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1.  Development of a 384-Well Colorimetric Assay to Quantify Hydrogen Peroxide Generated by the Redox Cycling of Compounds in the Presence of Reducing Agents 
We report here the development and optimization of a simple 384-well colorimetric assay to measure H2O2 generated by the redox cycling of compounds incubated with reducing agents in high-throughput screening (HTS) assay buffers. The phenol red-horseradish peroxidase (HRP) assay readily detected H2O2 either added exogenously or generated by the redox cycling of compounds in dithiothreitol (DTT). The generation of H2O2 was dependent on the concentration of both the compound and DTT and was abolished by catalase. Although both DTT and tris(2-carboxyethyl)-phosphine sustain the redox cycling generation of H2O2 by a model quinolinedione, 6-chloro-7-(2-morpholin-4-yl-ethylamino)-quinoline-5,8-dione (NSC 663284; DA3003-1), other reducing agents such as β-mercaptoethanol, glutathione, and cysteine do not. The assay is compatible with HTS. Once terminated, the assay signal was stable for at least 5 h, allowing for a reasonable throughput. The assay tolerated up to 20% dimethyl sulfoxide, allowing a wide range of compound concentrations to be tested. The assay signal window was robust and reproducible with average Z-factors of ≥0.8, and the redox cycling generation of H2O2 by DA3003-1 in DTT exhibited an average 50% effective concentration of 0.830 ± 0.068 μM. Five of the mitogen-activated protein kinase phosphatase (MKP) 1 inhibitors identified in an HTS were shown to generate H2O2 in the presence of DTT, and their inhibition of MKP-1 activity was shown to be time dependent and was abolished or significantly reduced by either 100 U of catalase or by higher DTT levels. A cross-target query of the PubChem database with three structurally related pyrimidotriazinediones revealed active flags in 36–39% of the primary screening assays. Activity was confirmed against a number of targets containing active site cysteines, including protein tyrosine phosphatases, cathepsins, and caspases, as well as a number of cellular cytotoxicity assays. Rather than utilize resources to conduct a hit characterization effort involving several secondary assays, the phenol red-HRP assay provides a simple, rapid, sensitive, and inexpensive method to identify compounds that redox cycle in DTT or tris(2-carboxyethyl)phosphine to produce H2O2 that may indirectly modulate target activity and represent promiscuous false-positives from a primary screen.
doi:10.1089/adt.2008.151
PMCID: PMC2752819  PMID: 18699726
2.  Profiling the NIH Small Molecule Repository for Compounds That Generate H2O2 by Redox Cycling in Reducing Environments 
We have screened the Library of Pharmacologically Active Compounds (LOPAC) and the National Institutes of Health (NIH) Small Molecule Repository (SMR) libraries in a horseradish peroxidase–phenol red (HRP-PR) H2O2 detection assay to identify redox cycling compounds (RCCs) capable of generating H2O2 in buffers containing dithiothreitol (DTT). Two RCCs were identified in the LOPAC set, the ortho-naphthoquinone β-lapachone and the para-naphthoquinone NSC 95397. Thirty-seven (0.02%) concentration-dependent RCCs were identified from 195,826 compounds in the NIH SMR library; 3 singleton structures, 9 ortho-quinones, 2 para-quinones, 4 pyrimidotriazinediones, 15 arylsulfonamides, 2 nitrothiophene-2-carboxylates, and 2 tolyl hydrazides. Sixty percent of the ortho-quinones and 80% of the pyrimidotriazinediones in the library were confirmed as RCCs. In contrast, only 3.9% of the para-quinones were confirmed as RCCs. Fifteen of the 251 arylsulfonamides in the library were confirmed as RCCs, and since we screened 17,868 compounds with a sulfonamide functional group we conclude that the redox cycling activity of the arylsulfonamide RCCs is due to peripheral reactive enone, aromatic, or heterocyclic functions. Cross-target queries of the University of Pittsburgh Drug Discovery Institute (UPDDI) and PubChem databases revealed that the RCCs exhibited promiscuous bioactivity profiles and have populated both screening databases with significantly higher numbers of active flags than non-RCCs. RCCs were promiscuously active against protein targets known to be susceptible to oxidation, but were also active in cell growth inhibition assays, and against other targets thought to be insensitive to oxidation. Profiling compound libraries or the hits from screening campaigns in the HRP-PR H2O2 detection assay significantly reduce the timelines and resources required to identify and eliminate promiscuous nuisance RCCs from the candidates for lead optimization.
doi:10.1089/adt.2009.0247
PMCID: PMC3098569  PMID: 20070233
3.  Phosphines are ribonucleotide reductase reductants that act via C-terminal cysteines similar to thioredoxins and glutaredoxins 
Scientific Reports  2014;4:5539.
Ribonucleotide reductases (RNRs) catalyze the formation of 2′-deoxyribonucleotides. Each polypeptide of the large subunit of eukaryotic RNRs contains two redox-active cysteine pairs, one in the active site and the other at the C-terminus. In each catalytic cycle, the active-site disulfide is reduced by the C-terminal cysteine pair, which in turn is reduced by thioredoxins or glutaredoxins. Dithiols such as DTT are used in RNR studies instead of the thioredoxin or glutaredoxin systems. DTT can directly reduce the disulfide in the active site and does not require the C-terminal cysteines for RNR activity. Here we demonstrate that the phosphines tris(2-carboxyethyl)phosphine (TCEP) and tris(3-hydroxypropyl)phosphine (THP) are efficient non-thiol RNR reductants, but in contrast to the dithiols DTT, bis(2-mercaptoethyl)sulfone (BMS), and (S)-(1,4-dithiobutyl)-2-amine (DTBA) they act specifically via the C-terminal disulfide in a manner similar to thioredoxin and glutaredoxin. The simultaneous use of phosphines and dithiols results in ~3-fold higher activity compared to what is achieved when either type of reductant is used alone. This surprising effect can be explained by the concerted action of dithiols on the active-site cysteines and phosphines on the C-terminal cysteines. As non-thiol and non-protein reductants, phosphines can be used to differentiate between the redox-active cysteine pairs in RNRs.
doi:10.1038/srep05539
PMCID: PMC4078304  PMID: 24986213
4.  Thiol dependent recovery of catalytic activity from oxidized protein tyrosine phosphatases 
Biochemistry  2013;52(37):6412-6423.
