High-affinity binding of NADPH to camel lens zeta-crystallin

Fluorescence spectrum of camel lens zeta-crystallin, a major protein in the lens of camelids and histicomorph rodents, showed maximum emission at 315 nm. This emission maximum is blue shifted compared to most proteins, including alpha-crystallin, and appeared to be due to tryptophan in highly hydrophobic environment. Interaction of NADPH with zeta-crystallin quenched the protein fluorescence and enhanced the fluorescence of bound NADPH. Analysis of fluorescence quenching suggested high-affinity interaction between NADPH and zeta-crystallin with an apparent Km<0.45 microM. This value is at least an order of magnitude lower than that suggested by activity measurements. Analysis of NADPH fluorescence showed a biphasic curve representing fluorescence of free- and bound-NADPH. The intersection between free- and bound-NADPH closely paralleled the enzyme concentration, suggesting one mole of NADPH was bound per subunit of the enzyme. Phenanthrenequinone (PQ), the substrate of zeta-crystallin, also was able to quench the fluorescence of zeta-crystallin, albeit weaker than NADPH. Quantitative analysis suggested that zeta-crystallin had low affinity for PQ in the absence of NADPH, and PQ binding induced significant conformational changes in zeta-crystallin.     

Quinone induced stimulation of hexose monophosphate shunt activity in the guinea pig lens: role of zeta-crystallin.

The response of the hexose monophosphate shunt (HMS) in organ-cultured guinea pig lens to 1,2-naphthoquinone and 5-hydroxy-1,4-naphthoquinone (juglone) has been investigated. Both these compounds, which are substrates of guinea pig lens zeta-crystallin (NADPH:quinone oxidoreductase), were found to cause increases in the rate of 14CO2 production from 1-14C-labelled glucose. Exposure of lenses to 15 microM 1,2-naphthoquinone or 20 microM juglone yielded 5.9- and 7-fold stimulation of HMS activity, respectively. Unlike hydrogen peroxide-induced stimulation of HMS activity, these effects were not abolished by preincubation with the glutathione reductase inhibitor, 1,3-bis(2-chloroethyl)-1 nitrosourea (BCNU). While hydrogen peroxide produced substantial decrements in lens glutathione (GSH) levels, incubation with quinones was not associated with a similar reduction in GSH concentration. Protein-bound NADPH content in quinone-exposed guinea pig lenses was decreased, with a concomitant increase in the amounts of free NADP+. This finding supported the involvement of zeta-crystallin bound NADPH in the in vivo enzymic reduction of quinones. Hydrogen peroxide, on the other hand, caused decreases in the level of free NADPH alone, serving to confirm our earlier inference that quinone stimulated increases in the guinea pig lens HMS could be mediated through zeta-crystallin NADPH:quinone oxidoreductase activity.     

Molecular cloning of rabbit hyaluronic acid synthases and their expression patterns in synovial membrane and articular cartilage

Interaction of camel lens zeta-crystallin with the hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid (ANS) enhanced the ANS fluorescence and quenched the protein fluorescence. Both of these events were concentration-dependent and showed typical saturation curves suggesting specific ANS-zeta-crystallin binding. Quantitative analysis indicated that 1 mole zeta-crystallin bound at most 1 mole ANS. NADPH but not 9,10-phenanthrenequinone (PQ) was able to displace zeta-crystallin-bound ANS. These results suggested the presence of a hydrophobic domain in zeta-crystallin, possibly at the NADPH binding site. alpha-Crystallin as well as NADPH protected zeta-crystallin against thermal inactivation suggesting the importance of this site for enzyme stability. The NADPH:quinone oxidoreductase activity of zeta-crystallin was inhibited by ANS with NADPH as electron donor and PQ as electron acceptor. Lineweaver-Burk plots indicated mixed-type inhibition with respect to NADPH, with a K(i) of 2.3 microM. Secondary plots of inhibition with respect to NADPH indicated a dissociation constant (K'I) of 12 microM for the zeta-crystallin-NADPH-ANS complex. The K(i) being smaller than K'I suggested that competitive inhibition at the NADPH binding site was predominant over non-competitive inhibition. Like ANS-zeta-crystallin binding, inhibition was dependent on ANS concentration but independent of incubation time.     

