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Ketoreductases play an indispensable role in the asymmetric synthesis of chiral drug intermediates, and an in-depth understanding of their substrate selectivity can improve the efficiency of enzyme engineering. In this endeavor, a new short-chain dehydrogenase/reductase (SDR) SsSDR1 identified from Sphingobacterium siyangense SY1 by gene mining method was successfully cloned and functionally expressed in Escherichia coli. Its activity against halogenated acetophenones has been tested and the results illustrated that SsSDR1-WT exhibits high activity for 3,5-bis(trifluoromethyl)acetophenone (1f), an important precursor in the synthesis of aprepitant. In addition, SsSDR1-WT showed obvious substrate preference for acetophenones without α-halogen substitution compared to their α-halogen analogs. To explore the structural basis of substrate selectivity, the X-ray crystal structures of SsSDR1-WT in its apo form and the complex structure with NAD were resolved. Taking 2-chloro-1-(3, 4-difluorophenyl) ethanone (1i) as the representative α-haloacetophenone, the key sites affecting substrate selectivity of SsSDR1-WT were identified and through the rational remodeling of the cavities C1 and C2 of SsSDR1, an excellent mutant I144A/S153L with significantly improved activity against α-halogenated acetophenones was obtained. The asymmetric catalysis of 1f and 1i was performed at the scale of 50 mL, and the space-time yields (STY) of the two were 1200 and 6000 g/L∙d, respectively. This study not only provides valuable biocatalysts for halogenated acetophenones, but also yields insights into the relationship between the substrate-binding pocket and substrate selectivity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Bioreductive capabilities of four morphologically different strains of cyanobacteria have been assessed in this work. Arthrospira maxima, Leptolyngbya foveolarum, Nodularia sphaerocarpa and Synechococcus bigranulatus were applied as catalysts for the reduction of acetophenone to the corresponding chiral phenylethyl alcohol. The process was modified regarding substrate concentration, duration of pre-cultivation period, duration of biotransformation, light regime and glucose addition to the culture media. Obtained results clearly showed that cyanobacteria were active towards acetophenone what resulted in the substrate reduction to (S)-1-phenylethanol with high enantiomeric excess. The reaction efficiency increased with the biotransformation time, but the higher concentration of substrate limited the process yield. Also, all tested strains performed reaction with the highest efficacy under continuous light regime. The most active strains - N. sphaerocarpa and S. bigranulatus carried out the conversion of 1 mM acetophenone with high efficiency of respectively 97.6% and 96.2% after 13 days of biotransformation. A. maxima reached 45.8% of conversion after 13 days of biotransformation whereas L. foveolarum did not exceed 20%. The enantiomeric excesses were respectively 98.8%- A. maxima, 91.7%- L. foveolarum, 72.6%- S. bigranulatus and N. sphaerocarpa 16.2%., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Lignin and lignin components of woody biomass have been identified as an attractive alternative to fossil fuels. However, the complex composition of this plant polymer is one of the drawbacks that limits its exploitation. Biocatalysis of lignin to produce platform chemicals has been receiving great attention as it presents a sustainable approach for lignin valorisation. Aligned with this area of research, in the present study we have applied ultra-high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to identify the preferred lignin substrates of a ligninolytic enzyme, a laccase produced by the terrestrial fungus Trametes versicolor. A commercial lignin was incubated with the laccase and acetosyringone (a laccase mediator) for up to 168 h and direct infusion electrospray FT-ICR MS enabled the identification of thousands of molecular species present in the complex lignin sample at different incubation time points. Significant changes in the chemical composition of lignin were detected upon laccase treatment, which resulted in a decrease in the molecular mass distribution of assigned species, consistent with laccase lytic activity. This reduction was predominantly in species classified as lignin-like (based on elemental ratios) and polymeric in nature (>400 Da). Of particular note was a fall in the number of species assigned containing sulfur. Changes in the chemical composition/structure of the lignin polymer were supported by FT-IR spectroscopy. We propose the use of FT-ICR MS as a rapid and efficient technique to support the biotechnological valorisation of lignin as well as the development and optimization of laccase-mediator systems for treating complex mixtures., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The existence of a substrate-sensitive equilibrium between high spin (S=5/2) and low spin (S=1/2) ferric iron is a well-established phenomenon in the cytochrome P450 (CYP) superfamily, although its origins are still a subject of discussion. A series of mutations that strongly perturb the spin state equilibrium in the camphor hydroxylase CYP101A1 were recently described (Colthart et al., Sci. Rep. 6, 22035 (2016)). Wild type CYP101A1 as well as some CYP101A1 mutants are herein shown to be capable of catalyzing the reduction of nitroacetophenones by NADH to the corresponding anilino compounds (nitroreductase or NRase activity). The distinguishing characteristic between those mutants that catalyze the reduction and those that cannot appears to be the extent to which residual high spin form exists in the absence of the native substrate d-camphor, with those showing the largest spin state shifts upon camphor binding also exhibiting NRase activity. Optical and EPR spectroscopy was used to further examine these phenomena. These results suggest that reduction of nitroaromatics may provide a useful probe of residual high spin states in the CYP superfamily. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Paeonol, an active constituent in the root bark of Paeonia suffruticosa Andrews, is used to treat inflammation, headache and other diseases in clinic. Though the data on pharmacological researches of paeonol abounds, its metabolic profile is not so clear. It is essential to systematically characterize the in vivo metabolites in order to better understand its mechanism of action. In this study, ultra performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-Q/TOF-MS) with an integrative strategy was developed for analysis of paeonol metabolites. As a result, based on seven reference substances isolated or synthesized, twenty-five metabolites were detected and identified in urine, feces, bile and plasma of rats after oral administration of paeonol. To the best of our knowledge, 14 of these metabolites have not been reported previously. In addition, the dominating metabolic fates were oxidation, demethylation, hydrogenation, glucuronic acid and sulfate conjugations, and hydrogenation of paeonol was reported for the first time. This research provides scientific and reliable support for full understanding of the metabolic profiling of paeonol., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Guizhi Fuling capsule (GFC), a prestigious traditional Chinese medicinal (TCM) prescription, is efficiently used to treat primary dysmenorrhea in the clinical practice. It's significant to explore the metabolic fate of multiple components in vivo which are responsible for the pharmacological effects but not fully investigated. A rapid and high-throughput method using ultra performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS/MS) was established for systematic investigation on GFC, including GFC chemical compositions, and their absorption and metabolism in rat plasma, urine, uterus and brain after oral administration of GFC. A total of 102 nonvolatile GFC phytochemistry components were identified based on the accurately measured mass value, fragmentation pattern and retention behavior. Compared to the previous GFC study, additional 47 different GFC components were detected. Furthermore 21, 9, 4 and 3 prototype compounds were separately observed in plasma, urine, uterus and brain samples with the support of in vitro GFC study. While 29, 33, 10 and 8 metabolites were also identified with the assistance of the MetaboLynx tool in these biological samples. The result indicated that the developed method was suitable for the components identification even in the complex matrix. The chemical and metabolic profiling of GFC provided an abundant substance foundation for the extensive GFC research, especially for the pharmacodynamic mechanisms research., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Seventy-nine strains of endophytic fungi isolated from the healthy leaves, twigs and fruits of Illicium verum were screened for the asymmetric reduction activities to acetophenone. Strain BYEF07, which showed relatively high reduction activities, has been classified as Neofusicoccum parvum, and the main product was confirmed to be (R)-(+)-1-phenylethanol by GC-MS and chiral HPLC methods. The bio-reduction conditions of acetophenone by cells of N. parvum BYEF07 were investigated in detail. Under the conditions of 1.8 g/L of acetophenone, 100 g/L of microorganism cells and 10 g/L of glucose in 40 mL Na2HPO4 KH2PO4 buffer solution at pH7.5, 30 °C and 150 rpm, after 48 h reaction, the production yield of 1-phenylethanol and enantiomeric excess value of (R)-(+)-1-phenylethanol were 78% and 96%, respectively., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The unusual glutathione S-transferase GSTO1 reduces, rather than conjugates, endo- and xenobiotics, and its role in diverse cellular processes has been proposed. GSTO1 has been assayed spectrophotometrically by measuring the disappearance of its substrate, S-(4-nitrophenacyl)glutathione (4-NPG), in the presence of 2-mercaptoethanol that regenerates GSTO1 from its mixed disulfide. To assay GSTO1 in rat liver cytosol, we have developed a high-performance liquid chromatography (HPLC)-based procedure with two main advantages: (i) it measures the formation of the 4-NPG reduction product 4-nitroacetophenone, thereby offering improved sensitivity and accuracy, and (ii) it can use glutathione, the physiological reductant of GSTO1, which is impossible to do with the spectrophotometric procedure. Using the new assay, we show that (i) the GSTO1-catalyzed reduction of 4-NPG in rat liver cytosol also yields 1-(4-nitrophenyl)ethanol, whose formation from 4-nitroacetophenone requires NAD(P)H; (ii) the two assays measure comparable activities with 2-mercaptoethanol or tris(2-carboxyethyl)phosphine used as reductant; (iii) the cytosolic reduction of 4-NPG is inhibited by GSTO1 inhibitors (KT53, 5-chloromethylfluorescein diacetate, and zinc), although the inhibitory effect is strikingly influenced by the type of reductant in the assay and by the sequence of reductant and inhibitor addition. Characterization of GSTO1 inhibitors with the improved assay provides better understanding of interaction of these chemicals with the enzyme., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Acetosyringone (3',5'-dimethoxy-4'-hydroxyacetophenone) is a well-known and very effective inducer of the virulence genes of Agrobacterium tumefaciens but the precise pathway of its biosynthesis in plants is still unknown. We have used two tobacco cell lines, cultured in suspension and exhibiting different patterns of accumulation of acetosyringone in their culture medium upon treatment with methyl jasmonate, to study different steps of acetosyringone biosynthesis. In the two cell lines studied, treatment with 100 μM methyl jasmonate triggered a rapid and transient increase in acetovanillone synthase activity followed by a progressive increase in S-adenosyl-L-methionine: 5-hydroxyacetovanillone 5-O-methyltransferase activity which paralleled the rise in acetosyringone concentration in the culture medium. This O-methyltransferase displayed Michaelis-Menten kinetics with an apparent Km value of 18 μM for 5-hydroxyacetovanillone and its activity was magnesium-independent. Its molecular mass was estimated by gel permeation on an FPLC column and was found to be of ca. 81 kDa. 5-Hydroxyacetovanillone was the best substrate among the different o-diphenolic compounds tested as methyl acceptors in the O-methyltransferase assay. No formation of 5-hydroxyacetovanillone could be detected in vitro from 5-hydroxyferuloyl-CoA and NAD in the extracts used to measure acetovanillone synthase activity, indicating that 5-hydroxyacetovanillone is probably formed by direct hydroxylation of acetovanillone rather than by β-oxidation of 5-hydroxyferulic acid. Taken together our results strongly support the hypothesis that acetosyringone biosynthesis in tobacco proceeds from feruloyl-CoA via acetovanillone and 5-hydroxyacetovanillone., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Facile and specific methods to quantify a nucleobase in biological samples are of great importance for diagnosing disorders in nucleic acid metabolism. In the present study, a novel fluorogenic reaction specific for uracil has been developed. The reaction was carried out in an alkaline medium containing benzamidoxime and K(3)[Fe(CN)(6)] which were heated for 2.0 min. Under the optimum reaction conditions, strong fluorescence was produced only from uracil, not from other many biogenic compounds tested such as cytosine, thymine, adenine, guanine, nucleobases, nucleosides, nucleotides, amino acids, saccharides, creatine, creatinine and urea. The sensitivity of this method was compared with a known fluorogenic reaction using phenacylbromide which does not react with uracil but reacts with cytosine, adenine and their analogues. The proposed uracil-specific reaction showed approximately 400-fold higher sensitivity than the phenacylbromide reaction. The lower detection limit of uracil by the present method was 100 pmol mL(-1), and a good linearity of the calibration curve was obtained up to 100 nmol mL(-1) uracil. Due to its high sensitivity and specificity, the quantitative determination of uracil was possible by the proposed fluorimetric method., (2010 Elsevier B.V. All rights reserved.)