Protein tyrosine phosphatases (PTPs) play an important role in the regulation of mammalian signal transduction. During some cell signaling processes, the generation of endogenous hydrogen peroxide inactivates selected PTPs via oxidation of the enzyme's catalytic cysteine thiolate group. Importantly, low molecular weight and protein thiols in the cell have the potential to regenerate the catalytically active PTPs. Here we examined the recovery of catalytic activity from two oxidatively-inactivated PTPs (PTP1B and SHP-2) by various low molecular weight thiols and the enzyme thioredoxin. All thiols examined regenerated the catalytic activity of oxidized PTP1B, with apparent rates constants that varied by a factor of approximately eight. In general, molecules bearing low pKa thiol groups were particularly effective. The biological thiol, glutathione repaired oxidized PTP1B with an apparent second-order rate constant of 0.023 ± 0.004 M−1 s−1, while the dithiol, DTT, displayed an apparent second-order rate constant of 0.325 ± 0.007 M−1 s−1. The enzyme thioredoxin regenerated the catalytic activity of oxidized PTP1B at a substantially faster rate than DTT. Thioredoxin (2 μM) converted oxidized PTP1B to the active form with an observed rate constant of 1.4 × 10−3 s−1. The rates at which these agents regenerated oxidized PTP1B followed the trend Trx > DTT > GSH, with comparable values observed at 2 μM Trx, 4 mM DTT and 60 mM GSH. Various disulfides that are byproducts of the reactivation process did not inactivate native PTP1B at concentrations of 1-20 mM. The common biochemical reducing agent tris(2-carboxyethyl)phosphine (TCEP) regenerates enzymatic activity from oxidized PTP1B somewhat faster than the thiol-based reagents, with a rate constant of 1.5 ± 0.5 M−1 s−1. We observed profound kinetic differences between the thiol-dependent regeneration of activity from oxidized PTP1B and SHP-2, highlighting the potential for structural differences in various oxidized PTPs to play a significant role in the rates at which low molecular weight thiols and thiol-containing enzymes such as thioredoxin and glutaredoxin return catalytic activity to these enzymes during cell signaling events.
doi:10.1021/bi400451m
PMCID: PMC4006132  PMID: 23957891
5.  Reducing agents affect inhibitory activities of compounds: Results from Multiple Drug Targets 
Analytical Biochemistry  2012;423(1):46-53.
High-throughput screening (HTS) of large compound libraries has become a commonly used method for the identification of drug leads, and non-physiological reducing agents have been widely used for HTS. However, a comparison of the difference in the HTS results based on the choice of reducing agent used and potency comparisons of selected inhibitors has not been done with the physiological reducing agent, reduced glutathione (GSH). Here, we compared the effects of three reducing agents: dithiothreitol (DTT), β-mercaptoethanol (β-MCE), and tris-(2-carboxyethyl)-phosphine (TCEP), in addition to GSH, against three drug target proteins. Approximately 100,000 compounds were computationally screened for each target protein, and experimental testing of high scoring compounds (~560 compounds) with the four reducing agents surprisingly produced many non-overlapping hits. More importantly, we find that various reducing agents alter inhibitor potency (IC50) from ~10 µM with one reducing agent to complete loss (IC50 > 200 µM) of inhibitory activity with another reducing agent. Therefore, the choice of reducing agent in a HTS is critical as this may lead to the pursuit of falsely identified active compounds or failure to identify the true active compounds. We demonstrate the feasibility of using GSH for in vitro HTS assays with these three target enzymes.
doi:10.1016/j.ab.2012.01.006
PMCID: PMC3299889  PMID: 22310499
Reducing agent; inhibitor screening; false positives; false negatives; potency comparison
6.  Flavin-linked Erv-family sulfhydryl oxidases release superoxide anion during catalytic turnover† 
Biochemistry  2011;51(1):265-272.
Typically, simple flavoprotein oxidases couple the oxidation of their substrates with the formation of hydrogen peroxide without release of significant levels of the superoxide ion. However, two evolutionarily-related single-domain sulfhydryl oxidases (Erv2p; a yeast endoplasmic reticulum resident protein and augmenter of liver regeneration, ALR, an enzyme predominantly found in the mitochondrial intermembrane) release up to ~30% of the oxygen they reduce as the superoxide ion. Both enzymes oxidize dithiol substrates via a redox-active disulfide adjacent to the flavin cofactor within the helix-rich Erv domain. Subsequent reduction of the flavin is followed by transfer of reducing equivalents to molecular oxygen. Superoxide release was initially detected using tris(3-hydroxypropyl)phosphine (THP) as an alternative reducing substrate to dithiothreitol (DTT). THP, and other phosphines, showed anomalously high turnover numbers with Erv2p and ALR in the oxygen electrode but oxygen consumption was drastically suppressed upon the addition of superoxide dismutase. The superoxide ion initiates a radical chain reaction promoting the aerobic oxidation of phosphines with the formation of hydrogen peroxide. Use of a known flux of superoxide generated by the xanthine/xanthine oxidase system showed that one superoxide ion stimulates the reduction of 27 and 4.5 molecules of oxygen using THP and tris(2-carboxyethyl)phosphine (TCEP) respectively. This superoxide-dependent amplification of oxygen consumption by phosphines provides a new kinetic method for the detection of superoxide. Superoxide release was also observed by a standard chemiluminescence method using a luciferin analog (MCLA) when 2 mM DTT was employed as a substrate of Erv2p and ALR. The percentage of superoxide released from Erv2p increased to ~65% when monomeric mutants of the normally homodimeric enzyme were used. In contrast, monomeric multi-domain Quiescin-sulfhydryl oxidase enzymes that also contain an Erv FAD-binding fold release only 1-5% of their total reduced oxygen species as the superoxide ion. Aspects of the mechanism and possible physiological significance of superoxide release from these Erv-domain flavoproteins are discussed.
doi:10.1021/bi201672h
PMCID: PMC3254808  PMID: 22148553
7.  Reducing conditions are the key for efficient production of active ribonuclease inhibitor in Escherichia coli 
Background
The eukaryotic RNase ribonuclease/angiogenin inhibitors (RI) are a protein group distinguished by a unique structure - they are composed of hydrophobic leucine-rich repeat motifs (LRR) and contain a high amount of reduced cysteine residues. The members of this group are difficult to produce in E. coli and other recombinant hosts due to their high aggregation tendency.