Zeta-crystallin displays strong selectivity for salicylic acid over aspirin

Interaction of camel lens zeta-crystallin with aspirin was investigated by activity and fluorescence measurements. Aspirin minimally inhibited the oxidoreductase activity of the enzyme and weakly quenched its fluorescence. However, significant fluorescence quenching of zeta-crystallin coincided with the appearance of a fluorescence signal characteristic of salicylic acid thereby raising the possibility that salicylic acid might have been the moiety responsible for inhibition and fluorescence quenching. Direct fluorescence measurements showed that zeta-crystallin had a much higher affinity for salicylic acid than aspirin (K(i) of about 24 microM for salicylic acid versus 630 microM for aspirin). Salicylic acid was also far more effective in inhibiting zeta-crystallin than aspirin (K(i) values were 23 microM versus 820 microM, respectively). Inhibition kinetics suggested that salicylic acid interacted with zeta-crystallin via a binding site that was distinct from that of NADPH. Salicylic acid also interacted with and quenched the fluorescence of camel lens alpha-crystallin suggesting a general mode of interaction with lens proteins. Within the normal therapeutic concentrations of salicylic acid or aspirin, only crystallin-salicylic acid interactions might be significant. These results showed that camel lens zeta- and alpha-crystallin exhibited remarkable selectivity for salicylic acid over aspirin, and thus, could be considered as salicylate-binding proteins.     

Identification and characterization of the enzymatic activity of zeta-crystallin from guinea pig lens. A novel NADPH:quinone oxidoreductase.

zeta-Crystallin is a major protein in the lens of certain mammals. In guinea pigs it comprises 10% of the total lens protein, and it has been shown that a mutation in the zeta-crystallin gene is associated with autosomal dominant congenital cataract. As with several other lens crystallins of limited phylogenetic distribution, zeta-crystallin has been characterized as an "enzyme/crystallin" based on its ability to reduce catalytically the electron acceptor 2,6-dichlorophenolindophenol. We report here that certain naturally occurring quinones are good substrates for the enzymatic activity of zeta-crystallin. Among the various quinones tested, the orthoquinones 1,2-naphthoquinone and 9,10-phenanthrenequinone were the best substrates whereas menadione, ubiquinone, 9,10-anthraquinone, vitamins K1 and K2 were inactive as substrates. This quinone reductase activity was NADPH specific and exhibited typical Michaelis-Menten kinetics. Activity was sensitive to heat and sulfhydryl reagents but was very stable on freezing. Dicumarol (Ki = 1.3 x 10(-5) M) and nitrofurantoin (Ki = 1.4 x 10(-5) M) inhibited the activity competitively with respect to the electron acceptor, quinone. NADPH protected the enzyme against inactivation caused by heat, N-ethylmaleimide, or H2O2. Electron paramagnetic resonance spectroscopy of the reaction products showed formation of a semiquinone radical. The enzyme activity was associated with O2 consumption, generation of O2- and H2O2, and reduction of ferricytochrome c. These properties indicate that the enzyme acts through a one-electron transfer process. The substrate specificity, reaction characteristics, and physicochemical properties of zeta-crystallin demonstrate that it is an active NADPH:quinone oxidoreductase distinct from quinone reductases described previously.     

High-affinity binding of NADPH to camel lens ζ-crystallin

Fluorescence spectrum of camel lens ζ-crystallin, a major protein in the lens of camelids and histicomorph rodents, showed maximum emission at 315 nm. This emission maximum is blue shifted compared to most proteins, including α-crystallin, and appeared to be due to tryptophan in highly hydrophobic environment. Interaction of NADPH with ζ-crystallin quenched the protein fluorescence and enhanced the fluorescence of bound NADPH. Analysis of fluorescence quenching suggested high-affinity interaction between NADPH and ζ-crystallin with an apparent K_m<0.45 μM. This value is at least an order of magnitude lower than that suggested by activity measurements. Analysis of NADPH fluorescence showed a biphasic curve representing fluorescence of free- and bound-NADPH. The intersection between free- and bound-NADPH closely paralleled the enzyme concentration, suggesting one mole of NADPH was bound per subunit of the enzyme. Phenanthrenequinone (PQ), the substrate of ζ-crystallin, also was able to quench the fluorescence of ζ-crystallin, albeit weaker than NADPH. Quantitative analysis suggested that ζ-crystallin had low affinity for PQ in the absence of NADPH, and PQ binding induced significant conformational changes in ζ-crystallin.     