A soluble enzyme, extracted from tobacco cell-suspension cultures 24h after treatment with 100 microM methyl jasmonate, has been shown to synthesize acetovanillone (apocynin) from feruloyl-CoA in the presence of NAD. The enzyme displayed Michaelis-Menten kinetics with apparent K(m) values of 5.6 microM for feruloyl-CoA and 260 microM for NAD and exhibited very high specificity for its substrates. The increase in acetovanillone synthase activity was followed by an increase in the concentration of both acetovanillone and acetosyringone in the culture medium. No intermediate could be detected when analysing the reaction medium by HPLC during the formation of acetovanillone in cell-free extracts. The apparent molecular mass estimated by gel permeation on an FPLC column was ca. 79 kDa. To our knowledge, this is the first report of an enzymic system catalysing the synthesis of an acetophenone. This work demonstrates that the biosynthesis of acetophenones in tobacco proceeds from hydroxycinnamic acids through a CoA-dependent beta-oxidation pathway. Interestingly in methyl jasmonate-treated cells, which synthesize very large amounts of hydroxycinnamoylputrescines, inhibition of the synthesis of these conjugates increased the concentration of acetovanillone and acetosyringone in the culture medium, suggesting that the two metabolic pathways can compete for their common precursors, i.e. hydroxycinnamoyl-CoA thioesters., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Reactive oxygen species (ROS) are increased in human abdominal aortic aneurysms (AAA). NADPH oxidases are the predominant source of superoxide anion (O(2)(-)) in the vasculature. Inducible nitric oxide synthase (iNOS) produces a significant amount of nitric oxide (NO) during inflammatory processes. We hypothesized that ROS produced by NADPH oxidases and iNOS played an important role in aneurysm formation. We examined this hypothesis using selective blockade of NADPH oxidases and iNOS in a murine model of AAA. Mice, including C57BL/6, iNOS knockout (iNOS(-/-)) mice, and its background matched control (C57BL/6), underwent AAA induction by periaortic application of CaCl(2). Aortic diameter was measured at aneurysm induction and harvest. Beginning 1 week prior to aneurysm induction and continuing to aortic harvest 6 weeks later, one group of the C57BL/6 mice were treated with orally administered apocynin (NADPH oxidase inhibitor). Control mice were given water. The mean diameter and change in diameter of each group were compared with concurrent controls. Aortic levels of the NO metabolite, NO(x) (NO(2) and NO(3)), were significantly increased in CaCl(2)-treated wild type mice. INOS(-/-) mice were partly resistant to aneurysm induction. This was associated with reduced expression of matrix metalloproteinase (MMP)-2 and MMP-9 and decreased production of NO(x) in the aortic tissues. Inhibition of NADPH oxidase by apocynin also blocked aneurysm formation. In conclusion, both iNOS deficiency and NADPH oxidase inhibition suppressed aneurysm formation in association with decreased NO(x) levels. These studies suggest that both NADPH oxidase and iNOS pathways contribute to ROS production and AAA development.