Results
In this work dithiothreitol (DTT) was successfully applied for improving the yield of correctly folded ribonuclease/angiogenin inhibitor in E. coli K12 periplasmic and cytoplasmic compartments. The feasibility of the in vivo folding concepts for cytoplasmic and periplasmic production were demonstrated at batch and fed-batch cultivation modes in shake flasks and at the bioreactor scale.
Firstly, the best secretion conditions of RI in the periplasmic space were evaluated by using a high throughput multifactorial screening approach of a vector library, directly with the Enbase fed-batch production mode in 96-well plates. Secondly, the effect of the redox environment was evaluated in isogenic dsbA+ and dsbA- strains at the various cultivation conditions with reducing agents in the cultivation medium. Despite the fusion to the signal peptide, highest activities were found in the cytoplasmic fraction. Thus by removing the signal peptide the positive effect of the reducing agent DTT was clearly proven also for the cytoplasmic compartment. Finally, optimal periplasmic and cytoplasmic RI fed-batch production processes involving externally added DTT were developed in shake flasks and scaled up to the bioreactor scale.
Conclusions
DTT highly improved both, periplasmic and cytoplasmic accumulation and activity of RI at low synthesis rate, i.e. in constructs harbouring weak recombinant synthesis rate stipulating genetic elements together with cultivation at low temperature. In a stirred bioreactor environment RI folding was strongly improved by repeated pulse addition of DTT at low aeration conditions.
doi:10.1186/1475-2859-10-31
PMCID: PMC3112386  PMID: 21554746
8.  Identification of a Disulfide Bridge Important for Transport Function of SNAT4 Neutral Amino Acid Transporter 
PLoS ONE  2013;8(2):e56792.
SNAT4 is a member of system N/A amino acid transport family that primarily expresses in liver and muscles and mediates the transport of L-alanine. However, little is known about the structure and function of the SNAT family of transporters. In this study, we showed a dose-dependent inhibition in transporter activity of SNAT4 with the treatment of reducing agents, dithiothreitol (DTT) and Tris(2-carboxyethyl)phosphine (TCEP), indicating the possible involvement of disulfide bridge(s). Mutation of residue Cys-232, and the two highly conserved residues Cys-249 and Cys-321, compromised the transport function of SNAT4. However, this reduction was not caused by the decrease of SNAT4 on the cell surface since the cysteine-null mutant generated by replacing all five cysteines with alanine was equally capable of being expressed on the cell surface as wild-type SNAT4. Interestingly, by retaining two cysteine residues, 249 and 321, a significant level of L-alanine uptake was restored, indicating the possible formation of disulfide bond between these two conserved residues. Biotinylation crosslinking of free thiol groups with MTSEA-biotin provided direct evidence for the existence of a disulfide bridge between Cys-249 and Cys-321. Moreover, in the presence of DTT or TCEP, transport activity of the mutant retaining Cys-249 and Cys-321 was reduced in a dose-dependent manner and this reduction is gradually recovered with increased concentration of H2O2. Disruption of the disulfide bridge also decreased the transport of L-arginine, but to a lesser degree than that of L-alanine. Together, these results suggest that cysteine residues 249 and 321 form a disulfide bridge, which plays an important role in substrate transport but has no effect on trafficking of SNAT4 to the cell surface.
doi:10.1371/journal.pone.0056792
PMCID: PMC3579933  PMID: 23451088
9.  Residues C123 and D58 of the 2-Methylisocitrate Lyase (PrpB) Enzyme of Salmonella enterica Are Essential for Catalysis 
Journal of Bacteriology  2003;185(16):4837-4843.
The prpB gene of Salmonella enterica serovar Typhimurium LT2 encodes a protein with 2-methylisocitrate (2-MIC) lyase activity, which cleaves 2-MIC into pyruvate and succinate during the conversion of propionate to pyruvate via the 2-methylcitric acid cycle. This paper reports the isolation and kinetic characterization of wild-type and five mutant PrpB proteins. Wild-type PrpB protein had a molecular mass of approximately 32 kDa per subunit, and the biologically active enzyme was comprised of four subunits. Optimal 2-MIC lyase activity was measured at pH 7.5 and 50°C, and the reaction required Mg2+ ions; equimolar concentrations of Mn2+ ions were a poor substitute for Mg2+ (28% specific activity). Dithiothreitol (DTT) or reduced glutathione (GSH) was required for optimal activity; the role of DTT or GSH was apparently not to reduce disulfide bonds, since the disulfide-specific reducing agent Tris(2-carboxyethyl)phosphine hydrochloride failed to substitute for DTT or GSH. The Km of PrpB for 2-MIC was measured at 19 μM, with a kcat of 105 s−1. Mutations in the prpB gene were introduced by site-directed mutagenesis based on the active-site residues deemed important for catalysis in the closely related phosphoenolpyruvate mutase and isocitrate lyase enzymes. Residues D58, K121, C123, and H125 of PrpB were changed to alanine, and residue R122 was changed to lysine. Nondenaturing polyacrylamide gel electrophoresis indicated that all mutant PrpB proteins retained the same oligomeric state of the wild-type enzyme, which is known to form tetramers. The PrpBK121A, PrpBH125A, and PrpBR122K mutant proteins formed enzymes that had 1,050-, 750-, and 2-fold decreases in kcat for 2-MIC lyase activity, respectively. The PrpBD58A and PrpBC123A proteins formed tetramers that displayed no detectable 2-MIC lyase activity indicating that both of these residues are essential for catalysis. Based on the proposed mechanism of the closely related isocitrate lyases, PrpB residue C123 is proposed to serve as the active site base, and residue D58 is critical for the coordination of a required Mg2+ ion.