Sequential inactivation of zeta-crystallin by o-phthalaldehyde

o-Phthalaldehyde, a bifunctional cross-linking reagent, is commonly used as a probe for the active site of enzymes. In this study, the interaction of o-phthalaldehyde with camel lens zeta-crystallin was examined by activity and fluorescence measurements. Predictably, the oxidoreductase activity of zeta-crystallin was inhibited irreversibly by o-phthalaldehyde in a time- and concentration-dependent manner, and the presence of NADPH with the enzyme appeared to provide a high degree of protection against o-phthalaldehyde inactivation. Interaction of o-phthalaldehyde with zeta-crystallin resulted in formation of isoindole adduct, which exhibited characteristic fluorescence at 415 nm. However, neither inactivation nor modification of the enzyme showed the expected pseudo-first-order kinetics; both events were highly sequential reaching different levels of saturation at different concentrations of o-phthalaldehyde. The modified enzyme had a maximum stoichiometry of 1 mol isoindole/subunit, and bound NADPH to nearly the same extent as unmodified enzyme. Gel filtration experiments suggested that o-phthalaldehyde-modified zeta-crystallin had higher apparent molecular weight than unmodified enzyme, even though the enzyme remained largely monomeric as revealed by electrophoresis on denaturing gel. These results suggested that modification by o-phthalaldehyde might have been so intrusive as to sequentially modify the tetrameric structure of zeta-crystallin.     

Zeta-crystallin from guinea pig lens is capable of functioning catalytically as an oxidoreductase

zeta-Crystallin, a major taxon-specific protein of the guinea pig lens, has been shown to be distantly related to the alcohol/polyol dehydrogenase family and to specifically bind NADPH. The capacity of zeta-crystallin to function catalytically was investigated in the present study. zeta-Crystallin exhibited an NADPH-dependent oxidoreductase activity with 2,6-dichlorophenolindophenol (DCIP). The NADPH:DCIP oxidoreductase activity of zeta-crystallin exhibits a linear response with increasing protein concentration, and saturation kinetics with NADPH and DCIP. This activity was abolished by heat inactivation and immunoadsorption of the protein. Dicumarol, Cibacron blue, manganese, and sulfhydryl reagents were inhibitory.     

Taxon-specific zeta -crystallin in Japanese tree frog (Hyla japonica) lens

The present study demonstrated that the 38-kDa protein, instead of rho-crystallin (36 kDa), is expressed taxon specifically in the lens of Japanese tree frog (Hyla japonica). The 38-kDa protein was distinguished from rho-crystallin expressed in the lenses of bullfrog (Rana catesbeiana) and European common frog (Rana temporaria) immunochemically. Although the N terminus of the 38-kDa protein was blocked, the analyses of partial amino acid sequences showed that the protein was zeta-crystallin. Analysis of cDNA sequence encoding zeta-crystallin of the tree frog lens demonstrated that the deduced protein consisted of 329 amino acids including initial methionine and having 62.2 and 62.9% identity with zeta-crystallin of camel and guinea pig lenses, respectively. The molecular mass of the deduced structure was calculated to be 35,564 Da. zeta-Crystallin of the tree frog lens exhibited the intrinsic enzymatic activity of quinone reductase (EC, NADPH:quinone oxidoreductase). The crystallin specifically catalyzed the reduction of 9,10-phenanthrenequinone (Km, 42 microm) using NADPH (Km, 60 microm) as a cofactor. The enzymatic activity was inhibited by dicumarol, anti-coagulant drug, with IC50 of 4 microm. On gel filtration chromatography, the crystallin was recovered as 150-kDa molecular mass complex, indicating that the crystallin was homotetramer consisting of 38-kDa subunits. The crystallin gene was expressed specifically in the lens. These results show that taxon-specific crystallins such as zeta- and rho-crystallins may be available for the biochemical discrimination of Hyla- and Rana groups among frogs.     

Inhibition of camel lens zeta-crystallin by aspirin and aspirin-like analgesics.