In the course of a project aimed to clarify the molecular mechanisms by which phorbol 12-myristate 13-acetate (PMA)-activated forms of protein kinase C (PKC) promote growth arrest in an MCF-7 cell line, we found that the PKCdelta inhibitor Rottlerin was able by itself to block cell proliferation. In the current study, we investigated further the antiproliferative response to Rottlerin. Western blotting analysis of cytoplasmic/nuclear extracts showed that the drug did not prevent either extracellular signal-regulated kinase (ERK) activation by PMA or Akt phosphorylation, but did interfere with the NFkappaB activation process (both basal and PMA-stimulated), by lowering the levels of phospho-IkappaBalpha and preventing p65 nuclear migration. The growth arrest evoked by Rottlerin was not mediated by cell-cycle inhibitors p21 and p27 but was accompanied by a dramatic fall in the cyclin-D1 protein, the levels of which were not altered by the pan-PKC inhibitor GF 109203X, thus excluding a PKC-mediated mechanism in the Rottlerin effect. The parallel drop in cyclin-D1 mRNA suggested a down-regulation of the gene caused by the inhibition of nuclear factor-kappa B (NFkappaB), which occurs via a PKC-, Akt-, ERK- and mitochondrial uncoupling-independent mechanism. We provide preliminary evidence that the interference on the NFkappaB activation process likely occurs at the level of calcium/calmodulin-dependent protein kinase II (CaMKII), a known Rottlerin target. Indeed the drug prevented calcium-induced CaMKII autophosphorylation which, in turn, led to decreased NFkappaB activation.