doi:10.1128/JB.185.16.4837-4843.2003
PMCID: PMC166468  PMID: 12897003
10.  On dithiothreitol (DTT) as a measure of oxidative potential for ambient particles: evidence for the importance of soluble transition metals 
The rate of consumption of dithiothreitol (DTT) is increasingly used to measure the oxidative potential of particulate matter (PM), which has been linked to the adverse health effects of PM. While several quinones are known to be very reactive in the DTT assay, it is unclear what other chemical species might contribute to the loss of DTT in PM extracts. To address this question, we quantify the rate of DTT loss from individual redox-active species that are common in ambient particulate matter. While most past research has indicated that the DTT assay is not sensitive to metals, our results show that seven out of the ten transition metals tested do oxidize DTT, as do three out of the five quinones tested. While metals are less efficient at oxidizing DTT compared to the most reactive quinones, concentrations of soluble transition metals in fine particulate matter are generally much higher than those of quinones. The net result is that metals appear to dominate the DTT response for typical ambient PM2.5 samples. Based on particulate concentrations of quinones and soluble metals from the literature, and our measured DTT responses for these species, we estimate that for typical PM2.5 samples approximately 80 % of DTT loss is from transition metals (especially copper and manganese), while quinones account for approximately 20 %. We find a similar result for DTT loss measured in a small set of PM2.5 samples from the San Joaquin Valley of California. Because of the important contribution from metals, we also tested how the DTT assay is affected by EDTA, a chelator that is sometimes used in the assay. EDTA significantly suppresses the response from both metals and quinones; we therefore recommend that EDTA should not be included in the DTT assay.
doi:10.5194/acpd-12-11317-2012
PMCID: PMC3564657  PMID: 23393494
11.  High Throughput Screening for Small Molecule Inhibitors of Heparin-induced Tau Fibril Formation 
A library of ∼51,000 compounds was interrogated by high throughput screening (HTS) using a heparin-induced tau fibrillization assay. HTS was conducted with bacterially expressed recombinant tau fragment K18 and the reaction was monitored by thioflavine T fluorescence. Hits meeting criteria set for selection in HTS were further evaluated in a panel of assays designed (a) to confirm the initial results and (b) to identify possible false positives arising from non-specific mechanisms or assay dependent artifacts. Two 2,3-di(furan-2-yl)-quinoxalines were confirmed as inhibitors of tau fibrillization with IC50s in the low micromolar range (l–3 μM). Among false positives hits, members of the pyrimidotriazines, benzofurans, porphyrins and anthraquinones, inhibited tau fibrillization by generating peroxides via catalytic redox cycles due to the reducing agent dithiothreitol (DTT) in the assay. This study delineates focused strategies for HTS of tau fibrillization inhibitors that are relevant to drug discovery for Alzheimer's disease and related tauopathies.
doi:10.1016/j.bbrc.2007.03.056
PMCID: PMC2646256  PMID: 17482143
12.  The vagina has reducing environment sufficient for activation of Trichomonas vaginalis cysteine proteinases. 
Genitourinary Medicine  1997;73(4):291-296.
BACKGROUND: Trichomonas vaginalis, a worldwide distributed sexually transmitted protozoan, is remarkable for synthesis of numerous, distinct cysteine proteinases, the significance of which is evidenced by the presence in vivo of soluble proteinases in secretions and antiproteinase antibody in serum of patients with trichomonosis. These proteinases purportedly play a role in host parasitism and immune evasion. OBJECTIVE: It is known that for cysteine proteinases to be functional, they must be activated by disulphide reducing reagents. Whether or not the host vaginal environment has the reducing environment essential for activation of the trichomonad cysteine proteinases is unknown. Our goal, therefore, was to determine whether or not vaginal secretions had sufficient reducing power to activate the trichomonad proteinases. METHODS: 48 vaginal washes (VWs) from patients were assayed for reducing equivalents and a score in dithiothreitol (DTT) reducing equivalents was assigned to each VW. Activation of trichomonad cysteine proteinases was then tested under the range of reducing equivalents detected from VWs. The possible protective effect of hydrogen peroxide, an oxidising agent produced by some Lactobacillus species, on proteinase activity was also determined. RESULTS: Nine of 48 VWs (18.7%) possessed < or = 10 microM DTT reducing equivalents, four VWs (8.3%) had from 20 microM DTT to 40 microM DTT reducing equivalents, and most (50%) were between 10 microM to 15 microM. Overall, the range in VWs was from approximately 10 microM to 40 microM reducing equivalents. Importantly, data suggest differential proteinase activation over this in vivo range of reducing level. Only two T vaginalis cysteine proteinase activities were stimulated at 2.5 microM DTT in contrast with all proteinase activities present at 40 microM DTT, albeit quantitatively diminished compared with the activity at 1 mM DTT, the concentration routinely used in vitro. Finally, hydrogen peroxide reversibly neutralised all trichomonad proteinases. CONCLUSIONS: These results show that the vagina of women has a reducing environment adequate for activation of trichomonad proteinases. The data underscore that the host environment plays a role in the host-parasite interrelation. Finally, hypotheses can now be formulated to help explain resistance and susceptibility to infection commonly reported among women and between men and women with trichomonosis.
Images
PMCID: PMC1195862  PMID: 9389953
13.  (−)-Epigallocatechin Gallate, A Major Constituent of Green Tea, Poisons Human Type II Topoisomerases† 
Chemical research in toxicology  2008;21(4):936-943.