Camel lens zeta-crystallin was reversibly inhibited to various degrees by aspirin (acetyl salicylic acid) and the aspirin-like analgesics: paracetamol (acetaminophen) and ibuprofen (2-(4-isobutyl phenyl)-propionic acid). Among these, aspirin was the most potent inhibitor, causing nearly complete inhibition in a dose-dependent, but time-independent manner. Analysis of inhibition kinetics revealed that aspirin was uncompetitive inhibitor (K(i) 0.64 mM) with respect to NADPH and non-competitive inhibitor (K(i) 1.6 mM) with respect to the substrate, 9,10-phenanthrenequinone (PQ). Multiple-inhibition analysis showed that aspirin and pyridoxal 5' phosphate (PAL-P), a lysine specific reagent, simultaneously bound to a critical lysine residue located towards the NADPH binding region. Consistent with this, NADPH was able to substantially protect zeta-crystallin against aspirin, whereas PQ did not provide any protection. The results suggested that an essential lysine residue was the locus of aspirin binding. The inhibition of zeta-crystallin by aspirin and aspirin-like analgesics was reversible thus eliminating acetylation as a mechanism for inhibition. Reversible binding of aspirin to this lysine may cause steric hindrance resulting in uncompetitive inhibition with respect to NADPH.     

Quinone-induced inhibition of urease: elucidation of its mechanisms by probing thiol groups of the enzyme

In this work we studied the reaction of four quinones, 1,4-benzoquinone (1,4-BQ), 2,5-dimethyl-1,4-benzoquinone (2,5-DM-1,4-BQ), tetrachloro-1,4-benzoquinone (TC-1,4-BQ) and 1,4-naphthoquinone (1,4-NQ) with jack bean urease in phosphate buffer, pH 7.8. The enzyme was allowed to react with different concentrations of the quinones during different incubation times in aerobic conditions. Upon incubation the samples had their residual activities assayed and their thiol content titrated. The titration carried out with use of 5,5'-di-thiobis(2-nitrobenzoic) acid was done to examine the involvement of urease thiol groups in the quinone-induced inhibition. The quinones under investigation showed two distinct patterns of behaviour, one by 1,4-BQ, 2,5-DM-1,4-BQ and TC-1,4-BQ, and the other by 1,4-NQ. The former consisted of a concentration-dependent inactivation of urease where the enzyme-inhibitor equilibrium was achieved in no longer than 10min, and of the residual activity of the enzyme being linearly correlated with the number of modified thiols in urease. We concluded that arylation of the thiols in urease by these quinones resulting in conformational changes in the enzyme molecule is responsible for the inhibition. The other pattern of behaviour observed for 1,4-NQ consisted of time- and concentration-dependent inactivation of urease with a nonlinear residual activity-modified thiols dependence. This suggests that in 1,4-NQ inhibition, in addition to the arylation of thiols, operative are other reactions, most likely oxidations of thiols provoked by 1,4-NQ-catalyzed redox cycling. In terms of the inhibitory strength, the quinones studied formed a series: 1,4-NQ approximately 2,5-DM-1,4-BQ<1,4-BQ相似文献   

A novel NADPH:diamide oxidoreductase activity in arabidopsis thaliana P1 zeta-crystallin.

The zeta-crystallin (ZCr) gene P1 of Arabidopsis thaliana, known to confer tolerance toward the oxidizing drug 1,1'-azobis(N, N-dimethylformamide) (diamide) to yeast [Babiychuk, E., Kushnir, S., Belles-Boix, E., Van Montagu, M. & Inzé, D. (1995) J. Biol. Chem. 270, 26224], was expressed in Escherichia coli to characterize biochemical properties of the P1-zeta-crystallin (P1-ZCr). Recombinant P1-ZCr, a noncovalent dimer, showed NADPH:quinone oxidoreductase activity with specificity to quinones similar to that of guinea-pig ZCr. P1-ZCr also catalyzed the divalent reduction of diamide to 1,2-bis(N,N-dimethylcarbamoyl)hydrazine, with a kcat comparable with that for quinones. Two other azodicarbonyl compounds also served as substrates of P1-ZCr. Guinea-pig ZCr, however, did not catalyze the azodicarbonyl reduction. Hence, plant ZCr is distinct from mammalian ZCr, and can be referred to as NADPH:azodicarbonyl/quinone reductase. The quinone-reducing reaction was accompanied by radical chain reactions to produce superoxide radicals, while the azodicarbonyl-reducing reaction was not. Specificity to NADPH, as judged by kcat/Km, was > 1000-fold higher than that to NADH both for quinones and diamide. N-Ethylmaleimide and p-chloromercuribenzoic acid inhibited both quinone-reducing and diamide-reducing activities. Both NADPH and NADP+ suppressed the inhibition, but NADH did not, suggesting that sulfhydryl groups reside in the binding site for the phosphate group on the adenosine moiety of NADPH. The diamide-reducing activity of P1-ZCr accounts for the tolerance of P1-overexpressing yeast to diamide. Other possible physiological functions of P1-ZCr in plants are discussed.     

