Pro-fluorescent probe with morpholine moiety and its reactivity towards selected biological oxidants.

Biological oxidants participate in many processes in the human body. Their excessive production causes organelle damage, which may result in the accumulation of cytotoxic mediators and cell degradation and may manifest itself in various diseases. Peroxynitrite (ONOO- ), hypochlorous acid (HOCl), hydrogen peroxide (H2 O2 ), and peroxymonocarbonate (HOOCO2 - ) are important oxidants in biology, toxicology, and various pathologies. Derivatives of coumarin, containing an oxidant-sensitive boronate group, have been recently developed for the fluorescent detection of inflammatory oxidants. Here, we report the synthesis and characterization of 4-[2-(morpholin-4-yl)-2-oxoethyl]-2-oxo-2H-chromen-7-yl boronic acid (MpC-BA) as a fluorescent probe for the detection of oxidants, with better solubility in water, high stability and fast response time toward peroxynitrite and hypochlorous acid. The effectiveness of the MpC-BA probe for the detection of peroxynitrite was measured by adding bolus ONOO- or using the co-generating superoxide and nitrogen oxide system. MpC-BA is oxidized by ONOO- to 7-hydroxy-4-[2-(morpholin-4-yl)-2-oxoethyl]-2H-chromen-2-one (MpC-OH). However, peroxynitrite-specific product (MpC-H) is formed in the minor reaction pathway. MpC-OH is also yielded in the reaction of MpC-BA with HOCl, and the subsequent formation of a chlorinated MpC-OH gives a specific product for HOCl (MpC-OHCl). H2 O2 slowly oxidizes MpC-BA. However, the addition of NaHCO3 increased the MpC-OH formation rate. We conclude that MpC-BA is potentially an improved fluorescent probe detecting peroxynitrite and hypochlorite in biological settings. Complementation of the fluorescence measurements by HPLC-based identification of chlorinated and reduced coumarin(s) will help identify the oxidants detected.

Evaluation of a novel pyridinium cation-linked styryl-based boronate probe for the detection of selected inflammation-related oxidants.

Reactive oxygen and nitrogen species (RONS) are a range of chemical individuals produced by living cells that contribute to the proper functioning of organisms. Cells under oxidative and nitrative stress show excessive production of RONS (including hydrogen peroxide, H2O2, hypochlorous acid, HOCl, and peroxynitrite, ONOO-) which may result in a damage proteins, lipids, and genetic material. Thus, the development of probes for in vivo detection of such oxidants is an active area of research, focusing on molecular redox sensors, including boronate-caged fluorophores. Here, we report a boronate-based styryl probe with a cationic pyridinium moiety (BANEP+) for the fluorescent detection of selected biological oxidants in vitro and in vivo. We compare the chemical reactivity of the BANEP+ probe toward H2O2, HOCl, and ONOO- and examine the influence of the major intracellular non-enzymatic antioxidant molecule, glutathione (GSH). We demonstrate that, at the physiologically relevant GSH concentration, the BANEP+ probe is efficiently oxidized by peroxynitrite, forming its phenolic derivative HNEP+. GSH does not affect the fluorescence properties of the BANEP+ and HNEP+ dyes. Finally, we report the identification of a novel type of molecular marker, with the boronate moiety replaced by the iodine atom, formed from the probe in the presence of HOCl and iodide anion. We conclude that the reported chemical reactivity and structural features of the BANEP+ probe may be a basis for the development of new red fluorescent probes for in vitro and in vivo detection of ONOO-.

Sensitive Detection of Various Forms of Hydrogen Sulfide via Highly Selective Naphthalimide-Based Fluorescent Probe.