(−)-Epigallocatechin gallate (EGCG) is the most abundant and biologically active polyphenol in green tea, and many of the therapeutic benefits of the beverage have been attributed to this compound. High concentrations of EGCG are cytotoxic and trigger genotoxic events in mammalian cells. Although this catechin affects a number of cellular systems, the genotoxic effects of several bioflavonoid-based dietary polyphenols are believed to be mediated, at least in part, by their actions on topoisomerase II. Therefore, the effects of green tea extract and EGCG on DNA cleavage mediated by human topoisomerase IIα and β were characterized. The extract and EGCG increased levels of DNA strand breaks generated by both enzyme isoforms. However, EGCG acted by a mechanism that was distinctly different from those of genistein, a dietary polyphenol, and etoposide, a widely prescribed anticancer drug. In contrast to these agents, EGCG exhibited all of the characteristics of a redox-dependent topoisomerase II poison that acts by covalently adducting to the enzyme. First, EGCG stimulated DNA scission mediated by both isoforms primarily at sites that were cleaved in the absence of compounds. Second, exposure of EGCG to the reducing agent dithiothreitol (DTT) prior to its addition to DNA cleavage assays abrogated the effects of the catechin on DNA scission. Third, once EGCG stimulated topoisomerase II-mediated DNA cleavage, exposure to DTT did not effect levels of DNA strand breaks. Finally, EGCG inhibited the DNA cleavage activities of topoisomerase IIα and β when incubated with either enzyme prior to the addition of DNA. Taken together, these results provide strong evidence that EGCG is a redox-dependent topoisomerase II poison, and utilizes a mechanism similar to that of 1,4-benzoquinone.
doi:10.1021/tx700434v
PMCID: PMC2893035  PMID: 18293940
14.  Cdc25B Dual-Specificity Phosphatase Inhibitors Identified in a High-Throughput Screen of the NIH Compound Library 
Abstract
The University of Pittsburgh Molecular Library Screening Center (Pittsburgh, PA) conducted a screen with the National Institutes of Health compound library for inhibitors of in vitro cell division cycle 25 protein (Cdc25) B activity during the pilot phase of the Molecular Library Screening Center Network. Seventy-nine (0.12%) of the 65,239 compounds screened at 10 μM met the active criterion of ≥50% inhibition of Cdc25B activity, and 25 (31.6%) of these were confirmed as Cdc25B inhibitors with 50% inhibitory concentration (IC50) values <50 μM. Thirteen of the Cdc25B inhibitors were represented by singleton chemical structures, and 12 were divided among four clusters of related structures. Thirteen (52%) of the Cdc25B inhibitor hits were quinone-based structures. The Cdc25B inhibitors were further characterized in a series of in vitro secondary assays to confirm their activity, to determine their phosphatase selectivity against two other dual-specificity phosphatases, mitogen-activated protein kinase phosphatase (MKP)-1 and MKP-3, and to examine if the mechanism of Cdc25B inhibition involved oxidation and inactivation. Nine Cdc25B inhibitors did not appear to affect Cdc25B through a mechanism involving oxidation because they did not generate detectable amounts of H2O2 in the presence of dithiothreitol, and their Cdc25B IC50 values were not significantly affected by exchanging the dithiothreitol for β-mercaptoethanol or reduced glutathione or by adding catalase to the assay. Six of the nonoxidative hits were selective for Cdc25B inhibition versus MKP-1 and MKP-3, but only the two bisfuran-containing hits, PubChem substance identifiers 4258795 and 4260465, significantly inhibited the growth of human MBA-MD-435 breast and PC-3 prostate cancer cell lines. To confirm the structure and biological activity of 4260465, the compound was resynthesized along with two analogs. Neither of the substitutions to the two analogs was tolerated, and only the resynthesized hit 26683752 inhibited Cdc25B activity in vitro (IC50 = 13.83 ± 1.0 μM) and significantly inhibited the growth of the MBA-MD-435 breast and PC-3 prostate cancer cell lines (IC50 = 20.16 ± 2.0 μM and 24.87 ± 2.25 μM, respectively). The two bis-furan-containing hits identified in the screen represent novel nonoxidative Cdc25B inhibitor chemotypes that block tumor cell proliferation. The availability of non-redox active Cdc25B inhibitors should provide valuable tools to explore the inhibition of the Cdc25 phosphatases as potential mono- or combination therapies for cancer.
doi:10.1089/adt.2008.186
PMCID: PMC2956648  PMID: 19530895
15.  Redox cycling compounds generate H2O2 in HTS buffers containing strong reducing reagents – real hits or promiscuous artifacts? 
Redox cycling compounds (RCCs) generate µM concentrations of hydrogen peroxide (H2O2) in the presence of strong reducing agents, common buffer components used to maintain the catalytic activity and/or folding of target proteins for high throughput screening (HTS) assays. H2O2 generated by RCCs can indirectly inhibit the catalytic activity of proteins by oxidizing accessible cysteine, tryptophan, methionine, histidine or selenocysteine residues, and indeed several important classes of protein targets are susceptible to H2O2-mediated inactivation; protein tyrosine phosphatases, cysteine proteases, and metalloenzymes. The main sources of H2O2 in cells are the Nox enzyme/SOD systems, peroxisome metabolism, and the autoxidation of reactive chemicals by enzyme mediated redox cycling at both the microsomal and mitochondrial sites of electron transport. Given the role of H2O2 as a second messenger involved in the regulation of many signaling pathways it is hardly surprising that compounds which can generate intracellular H2O2 by enzyme mediated redox cycling would have pleiotropic effects. RCCs can therefore have serious negative consequences for the probe and/or lead generation process: primary HTS assay hit rates may be inflated by RCC false positives; critical resources will be diverted to develop and implement follow up assays to distinguish RCCs from real hits; and screening databases will become annotated with the promiscuous activity of RCCs. In an attempt to mitigate the serious impact of RCCs on probe and lead generation, two groups have independently developed assays to indentify RCCs.