Multinucleated giant cells are a classic cellular feature of chronic inflammation, although the mechanism of macrophage fusion leading to their formation is not well understood. Here, we investigate the participation of protein kinase C (PKC) in the interleukin (IL)-4-induced fusion of human monocyte-derived macrophages and foreign body giant cell (FBGC) formation in vitro. The PKC inhibitors H-7 and calphostin C attenuated macrophage fusion, whereas H-8, which is more selective for PKA and PKG, did not. Macrophage fusion was also prevented by the phospholipase C inhibitor, Et-18-OCH(3), the PKC isoform inhibitors GO6983 or rottlerin and by peptide inhibitors for PKC (20-28), PKCbeta, or PKCzeta but not by HBDDE or peptide inhibitors for PKCvarepsilon or PKA. In cultures of fusing macrophages/FBGC, we detected only PKCalpha, beta, delta, and zeta by immunoprecipitation and immunoblotting, and we also observed strong expression of these isoforms by immunocytochemistry. Our collective results suggest that the gamma, epsilon, eta, mu, theta, or iota PKC isoforms are not required in the mechanism of IL-4-induced macrophage fusion; whether PKCalpha is required is unclear. However, new evidence is provided that FBGC formation is supported by PKCbeta, PKCdelta, and PKCzeta in combined diacylglycerol-dependent (PKCbeta and PKCdelta) and -independent (PKCzeta) signaling pathways.