Hydrogen sulfide (H2S) is an important gasotransmitter, but only a few methods are available for real-time detection. Fluorescent probes are attractive tools for biological applications because of their high sensitivity, convenience, rapid implementation, noninvasive monitoring capability, and simplicity in fluorescent imaging of living cells and tissues. Herein, we report on a pro-fluorescent probe, NAP-Py-N3 based on naphthalimide derivative, which was found to show high selectivity toward H2S over various other analytes, including biothiols, making it feasible to detect H2S. After reaction with H2S, this probe showed rapid and significant turn-on green fluorescent enhancement at 553 nm (about 54-fold, k2 = 9.62 M-1s-1), high sensitivity (LOD: 15.5 nM), significant Stokes shift (118 nm), and it was found that the fluorescence quantum yield of fluorescence product can reach 0.36. Moreover, the probe has also been successfully applied to detect the gaseous H2S and to confirm the presence of H2S released from modern organic donors, which in recent years have been commonly used to investigate the role of H2S in biological systems. All the results indicate that this probe is excellent and highly valuable.

Comparison of Reactive Sites in 2(1H)-Quinolone Derivatives for the Detection of Biologically Important Sulfur Compounds.

Novel fluorescent probes based on 2(1H)-quinolone skeleton containing a malonate group (Q1-Q3) were synthesized and proposed for biothiols detection. Their chemical reactivity toward thiols was compared to the reactivity of derivative having a dicyanovinyl group (Q4) as a reactive site. The detailed photophysical properties of these compounds were assessed through the determination of absorption and fluorescence spectra, fluorescence quantum yield, and fluorescence lifetime. In the presence of biothiols, an increase in the fluorescence intensity of compounds Q1-Q3 and a hypsochromic shift in their emission bands were observed. In contrast, the compound with the dicyanovinyl group (Q4) in the presence of biothiols and cyanide ion showed the quenching of fluorescence, while a fluorescence "turn on" effect was observed toward reactive sulfur species.

A New and Fast-Response Fluorescent Probe for Monitoring Hypochlorous Acid Derived from Myeloperoxidase.

Hypochlorous acid (HOCl) has been implicated in numerous pathologies associated with an inflammatory component, but its selective and sensitive detection in biological settings remains a challenge. In this report, imaging of HOCl was realized with a thiomorpholine-based probe as derivative of nitrobenzothiadiazole (NBD-S-TM). The fluorescence is based on photoinduced electron transfer by using nitrobenzothiadiazole core as a donor and thiomorpholine substituent as an acceptor. NBD-S-TM showed high sensitivity and a fast response to HOCl k = (2.6 ± 0.2) × 107 M-1s-1 with a 1:1 stoichiometry. The detection limit for HOCl was determined to be 60 nM. Furthermore, the desirable features of NBD-S-TM for the detection of HOCl in aqueous solutions, such as its reliability at physiological pH, rapid fluorescence response, and biocompatibility, enabled its application in the detection of HOCl in myeloperoxidase enzymatic system. Moreover, NBD-S-TM exhibited excellent selectivity and sensitivity for HOCl over other biologically relevant species, such as hydrogen peroxide and peroxynitrite. The fluorescent S-oxidized product (NBD-S-TSO) is only formed in the presence of HOCl. Probing with NBD-S-TM may be helpful to further the development of high throughput screening assays to monitor the activity of myeloperoxidase.

A thiomorpholine-based fluorescent probe for the far-red hypochlorous acid monitoring.

A simple thiomorpholine-based fluorescent probe was designed and synthesized by combining thiomorpholine (TM) and nitrobenzenoselenadiazoles fluorophore (NBD-Se). The thiomorpholine group quenches the fluorescence of NBD-Se efficiently through the photoinduced electron transfer (PET) effect. Hypochlorous acid (HOCl) oxidizes the NBD-Se-TM probe to its fluorescent S-oxide (NBD-Se-TSO) with a 1:1 stoichiometry. The desirable features of NBD-Se-TM for detecting HOCl in aqueous solutions, such as its high sensitivity and selectivity, reliability at physiological pH, and rapid fluorescence response, enabled its application in the detection of HOCl produced by myeloperoxidase. The results proved that NBD-Se-TM is a promising fluorescent probe that can be used in screening assays for MPO inhibitors. Its high reaction rate constant with HOCl (2k = 2.0 × 107M-1s-1) indicates the possibility of application in more complex biological systems.