doi:10.1016/j.cbpa.2010.10.022
PMCID: PMC3040250  PMID: 21075044
16.  Intracellular Redox State Alters N-Methyl D-Aspartate Receptor Response during Aging through Ca2+/Calmodulin-Dependent Protein Kinase II 
The contribution of the N-methyl D-aspartate receptors (NMDARs) to synaptic plasticity declines during aging and the decline is thought to contribute to memory deficits. Here, we demonstrate that an age-related shift in intracellular redox state contributes to the decline in NMDAR responses through Ca2+/calmodulin-dependent protein kinase II (CaMKII). The oxidizing agent xanthine/xanthine oxidase (X/XO) decreased the NMDAR mediated synaptic responses at hippocampal CA3-CA1 synapses in slices from young (3–8 mo), but not aged (20–25 mo) rats. Conversely, the reducing agent dithiothreitol (DTT) selectively enhanced NMDAR response to a greater extent in aged hippocampal slices. The enhancement of NMDAR responses facilitated induction of long-term potentiation (LTP) in aged but not young animals. The DTT-mediated growth in the NMDAR response was not observed for the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) mediated synaptic responses. A similar increase was observed by intracellular application of the membrane impermeable reducing agent, L-glutathione (L-GSH), through the intracellular recording pipette, indicating the increased NMDAR response was dependent on intracellular redox state. DTT enhancement of the NMDAR response was dependent on CaMKII activity and was blocked by the CaMKII inhibitor – myristoylated autocamtide-2 related inhibitory peptide (myr-AIP), but not by inhibition of the activity of protein phosphatases - PP1 and calcineurin (CaN/PP2B) or protein kinase C. CaMKII activity assays established that DTT increased CaMKII activity in CA1 cytosolic extracts in aged but not in young animals. These findings indicate a link between oxidation of CaMKII during aging, a decline in NMDAR responses, and altered synaptic plasticity.
doi:10.1523/JNEUROSCI.5485-09.2010
PMCID: PMC2853968  PMID: 20130200
NMDA receptor; aging; hippocampus; ROS; oxidative stress; CaMKII; long-term potentiation
17.  Effect of sputum processing with dithiothreitol on the detection of inflammatory mediators in chronic bronchitis and bronchiectasis 
Thorax  2002;57(8):667-671.
Background: Sputum analysis is used increasingly to assess airway inflammation in patients with chronic obstructive pulmonary disease, including those with chronic bronchitis and bronchiectasis. However, it is not known whether dithiothreitol (DTT), a reducing mucolytic agent regularly used to homogenise sputum, affects the detection of inflammatory mediators in the sputum soluble phase from such patients.
Methods: Thirty two spontaneous sputum samples were collected from 13 patients with chronic bronchitis and 17 with bronchiectasis. An aliquot from each sample was treated with either freshly prepared 0.1% DTT plus normal saline (NaCl) or NaCl alone, then ultracentrifuged to obtain the sputum sol phase. Interleukin (IL)-1ß, IL-6, IL-8, leukotriene B4 (LTB4), secretory leukoprotease inhibitor (SLPI), alpha-1-antitrypsin (α1-AT), and tumour necrosis factor alpha (TNFα) were measured by ELISA, and neutrophil elastase (NE) and myeloperoxidase (MPO) by chromogenic substrate assay. The effect of DTT on the detection of assay standards was also determined.
Results: Median levels of IL-1ß, IL-6, IL-8, SLPI, and NE were similar in the DTT and NaCl treated samples. There was a significant reduction in median (IQR) levels of detectable TNFα (0.07 (0.00–0.47) pM v 0.90 (0.06–6.98) pM, p<0.001), LTB4 (1.67 (1.31–2.64) nM v 2.29 (0.95–4.22) nM, p<0.05) and MPO (0.00 (0.00–0.00) mg/l v 4.48 (0.00–33.66) mg/l, p<0.001) and a small increase in the median α1-AT concentration (0.05 (0.03–0.08) nM v 0.03 (0.02–0.08) nM, p<0.01) in the DTT treated samples. DTT had no effect on the assay standards for IL-1ß, IL-8 or TNFα, but at higher concentrations it did affect IL-6, SLPI, NE, and LTB4 standards (43%, 70%, 76% and 643% of control value for top standard, respectively) and at all concentrations DTT completely abolished MPO activity.
Conclusions: Sputum processing with DTT significantly reduces the detectable concentration of TNFα, LTB4 and MPO, and produces a small but significant increase in median α1-AT levels. To avoid this problem we recommend that an untreated aliquot of sputum be retained for cytokine analysis, unless the assay has been specifically validated.
doi:10.1136/thorax.57.8.667
PMCID: PMC1746392  PMID: 12149524
18.  Identification and Characterization of 3-Substituted Pyrazolyl Esters as Alternate Substrates for Cathepsin B. The Confounding Effects of DTT and Cysteine in Biological Assays 
Substituted pyrazole esters were identified as hits in a high throughput screen (HTS) of the NIH Molecular Libraries Small Molecule Repository (MLSMR) to identify inhibitors of the enzyme cathepsin B. Members of this class, along with functional group analogs, were synthesized in an effort to define the structural requirements for activity. Analog characterization was hampered by the need to include a reducing agent such as dithiothreitol (DTT) or cysteine in the assay, highlighting the caution required in interpreting biological data gathered in the presence of such nucleophiles. Despite the confounding effects of DTT and cysteine, our studies demonstrate that the pyrazole 1 acts as alternate substrate for cathepsin B, rather than as an inhibitor.
doi:10.1016/j.bmcl.2007.06.091
PMCID: PMC2041802  PMID: 17656088
19.  New colorimetric screening assays for the directed evolution of fungal laccases to improve the conversion of plant biomass 
BMC Biotechnology  2013;13:90.
Background
Fungal laccases are multicopper oxidases with huge applicability in different sectors. Here, we describe the development of a set of high-throughput colorimetric assays for screening laccase libraries in directed evolution studies.
Results
Firstly, we designed three colorimetric assays based on the oxidation of sinapic acid, acetosyringone and syringaldehyde with λmax of 512, 520 and 370 nm, respectively. These syringyl-type phenolic compounds are released during the degradation of lignocellulose and can act as laccase redox mediators. The oxidation of the three compounds by low and high-redox potential laccases evolved in Saccharomyces cerevisiae produced quantifiable and linear responses, with detection limits around 1 mU/mL and CV values below 16%. The phenolic substrates were also suitable for pre-screening mutant libraries on solid phase format. Intense colored-halos were developed around the yeast colonies secreting laccase. Furthermore, the oxidation of violuric acid to its iminoxyl radical (λmax of 515 nm and CV below 15%) was devised as reporter assay for laccase redox potential during the screening of mutant libraries from high-redox potential laccases. Finally, we developed three dye-decolorizing assays based on the enzymatic oxidation of Methyl Orange (470 nm), Evans Blue (605 nm) and Remazol Brilliant Blue (640 nm) giving up to 40% decolorization yields and CV values below 18%. The assays were reliable for direct measurement of laccase activity or to indirectly explore the oxidation of mediators that do not render colored products (but promote dye decolorization). Every single assay reported in this work was tested by exploring mutant libraries created by error prone PCR of fungal laccases secreted by yeast.