Apocynin has been used as an efficient inhibitor of the NADPH oxidase complex and its mechanism of inhibition is linked to prior activation through the action of peroxidases. Here we studied the oxidation of apocynin catalyzed by myeloperoxidase (MPO) and activated neutrophils. We found that apocynin is easily oxidized by MPO/H2O2 or activated neutrophils and has as products dimer and trimer derivatives. Since apocynin impedes the migration of the cytosolic component p47phox to the membrane and this effect could be related to its conjugation with essential thiol groups, we studied the reactivity of apocynin and its MPO-catalyzed oxidation products with glutathione (GSH). We found that apocynin and its oxidation products do not react with GSH. However, this thiol compound was efficiently oxidized by the apocynin radical during the MPO-catalyzed oxidation. We suggest that the reactivity of apocynin radical with thiol compounds could be involved in the inhibitory effect of this methoxy-catechol on NADPH oxidase complex.

Apparently conflicting observations indicated that protein kinase C both may block and support the assembly of tight junctions. We therefore tested the hypothesis that different isoenzymes antagonistically affect tight junction proteins and function. Thus, by using specific inhibitors we investigated the involvement of conventional and novel protein kinase C of kidney tubule cells in tight junction assembly. In low Ca2+ medium, the application of pan-protein kinase C inhibitor GF-109203X blocked the formation of tight junctions induced by protein kinase C agonist diacyglycerol. Gö6976, inhibitor of conventional protein kinase C, promoted the formation of tight junctions and occludin phosphorylation in cells cultivated in low Ca2+ medium and attenuated the disruption of tight junction complex induced by the switch to low Ca2+ medium. In addition, Gö6976 accelerated the occludin phosphorylation and the formation of tight junction barrier during assembly of tight junctions induced by Ca2+ re-addition. This phosphorylation was accompanied by accelerated occludin incorporation into newly forming tight junctions and by reducing the paracellular permeability. In contrast, inhibitor of novel protein kinase C rottlerin blocked the occludin phosphorylation and the formation of tight junction barrier, both caused by re-addition of normal Ca2+ medium. It is concluded that the conventional protein kinase C alpha participates in tight junction disassembly while the novel protein kinase C epsilon plays a role in tight junction formation of kidney epithelial cells. The discovered antagonism contributes to a better understanding of the regulation of the structure and function of tight junctions and hence to that of the epithelial barrier.

In this study, acetosyringone was identified as one of the major extracellular phenolics in tobacco suspension cells and was shown to have bioactive properties that influence early events in plant-bacterial pathogenesis. In our model system, tobacco cell suspensions treated with bacterial isolate Pseudomonas syringae WT (HR+) undergo a resistant interaction characterized by a burst in oxygen uptake several hours after inoculation. When the extracellular concentration of acetosyringone in tobacco cell suspensions was supplemented with exogenous acetosyringone, the burst in oxygen uptake occurred as much as 1.5h earlier. The exogenous acetosyringone had no effect on tobacco suspensions undergoing susceptible interactions with Pseudomonas tabaci or a non-resistant interaction with a near-isogenic mutant derivative of isolate P. syringae WT (HR+). Resistant interactions with isolate P. syringae WT (HR+) also produce an oxidative burst which oxidizes the extracellular acetosyringone. This study demonstrates that acetosyringone, and likely other extracellular phenolics, may have bioactive characteristics that can influence plant-bacterial pathogenesis.

Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) catalyzes the hydrolysis of glycerophospholipids at the sn-2 position to liberate fatty acids. Although cPLA(2)alpha has been implicated in various cellular processes, the detailed mechanism of its expression remains to be elucidated. Here we report that phorbol 12-myristate 13-acetate (PMA) up-regulates cPLA(2)alpha in A549 airway epithelium cells, and that this effect is sensitive to rottlerin, a potent inhibitor of protein kinase Cdelta (PKCdelta). Consistent with this observation, a dominant negative mutant of PKCdelta reduced cPLA(2)alpha induction in response to PMA. Up-regulation of cPLA(2)alpha by PMA was also inhibited by PDTC, an inhibitor of nuclear factor-kappa B (NF-kappaB), and degradation of IkappaB and subsequent activation of NF-kappaB occurred in response to PMA treatment. These findings indicate that PMA induces expression of cPLA(2)alpha at the transcriptional level via an NF-kappaB-dependent mechanism. In addition, activation of the NF-kappaB promoter by PMA was diminished by pretreatment with DPI, a flavoenzyme inhibitor as well as by rottlerin, suggesting a role for reactive oxygen species (ROS) as well as PKCdelta. Consistent with this, PMA stimulated the production of ROS and this was blocked by inhibiting PKCdelta. Our results suggest that PKCdelta and ROS lie upstream of NF-kappaB, and we conclude that a PKCdelta-ROS-NF-kappaB cascade plays a pivotal role in cPLA(2)alpha induction by PMA.

The kinase activity of DNA-dependent protein kinase (DNA-PK) is required for efficient repair of DNA double-strand breaks (DSB) by non-homologous end joining (NHEJ). DNA-PK also participates in protection of mammalian telomeres, the natural ends of chromosomes. Here we investigate whether the kinase activity of DNA-PK is similarly required for effective telomere protection. DNA-PK proficient mouse cells were exposed to a highly specific inhibitor of DNA-PK phosphorylation designated IC86621. Chromosomal end-to-end fusions were induced in a concentration-dependent manner, demonstrating that the telomere end-protection role of DNA-PK requires its kinase activity. These fusions were uniformly chromatid-type, consistent with a role for DNA-PK in capping telomeres after DNA replication. Additionally, fusions involved exclusively telomeres produced via leading-strand DNA synthesis. Unexpectedly, the rate of telomeric fusions induced by IC86621 exceeded that which occurs spontaneously in DNA-dependent protein kinase catalytic subunit (DNA-PKcs) mutant cells by up to 110-fold. One explanation, that IC86621 might inhibit other, as yet unknown proteins, was ruled out when the drug failed to induce fusions in DNA-PKcs knock-out mouse cells. IC86621 did not induce fusions in Ku70 knock-out cells suggesting the drug requires the holoenzyme to be effective. ATM also is required for effective chromosome end protection. IC86621 increased fusions in ATM knock-out cells suggesting DNA-PK and ATM act in different telomere pathways. These results indicate that the kinase activity of DNA-PK is crucial to reestablishing a protective terminal structure, specifically on telomeres replicated by leading-strand DNA synthesis., (Copyright 2003 Elsevier B.V.)

The enzyme poly(adenosine 5'-diphosphate (ADP)-ribose) polymerase (PARP-1) catalyzes the formation of (ADP)-ribose polymers on a variety of protein acceptors in a NAD+ -dependent manner. While PARP-1 is activated by DNA damage and plays a critical role in cellular survival mechanisms, its overactivation leads to a depletion of NAD+/ATP energy stores and ultimately to necrotic cell death. Due to this dual role of PARP in the cell, small-molecule inhibitors of the PARP family of enzymes have been widely investigated for use as potentiators of anticancer therapies and as inhibitors of neurodegeneration and ischemic injuries. Unfortunately, standard assays for PARP inhibition are not optimal for the high-throughput screening of compound collections or combinatorial libraries. Described herein is a highly sensitive, inexpensive, and operationally simple assay for the rapid assessment of PARP activity that relies on the conversion of NAD+ into a highly fluorescent compound. We demonstrate that this assay can readily detect PARP inhibitors in a high-throughput screen using 384-well plates. In addition, the assay can be used to determine IC50 values for PARP inhibitors that have a range of inhibitory properties. As existing PARP assays utilize specialized reagents such as radiolabeled/biotinylated NAD+ or antibodies to poly(ADP-ribose), the chemical quantitation method described herein offers a highly sensitive and convenient alternative for rapidly screening compound collections for PARP inhibition.