Expression of recombinant zeta-crystallin in Escherichia coli with the help of GroEL/ES and its purification

zeta-Crystallin is a taxon-specific crystallin found in the eye lens of guinea pig and other hystricomorph rodents and camelids. It is an NADPH:quinone oxidoreductase and is also present in low amounts in other tissues where it might act as a detoxifying enzyme. A lens-specific promoter confers lens-specific expression of the gene in high amounts where it is speculated to play a structural role in maintaining the transparency of the lens ensemble. A deletion mutation leads to autosomal dominant congenital cataract and also results in the loss of NADPH binding. In order to perform structural studies with the protein with an aim to delineate the cause of cataract in these mutant guinea pigs, recombinant zeta-crystallin was cloned and expressed in Escherichia coli. The overexpression of the protein in E. coli resulted in a major fraction of it partitioning into inclusion bodies. The co-overexpression of the bacterial chaperone system GroEL/ES along with zeta-crystallin could significantly enhance the yield of soluble protein. Active zeta-crystallin could then be purified from the E. coli using Mono Q anion exchange FPLC and was found to be identical to the native zeta-crystallin isolated from the guinea pig lens with respect to size, spectral properties, and activity.     

Plastoquinone B

A compound found in spinach and other higher plants previously referred to as R 263 has now been found to be a breakdown product of plastoquinone B. This quinone, PQ B, is found with 8 other quinones in spinach chloroplasts. These 9 quinones are PQ A, PQ B, PQ C, PQ D (7, 8, 15) Vitamin K₁ (10, 12), an unknown naphthoquinone (13) and α-, β- and γ-tocopherylquinones (7, 12). An improved method for purification of plastoquinone B is described. Previous confusion of this compound with other quinoid material on silica gel is described and corrected R_F values are given. The activity of PQ B is similar to the activity of PQ C in restoration studies of the photo-reduction of ferricyanide and indophenol.     

Chemical knockdown of protein-tyrosine phosphatase 1B by 1,2-naphthoquinone through covalent modification causes persistent transactivation of epidermal growth factor receptor

1,2-Naphthoquinone (1,2-NQ), an atmospheric contaminant, causes the contraction of guinea pig trachea through the activation of epidermal growth factor receptor (EGFR) by inhibiting protein-tyrosine phosphatases (PTPs). Phosphorylation of EGFR is negatively regulated by PTPs, but details of the mechanism by which 1,2-NQ inhibits PTPs have not been elucidated. Results described in this report demonstrate that 1,2-NQ forms covalent bonds with PTP1B after exposure to human epithelial A431 cells. In this study, a concentration-dependent phosphorylation of EGFR was found to be coupled to the reduction of PTP activity in the cells. The reduction in PTP activity was due to the irreversible modification of PTP1B, and when PTP1B was overexpressed by the cells, the 1,2-NQ-mediated EGFR phosphorylation was suppressed. Studies with purified PTP1B and 1,2-NQ showed that the reduction in enzyme activity was due to a nucleophilic attack by the quinone on the enzyme, to form covalent bonds. Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry analysis and mutation experiments revealed that PTP1B inactivation was primarily due to covalent attachment of the quinone to Cys-121 of the enzyme, with binding to His-25 and Cys-215 as well. Collectively, the results show that covalent attachment of 1,2-NQ to PTP1B is at least partially responsible for the reduction of PTP activity, which leads to prolonged transactivation of EGFR in the cells.     

zeta-Crystallin is a major protein in the lens of Camelus dromedarius

Camel (Camelus dromedarius) lenses contain a protein with an apparent subunit Mr 38,000 that constitutes approximately 8-13% of the total protein. The protein has been purified and has a native Mr 140,000 as determined by gel filtration. This is consistent with its being a tetramer. The protein reacts with antibodies raised against both guinea pig zeta-crystallin and peptides corresponding to amino acids 1-10 and 295-308, but not to antibodies raised against amino acids 320-328 of zeta-crystallin. Based on these criteria it is concluded that this protein, which is a major constituent of camel lens, is zeta-crystallin. This may be the first example of a protein (enzyme) being independently utilized as a crystallin in the lens of species from two mammalian orders.     