Conclusions
The high-throughput screening methods reported in this work could be useful for engineering laccases for different purposes. The assays based on the oxidation of syringyl-compounds might be valuable tools for tailoring laccases precisely enhanced to aid biomass conversion processes. The violuric assay might be useful to preserve the redox potential of laccase whilst evolving towards new functions. The dye-decolorizing assays are useful for engineering ad hoc laccases for detoxification of textile wastewaters, or as indirect assays to explore laccase activity on other natural mediators.
doi:10.1186/1472-6750-13-90
PMCID: PMC4015961  PMID: 24159930
High-throughput screening; Laccase; Lignocellulose; S-type phenolic mediators; Dyes; Violuric acid
20.  Mechanochemistry: One Bond at a Time 
ACS nano  2009;3(7):1628-1645.
Single-molecule force clamp spectroscopy offers a novel platform for mechanically denaturing proteins by applying a constant force to a polyprotein. A powerful emerging application of the technique is that, by introducing a disulfide bond in each protein module, the chemical kinetics of disulfide bond cleavage under different stretching forces can be probed at the single-bond level. Even at forces much lower than that can rupture the chemical bond, the breaking of the S-S bond at the presence of various chemical reducing agents is significantly accelerated. Our previous work demonstrated that the rate of thiol/disulfide exchange reaction is force-dependent, and well described by an Arrhenius term of the form: r = A(exp((FΔxr-Ea)/kBT)[nucleophile]). From Arrhenius fits to the force dependency of the reduction rate we measured the bond elongation parameter, Δxr, along the reaction coordinate to the transition state of the SN2 reaction cleaved by different nucleophiles and enzymes, never before observed by any other technique. For S-S cleavage by various reducing agents, obtaining the Δxr value can help depicting the energy landscapes and elucidating the mechanisms of the reactions at the single-molecule level. Small nucleophiles, such as 1, 4-DL-dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP) and L-cysteine, react with the S-S bond with monotonically increasing rates under the applied force; while thioredoxin enzymes exhibit both stretching-favored and —resistant reaction-rate regimes. These measurements demonstrate the power of single-molecule force clamp spectroscopy approach in providing unprecedented access to chemical reactions.
doi:10.1021/nn900294n
PMCID: PMC2891658  PMID: 19572737
single-molecule force spectroscopy; atomic force microscopy (AFM); disulfide bond; protein; force-clamp spectroscopy; thioredoxin; bimolecular nucleophilic substitution (SN2)
21.  Redox-Based Inactivation of Cysteine Cathepsins by Compounds Containing the 4-Aminophenol Moiety 
PLoS ONE  2011;6(11):e27197.
Background
Redox cycling compounds have been reported to cause false positive inhibition of proteases in drug discovery studies. This kind of false positives can lead to unusually high hit rates in high-throughput screening campaigns and require further analysis to distinguish true from false positive hits. Such follow-up studies are both time and resource consuming.
Methods and Findings
In this study we show that 5-aminoquinoline-8-ol is a time-dependent inactivator of cathepsin B with a kinact/KI of 36.7±13.6 M−1s−1 using enzyme kinetics. 5-Aminoquinoline-8-ol inhibited cathepsins H, L and B in the same concentration range, implying a non-specific mechanism of inhibition. Further analogues, 4-aminonaphthalene-1-ol and 4-aminophenol, also displayed time-dependent inhibition of cathepsin B with kinact/KI values of 406.4±10.8 and 36.5±1.3 M−1s−1. No inactivation occurred in the absence of either the amino or the hydroxyl group, suggesting that the 4-aminophenol moiety is a prerequisite for enzyme inactivation. Induction of redox oxygen species (ROS) by 4-aminophenols in various redox environments was determined by the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate. Addition of catalase to the assay buffer significantly abrogated the ROS signal, indicating that H2O2 is a component of the ROS induced by 4-aminophenols. Furthermore, using mass spectrometry, active site probe DCG-04 and isoelectric focusing we show that redox inactivation of cysteine cathepsins by 5-aminoquinoline-8-ol is active site directed and leads to the formation of sulfinic acid.
Conclusions
In this study we report that compounds containing the 4-aminophenol moiety inactivate cysteine cathepsins through a redox-based mechanism and are thus likely to cause false positive hits in the screening assays for cysteine proteases.
doi:10.1371/journal.pone.0027197
PMCID: PMC3208577  PMID: 22073285
22.  Erv2p: characterization of the redox behavior of a yeast sulfhydryl oxidase† 
Biochemistry  2007;46(11):3246-3254.
The FAD prosthetic group of the ERV/ALR family of sulfhydryl oxidases is housed at the mouth of a 4-helix bundle and communicates with a pair of juxtaposed cysteine residues that form the proximal redox active disulfide. Most of these enzymes have one or more additional distal disulfide redox centers that facilitate the transfer of reducing equivalents from the dithiol substrates of these oxidases to the isoalloxazine ring where the reaction with molecular oxygen occurs. The present study examines yeast Erv2p and compares the redox behavior of this ER luminal protein with the augmenter of liver regeneration, a sulfhydryl oxidase of the mitochondrial intermembrane space, and a larger protein containing the ERV/ALR domain, quiescin-sulfhydryl oxidase (QSOX). Dithionite and photochemical reductions of Erv2p show full reduction of the flavin cofactor after the addition of 4-electrons with a mid-point potential of -200 mV at pH 7.5. A charge-transfer complex between a proximal thiolate and the oxidized flavin is not observed in Erv2p consistent with a distribution of reducing equivalents over the flavin and distal disulfide redox centers. Upon coordination with Zn2+, full reduction of Erv2p requires 6-electrons. Zn2+ also strongly inhibits Erv2p when assayed using tris(2-carboxyethyl)phosphine (TCEP) as the reducing substrate of the oxidase. In contrast to QSOX, Erv2p shows a comparatively low turnover with a range of small thiol substrates, with reduced Escherichia coli thioredoxin and with unfolded proteins. Rapid reaction studies confirm that reduction of the flavin center of Erv2p is rate-limiting during turnover with molecular oxygen. This comparison of the redox properties between members of the ERV/ALR family of sulfhydryl oxidases provides insights into their likely roles in oxidative protein folding.
doi:10.1021/bi602499t
PMCID: PMC2573868  PMID: 17298084
23.  Microfluidic Electrochemical Sensor for On-line Monitoring of Aerosol Oxidative Activity 
Journal of the American Chemical Society  2012;134(25):10562-10568.
