD+ auxotrophic mutant YJE003 by disrupting the essential NAD+ biosynthesis gene nadE in cells with an NTT4 expression background. The minimal NAD+ level was determined in M9 medium by proliferating YJE003 cells that were fed with exogenous NAD+. The maximal NAD(H) level was determined by exposing the cells to high concentrations of exogenous NAD(H). Compared with supplementation of NADH, cells grew faster and had a higher intracellular NAD(H) level when NAD+ was fed. The intracellular NAD(H) level increased with the increase of exogenous NAD+ concentration until it reached a plateau. Thus, a minimal NAD(H) level of 0.039 mM and a maximum of 8.49 mM were determined, which were 0.044- and 9.6-fold amounts of those of the wide-type cells, respectively. Finally, the potential application of this strategy in biotechnology was briefly discussed.
Auxotrophy
Glycerol-3-phosphate dehydrogenase
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Regeneration of NAD(P)(superscript +) and NAD(P)H captures more and more attention in recent years due to the following considerations: most oxidoreductase-catalyzed reactions need NAD(P)(superscript +) or NAD(P)H as oxidants or reductants, oxidoreductases are used extensively, and NAD(P)(superscript +) and NAD(P)H are quite expensive. This article reviews recent progress in regeneration of NAD(P)(superscript +) and NAD(P)H by enzymatic, electrochemical and photochemical methods. The application and development status of regeneration technology are also illustrated.
Glycerol-3-phosphate dehydrogenase
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The oxidoreductase inhibitor is not formed from NADH as previously thought, but only from NAD under alkaline conditions. Analogues of NAD (e.g. NADP) and components of the NAD molecule (e.g. ADP) have no effect on the formation of the inhibitor. The most favourable pH, temperature, duration of incubation, type of buffer and NAD concentration for the formation of the inhibitor were investigated. The method for the formation and chromatographic isolation of the oxidoreductase inhibitor is briefly described.
Glycerol-3-phosphate dehydrogenase
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The reaction of a number of easily prepared hydroxy-derivatives containing the thiocarbonyl or selenocarbonyl group with soft electrophilic reagents has been studied. The products are derivatives with carbon bonded to the anionic fragment of the electrophile. The reactions occur under mild, neutral conditions and, in appropriate cases, afford good yields.
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Carbon fibers
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NAD+ is mainly synthesized from nicotinamide (Nam) by the rate-limiting enzyme Nam phosphoribosyltransferase (Nampt) and degraded to Nam by NAD+-degrading enzymes in mammals. Numerous studies report that tissue NAD+ levels decrease during aging and age-related diseases and suggest that NAD+ replenishment promotes healthy aging. Although increased expression of Nampt might be a promising intervention for healthy aging, forced expression of Nampt gene, inducing more than 10-fold increases in the enzyme protein level, has been reported to elevate NAD+ levels only 40–60% in mammalian cells. Mechanisms underlying the limited increases in NAD+ levels remain to be determined. Here we show that Nampt is inhibited in cells and that enhanced expression of Nampt activates NAD+ breakdown. Combined with the measurement of each cell's volume, we determined absolute values (μM/h) of the rates of NAD+ synthesis (RS) and breakdown (RB) using a flux assay with a 2H (D)-labeled Nam, together with the absolute NAD+ concentrations in various mammalian cells including primary cultured cardiomyocytes under the physiological conditions and investigated the relations among total cellular Nampt activity, RS, RB, and the NAD+ concentration. NAD+ concentration was maintained within a narrow range (400–700 μM) in the cells. RS was much smaller than the total Nampt activity, indicating that NAD+ synthesis from Nam in the cells is suppressed. Forced expression of Nampt leading to 6-fold increase in total Nampt activity induced only a 1.6-fold increase in cellular NAD+ concentration. Under the conditions, RS increased by 2-fold, while 2-fold increase in RB was also observed. The small increase in cellular NAD+ concentration is likely due to both inhibited increase in the NAD+ synthesis and the activation of its breakdown. Our findings suggest that cellular NAD+ concentrations do not vary dramatically by the physiological fluctuation of Nampt expression and show the tight link between the NAD+ synthesis and its breakdown.