The crystal structure of the apo-form of an R-specific alcohol dehydrogenase from Lactobacillus brevis (LB-RADH) was solved and refined to 1.8A resolution. LB-RADH is a member of the short-chain dehydrogenase/reductase (SDR) enyzme superfamily. It is a homotetramer with 251 amino acid residues per subunit and uses NADP(H) as co-enzyme. NADPH and the substrate acetophenone were modelled into the active site. The enantiospecificity of the enzyme can be explained on the basis of the resulting hypothetical ternary complex. In contrast to most other SDR enzymes, the catalytic activity of LB-RADH depends strongly on the binding of Mg(2+). Mg(2+) removal by EDTA inactivates the enzyme completely. In the crystal structure, the Mg(2+)-binding site is well defined. The ion has a perfect octahedral coordination sphere and occupies a special position concerning crystallographic and molecular point symmetry, meaning that each RADH tetramer contains two magnesium ions. The magnesium ion is no direct catalytic cofactor. However, it is structurally coupled to the putative C-terminal hinge of the substrate-binding loop and, via an extended hydrogen bonding network, to some side-chains forming the substrate binding region. Therefore, the presented structure of apo-RADH provides plausible explanations for the metal dependence of the enzyme.

Inhibitors of human immunodeficiency virus (HIV) reverse transcriptase (RT) are widely used in the treatment of HIV infection. Loviride (an alpha-APA derivative) and HBY 097 (a quinoxaline derivative) are two potent non-nucleoside RT inhibitors (NNRTIs) that have been used in human clinical trials. A major problem for existing anti-retroviral therapy is the emergence of drug-resistant mutants with reduced susceptibility to the inhibitors. Amino acid residue 103 in the p66 subunit of HIV-1 RT is located near a putative entrance to a hydrophobic pocket that binds NNRTIs. Substitution of asparagine for lysine at position 103 of HIV-1 RT is associated with the development of resistance to NNRTIs; this mutation contributes to clinical failure of treatments employing NNRTIs. We have determined the structures of the unliganded form of the Lys103Asn mutant HIV-1 RT and in complexes with loviride and HBY 097. The structures of wild-type and Lys103Asn mutant HIV-1 RT in complexes with NNRTIs are quite similar overall as well as in the vicinity of the bound NNRTIs. Comparison of unliganded wild-type and Lys103Asn mutant HIV-1 RT structures reveals a network of hydrogen bonds in the Lys103Asn mutant that is not present in the wild-type enzyme. Hydrogen bonds in the unliganded Lys103Asn mutant but not in wild-type HIV-1 RT are observed between (1) the side-chains of Asn103 and Tyr188 and (2) well-ordered water molecules in the pocket and nearby pocket residues. The structural differences between unliganded wild-type and Lys103Asn mutant HIV-1 RT may correspond to stabilization of the closed-pocket form of the enzyme, which could interfere with the ability of inhibitors to bind to the enzyme. These results are consistent with kinetic data indicating that NNRTIs bind more slowly to Lys103Asn mutant than to wild-type HIV-1 RT. This novel drug-resistance mechanism explains the broad cross-resistance of Lys103Asn mutant HIV-1 RT to different classes of NNRTIs. Design of NNRTIs that make favorable interactions with the Asn103 side-chain should be relatively effective against the Lys103Asn drug-resistant mutant., (Copyright 2001 Academic Press.)

This paper describes the isolation and primary structure analysis of a new phospholipase A2 with platelet-aggregation-inhibiting activity from the venom of Bothrops jararaca. The protein, named BJ-PLA2, was isolated by means of ammonium sulfate precipitation and anion-exchange and reversed-phase chromatographies and behaved as a homogeneous single-chain protein on SDS-PAGE. Its amino acid sequence was determined by N-terminal sequencing and analysis of overlapped chemical and proteolytic fragments by automated Edman degradation and mass spectometry determination. BJ-PLA2 consists of 124 amino acid residues and has the structural features of snake venom class II phospholipases A2. Chemical modification with p-bromophenacylbromide caused complete loss of enzymatic activity and partially affected the platelet-aggregation-inhibiting activity of BJ-PLA2., (Copyright 1999 Academic Press.)