Particulate matter (PM) air pollution has a significant impact on human morbidity and mortality; however, the mechanisms of PM-induced toxicity are poorly defined. A leading hypothesis states that airborne PM induces harm by generating reactive oxygen species (ROS) in and around human tissues, leading to oxidative stress. We report here, a system employing a microfluidic electrochemical sensor coupled directly to a Particle-into-Liquid-Sampler (PILS) system to measure aerosol oxidative activity in an on-line format. The oxidative activity measurement is based on the dithiothreitol assay (DTT assay) where after oxidized by PM, the remaining reduced DTT was analyzed by the microfluidic sensor. The sensor consists of an array of working, reference, and auxiliary electrodes fabricated in a poly(dimethylsiloxane) (PDMS)-based microfluidic device. Cobalt (II) phthalocyanine (CoPC)-modified carbon paste was used as the working electrode material allowing selective detection of reduced DTT. The electrochemical sensor was validated off-line against the traditional DTT assay using filter samples taken from urban environments and biomass burning events. After off-line characterization, the sensor was coupled to a PILS to enable on-line sampling/analysis of aerosol oxidative activity. Urban dust and industrial incinerator ash samples were aerosolized in an aerosol chamber and analyzed for their oxidative activity. The on-line sensor reported DTT consumption rates (oxidative activity) in good correlation with aerosol concentration (R2 from 0.86–.97) with a time-resolution of approximately 3 minutes.
doi:10.1021/ja3031104
PMCID: PMC3397383  PMID: 22651886
24.  A fast, sensitive and easy colorimetric assay for chitinase and cellulase activity detection 
Background
Most of the current colorimetric methods for detection of chitinase or cellulase activities on the insoluble natural polymers chitin and cellulose depend on a chemical redox reaction. The reaction involves the reducing ends of the hydrolytic products. The Schales’ procedure and the 3,5-dinitrosalicylic acid (DNS) method are two examples that are commonly used. However, these methods lack sensitivity and present practical difficulties of usage in high-throughput screening assays as they require boiling or heating steps for color development.
Results
We report a novel method for colorimetric detection of chitinase and cellulase activity. The assay is based on the use of two oxidases: wild-type chito-oligosaccharide oxidase, ChitO, and a mutant thereof, ChitO-Q268R. ChitO was used for chitinase, while ChitO-Q268R was used for cellulase activity detection. These oxidases release hydrogen peroxide upon the oxidation of chitinase- or cellulase-produced hydrolytic products. The hydrogen peroxide produced can be monitored using a second enzyme, horseradish peroxidase (HRP), and a chromogenic peroxidase substrate. The developed ChitO-based assay can detect chitinase activity as low as 10 μU within 15 minutes of assay time. Similarly, cellulase activity can be detected in the range of 6 to 375 mU. A linear response was observed when applying the ChitO-based assay for detecting individual chito-oligosaccharides and cello-oligosaccharides. The detection limits for these compounds ranged from 5 to 25 μM. In contrast to the other commonly used methods, the Schales’ procedure and the DNS method, no boiling or heating is needed in the ChitO-based assays. The method was also evaluated for detecting hydrolytic activity on biomass-derived substrates, that is, wheat straw as a source of cellulose and shrimp shells as a source of chitin.
Conclusion
The ChitO-based assay has clear advantages for the detection of chitinase and cellulase activity over the conventional Schales’ procedure and DNS method. The detection limit is lower and there is no requirement for harsh conditions for the development of the signal. The assay also involves fewer and easier handling steps. There is no need for boiling to develop the color and results are available within 15 minutes. These aforementioned features render this newly developed assay method highly suitable for applications in biorefinery-related research.
doi:10.1186/1754-6834-7-37
PMCID: PMC3975300  PMID: 24612932
Chitinase; Cellulase; Chito-oligosaccharide oxidase; High-throughput screening; Chitin; DNS; Schales’ procedure; Cellulose; Colorimetric assay
25.  Ultrafine particulate pollutants induce oxidative stress and mitochondrial damage. 
Environmental Health Perspectives  2003;111(4):455-460.
The objectives of this study were to determine whether differences in the size and composition of coarse (2.5-10 micro m), fine (< 2.5 microm), and ultrafine (< 0.1 microm) particulate matter (PM) are related to their uptake in macrophages and epithelial cells and their ability to induce oxidative stress. The premise for this study is the increasing awareness that various PM components induce pulmonary inflammation through the generation of oxidative stress. Coarse, fine, and ultrafine particles (UFPs) were collected by ambient particle concentrators in the Los Angeles basin in California and used to study their chemical composition in parallel with assays for generation of reactive oxygen species (ROS) and ability to induce oxidative stress in macrophages and epithelial cells. UFPs were most potent toward inducing cellular heme oxygenase-1 (HO-1) expression and depleting intracellular glutathione. HO-1 expression, a sensitive marker for oxidative stress, is directly correlated with the high organic carbon and polycyclic aromatic hydrocarbon (PAH) content of UFPs. The dithiothreitol (DTT) assay, a quantitative measure of in vitro ROS formation, was correlated with PAH content and HO-1 expression. UFPs also had the highest ROS activity in the DTT assay. Because the small size of UFPs allows better tissue penetration, we used electron microscopy to study subcellular localization. UFPs and, to a lesser extent, fine particles, localize in mitochondria, where they induce major structural damage. This may contribute to oxidative stress. Our studies demonstrate that the increased biological potency of UFPs is related to the content of redox cycling organic chemicals and their ability to damage mitochondria.
PMCID: PMC1241427  PMID: 12676598

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