Inhibition of naphthohydroquinone autoxidation by DT-diaphorase (NAD(P)H:[quinone acceptor] oxidoreductase)

Summary

It has been reported that little redox cycling occurs during the reduction of 2-methyl-1,4-naphthoquinone by DT-diaphorase, suggesting that the reduction product, 2-methyl-1,4-naphthohydroquinone, does not readily undergo autoxidation. In the present study, however, it has been shown that DT-diaphorase, by virtue of its ability to re-reduce the naphthoquinone formed in the oxidation reaction, decreases the rate of autoxidation of 2-methyl-1,4- naphthohydroquinone. Therefore, the low rate of redox cycling observed does not reflect an intrinsic stability of the hydroquinone but inhibition of its autoxidation by the enzyme. Redox cycling of 2,3-dimethyl-, 2,3-dimethoxy- and 2-methoxy-1,4-naphthoquinone, and the autoxidation of their respective hydroquinones, were similarly inhibited by diaphorase. The concentration of the enzyme required for inhibition varied widely among the different compounds, and this was related to the autoxidation rate of the hydroquinone and the rate at which the corresponding quinone was reduced by diaphorase. The behaviour of 2-hydroxy-1,4-naphthoquinone was exceptional in that the rate of redox cycling increased with increasing levels of diaphorase and no inhibition of the autoxidation of the hydroquinone derived from this substance could be demonstrated, even at very high enzyme concentrations. The results of the present experiments indicate that the relative stability of naphthohydroquinones cannot be judged on the basis of studies involving reduction of the quinone by DT-diaphorase and suggest that current concepts on the role of this enzyme in the detoxification of quinones may need revision.     

Purification and characterization of zeta-crystallin/quinone reductase from guinea pig liver.

zeta-Crystallin, a major lens protein of certain mammalian species, has recently been characterized as a novel and active NADPH:quinone oxidoreductase. Here we report the purification of this protein from guinea pig liver by utilizing sequentially: ammonium sulphate precipitation, Blue Sepharose affinity, cation exchange and hydrophobic chromatography steps. This four-step isolation procedure yielded 118-fold purification and a specific activity of 6 U/mg protein when assayed in the presence of 9,10-phenanthrenequinone. Kinetic, immunological and physical properties of this protein have been found to be identical with those of guinea pig lens zeta-crystallin. Western blot analysis using antibodies raised against zeta-crystallin peptides demonstrated the presence of substantial amounts of this protein in human liver homogenates.     

Purification and some properties of two isofunctional juglone hydroxylases from Pseudomonas putida J1

Juglone-induced cells of Pseudomonas putida J 1 were shown to contain two isofunctional juglone hydroxylases. Both enzymes were purified about 125-fold to homogeneity in polyacrylamide gel electrophoresis. The molecular masses of the native enzymes, as determined by Sephacryl S-200 gel filtration were 59 000 Da for enzyme 1 and 56 000 Da for enzyme 2. The molecular masses of the subunits were determined by dodecyl sulfate polyacrylamide gel electrophoresis as 25 000 Da (enzyme 1) and 23 500 Da (enzyme 2). Both enzymes hydroxylated juglone, naphthazarin, 1,4-naphthoquinone and 2-chloro-1,4-naphthoquinone, but they were completely inactive against naphtholes. The activity of both hydroxylases was not affected by chelating agents, thiol reagents, however, were found to be strong inhibitors. No external cofactors such as Fe2, NADH, NADPH, FAD, FMN were required for activity. Concomitant with the hydroxylation of juglone the consumption of oxygen in a molar ratio 2: 1 (juglone: oxygen) was observed but none of the enzymes incorporated 18O2 into the substrate juglone. By activity staining enzyme 1 was found to be present in induced and non-induced cells of P. putida J 1, enzyme 2, however, only in juglone-induced cells.     

Parallel synthesis of a library of 1,4-naphthoquinones and automated screening of potential inhibitors of trypanothione reductase from Trypanosoma cruzi

Solid- and solution-phase parallel syntheses of 1,4-naphthoquinones (1,4-NQ) are described. A library of 1360 amides was constructed from the combination of 12 newly synthesised 1,4-NQ carboxylic acid and 120 amines, and was screened for inhibition of trypanothione reductase (TR) from Trypanosoma cruzi. The most active hits from a primary screening were re-synthesised and confirmed. This approach proves that it is possible to design potent and highly specific TcTR inhibitors deriving from menadione, juglone and plumbagin.