pH dependence of the chemical reaction rates of p-bromophenacyl bromide (BPB) and of the binding constants of Ca2+ to bovine pancreatic active- and pro-phospholipases A2 (PLA2s) was studied at 25 degrees C and ionic strength 0.2. The pH dependence curves of the reaction rates of BPB with both enzymes were biphasic. The amino acid residues participating in the two transitions were ascribed to His 48 and the N-terminal alpha-amino group for the active enzyme and to His 48 and Arg -1 for the proenzyme. The pH dependence curve of Ca2+ binding to the active enzyme was interpreted in terms of participation of Asp 49, His 48, and the alpha-amino group. On the other hand, the curve for the proenzyme was interpreted in terms of participation of Asp 49, His 48, and Arg -1. The Ca2+ and pH dependence of the binding constant of a potent competitive inhibitor, monodispersed (R)-2-dodecanoylamino-1-hexanol-phosphocholine (amide-PC), to bovine pancreatic active-PLA2 was also studied. The binding of amide-PC was markedly facilitated by Ca2+ binding to the enzyme, whereas that of a genuine substrate, monodispersed 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), was independent of Ca2+ binding. The pH dependence curve of the binding constant of the amide-PC showed one transition, and this was interpreted in terms of participation of His 48, whereas the binding of the diC6PC was independent of the ionization state of His 48. The difference in the Ca2+ dependence for the bindings of the diC6PC and amide-PC was considered to arise from the fact that the amide group of amide-PC can form a hydrogen bond with His 48, whereas the genuine substrate cannot form such a hydrogen bond.

The study focused on the effects on various redox states of hemoglobin during NADPH: cytochrome P-450 reductase-catalyzed redox cycling of quinones and nitrocompounds. The following reactions involving quinone/semiquinone and methemoglobin/oxyhemoglobin redox couples were observed: (i) the direct oxidation of oxyhemoglobin by quinones, (ii) the reduction of methemoglobin by quinones, (ii) the reduction of methemoglobin during redox cycling of quinones and nitrocompounds was partially inhibited by superoxide dismutase, and (iii) the reoxidation of oxyhemoglobin by hydrogen peroxide, formed during redox cycling was accompanied by the formation of choleglobin. Hydrogen peroxide was produced during redox cycling, and upon depletion of hydrogen peroxide by catalase, the reduction of methemoglobin significantly prevailed over oxidation of oxyhemoglobin. Furthermore, the reduction of ferrylhemoglobin to oxyhemoglobin during redox cycling was about twice as slow as the reduction of methemoglobin. For a series of compounds possessing a single-electron reduction potential (E1(7)) between 0.01 and -0.355 V, the rate constants for methemoglobin reduction by their corresponding radicals was estimated to range from 4.1 x 10(5) to 7.6 x 10(7) M-1 S-1. Radicals of the nitrocompounds were approximately 10 times less reactive as compared to quinones possessing similar E1(7) values.

A high-performance liquid chromatographic assay for the determination of endogenous plasma and urine concentrations of N1-methylnicotinamide was developed. N1-Methylnicotinamide and N1-ethylnicotinamide (internal standard) are reacted with acetophenone in a strong base at 0 degree C, formic acid is added, and the reaction mixture is heated in a boiling water bath, resulting in the formation of fluorescent derivatives. These derivatives were chromatographed on a C18 reverse-phase column using a mobile phase of acetonitrile-triethylamine and 0.01 M heptanesulfonic acid adjusted to pH 3.2. Fluorescent detection was achieved using 366-nm excitation and 418-nm emission filters. Precision and accuracy were generally greater than 90%, interfering peaks did not cochromatograph, and the limit of quantification was 2 ng/ml in plasma using a 0.2-ml sample. The method was used to examine the concentrations of endogenous N1-methylnicotinamide in the plasma of 36 subjects with various pathology. The mean concentration was 18 ng/ml and the range was 6.2 to 116.7 ng/ml. The assay represents a marked improvement on previous methods and is suitable for routine clinical monitoring.

Mechanistic mode for the oxygenation of sulfides with the pig liver microsomal FAD-containing monooxygenase(EC 1.14.13.8) has been conveniently distinguished from that with the phenobarbital induced liver microsomal cytochrome P-450 by analyzing products of the oxygenation of phenacyl phenyl sulfide. Upon oxygenation of phenacyl phenyl sulfide, the FAD-containing monooxygenase gave solely phenacyl phenyl sulfoxide in contrast to the cytochrome P-450 promoted oxygenation which is known to give substantial amounts of C-S bond fission products. The observation suggests that the oxygenation of sulfide with FAD-containing monooxygenase involves the nucleophilic attack of the divalent sulfur on the reactive oxygen atom involved at the enzyme active site, namely electrophilic oxygenation of sulfide, though the oxygenation with the cytochrome P-450 is initiated by a single electron transfer from the sulfide to the enzyme active species.