Nicotinamide phosphoribosyltransferase
Glycerol-3-phosphate dehydrogenase
Nicotinamide mononucleotide
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NAD (NAD(+)) and its reduced form (NADH) are omnipresent cofactors in biological systems. However, it is difficult to determine the extremes of the cellular NAD(H) level in live cells because the NAD(+) level is tightly controlled by a biosynthesis regulation mechanism. Here, we developed a strategy to determine the extreme NAD(H) levels in Escherichia coli cells that were genetically engineered to be NAD(+) auxotrophic. First, we expressed the ntt4 gene encoding the NAD(H) transporter in the E. coli mutant YJE001, which had a deletion of the nadC gene responsible for NAD(+) de novo biosynthesis, and we showed NTT4 conferred on the mutant strain better growth in the presence of exogenous NAD(+). We then constructed the NAD(+)-auxotrophic mutant YJE003 by disrupting the essential gene nadE, which is responsible for the last step of NAD(+) biosynthesis in cells harboring the ntt4 gene. The minimal NAD(+) level was determined in M9 medium in proliferating YJE003 cells that were preloaded with NAD(+), while the maximal NAD(H) level was determined by exposing the cells to high concentrations of exogenous NAD(H). Compared with supplementation of NADH, cells grew faster and had a higher intracellular NAD(H) level when NAD(+) was fed. The intracellular NAD(H) level increased with the increase of exogenous NAD(+) concentration, until it reached a plateau. Thus, a minimal NAD(H) level of 0.039 mM and a maximum of 8.49 mM were determined, which were 0.044× and 9.6× those of wild-type cells, respectively. Finally, the potential application of this strategy in biotechnology is briefly discussed.
Auxotrophy
Glycerol-3-phosphate dehydrogenase
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The Kelch-like ECH-associating protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway is the major regulator of cytoprotective responses to oxidative and electrophilic stress. The Cul3/Keap1 E3 ubiquitin ligase complex interacts with Nrf2, leading to Nrf2 ubiquitination and degradation. In this study, we focused on the disruption of the Keap1-Nrf2 interaction to upregulate Nrf2 expression and the transcription of ARE-controlled cytoprotective oxidative stress response enzymes, such as HO-1. We completed a drug-repositioning screening for inhibitors of Keap1-Nrf2 protein-protein interactions using a newly established fluorescence correlation spectroscopy (FCS) screening system. The binding reaction between Nrf2 and Keap1 was successfully detected with a KD of 2.6 μM using our FCS system. The initial screening of 1,633 drugs resulted in 12 candidate drugs. Among them, 2 drugs significantly increased Nrf2 protein levels in HepG2 cells. These two promising drugs also upregulated ARE gene promoter activity and increased HO-1 mRNA expression, which confirms their ability to dissociate Nrf2 and Keap1. Thus, drug-repositioning screening for Keap1-Nrf2 binding inhibitors using FCS enabled us to find two promising known drugs that can induce the activation of the Nrf2-ARE pathway.
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SUMMARY The adenohypophysis of the pig was examined histochemically for the presence of 11 oxidative enzymes, namely: 1.1.1.27 l -lactate: NAD oxidoreductase, 1.1.1.30 d -3-hydroxybutyrate: NAD oxidoreductase, 1.1.1.37 l -malate: NAD oxidoreductase, 1.1.1.41 threo- d s -isocitrate: NAD oxidoreductase (decarboxylating), 1.1.1.42 threo- d s -isocitrate: NADP oxidoreductase (decarboxylating), 1.1.1.49 d -glucose-6-phosphate: NADP oxidoreductase, 1.1.99.5 l -glycerol-3-phosphate: (acceptor) oxidoreductase, 1.3.99.1 succinate: (acceptor) oxidoreductase, 1.4.1.2 l -glutamate: NAD oxidoreductase (deaminating), 1.6.99.1 reduced-NADP: (acceptor) oxidoreductase, 1.6.99.3 reduced-NAD: (acceptor) oxidoreductase. With the exception of 1.1.1.30 d -3-hydroxybutyrate: NAD oxidoreductase, activity was found throughout the adenohypophysis for all these enzymes. A comparison was made with the activity for these enzymes in liver. In the adenohypophysis, the pars tuberalis exhibited the highest activity for all enzymes, generally equal to or greater than that shown by the liver. The pars intermedia and the pars anterior showed similar activity for these enzymes, in general of a lower order than that given by the liver. The pattern of enzyme distribution in the pars intermedia is described; high activity for 1.1.1.37 l -malate: NAD oxidoreductase, 1.1.1.27 l -lactate: NAD oxidoreductase, 1.6.99.3 reduced-NAD: (acceptor) oxidoreductase, 1.6.99.1 reduced-NADP: (acceptor) oxidoreductase was shown by cells lining cysts and the pituitary cleft. The findings are discussed in relation to the possible association of these enzymes with secretory function.
Glycerol-3-phosphate dehydrogenase
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