HPLC Study of Product Formed in the Reaction of NBD-Derived Fluorescent Probe with Hydrogen Sulfide, Cysteine, N-acetylcysteine, and Glutathione.

Hydrogen sulfide (H2S) and its bioderivatives analogs, such as L-cysteine (L-Cys) and glutathione (GSH), are ubiquitous biological thiols in the physiological and pathological processes of living systems. Their aberrant concentration levels are associated with many diseases. Although several NBD-based fluorescence probes have been developed to detect biological thiols, the HPLC-detection of H2S, GSH, L-Cys, and N-acetylcysteine-specific products has not been described. Herein, a novel NBD-derived pro-coumarin probe has been synthesized and used to develop a new strategy for the triple mode detection of H2S and such thiols as GSH, L-Cys, and NAC. Hydrogen sulfide and those biothiols at physiological pH release fluorescent coumarin from the probe and cause a significant fluorescence enhancement at 473 nm. The appropriate NBD-derived product for H2S, L-Cys, GSH, and NAC has a different color and retention time that allows distinguishing these biological thiols meaning the probe has a great possibility in the biological application. Fluorescent imaging combined with colorimetric and HPLC detection of H2S/biothiol-specific product(s) brings a potential tool for confirming the presence of biological thiols and determining concentrations in various aqueous biological samples.

Synthesis and Spectroscopic Characterization of Selected Phenothiazines and Phenazines Rationalized Based on DFT Calculation.

Two unique structures were isolated from the phosphorylation reaction of 10H-phenothiazine. The 5,5-dimethyl-2-(10H-phenothiazin-10-yl)-1,3,2-dioxaphosphinane 2-oxide (2a) illustrates the product of N-phosphorylation of phenothiazine. Moreover, a potential product of 2a instability, a thiophosphoric acid 2b, was successfully isolated and structurally characterized. Molecule 2a, similarly to sulfoxide derivative 3, possesses interesting phosphorescence properties due to the presence of d-pπ bonds. The X-ray, NMR, and DFT computational studies indicate that compound 2a exhibits an anomeric effect. Additionally, the syntheses of selected symmetrical and unsymmetrical pyridine-embedded phenazines were elaborated. To compare the influence of phosphorus and sulfur atoms on the structural characteristics of 10H-phenothiazine derivatives, the high-quality crystals of (4a,12a-dihydro-12H-benzo[5,6][1,4]thiazino[2,3-b]quinoxalin-12-yl)(phenyl)methanone (1) and selected phenazines 5,12-diisopropyl-3,10-dimethyldipyrido[3,2-a:3',2'-h]phenazine (5) and 5-isopropyl-N,N,3-trimethylpyrido[3,2-a]phenazin-10-amine (6a) were obtained. The structures of molecules 1, 2a, 2-mercapto-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (2b), 3,7-dinitro-10H-phenothiazine 5-oxide (3), 5 and 6a were determined by single-crystal X-ray diffraction measurements.

Fluorescent probes for monitoring myeloperoxidase-derived hypochlorous acid: a comparative study.

MPO-derived oxidants including HOCl contribute to tissue damage and the initiation and propagation of inflammatory diseases. The search for small molecule inhibitors of myeloperoxidase, as molecular tools and potential drugs, requires the application of high throughput screening assays based on monitoring the activity of myeloperoxidase. In this study, we have compared three classes of fluorescent probes for monitoring myeloperoxidase-derived hypochlorous acid, including boronate-, aminophenyl- and thiol-based fluorogenic probes and we show that all three classes of probes are suitable for this purpose. However, probes based on the coumarin fluorophore turned out to be not reliable indicators of the inhibitors' potency. We have also determined the rate constants of the reaction between HOCl and the probes and they are equal to 1.8 × 104 M-1s-1 for coumarin boronic acid (CBA), 1.1 × 104 M-1s-1 for fluorescein based boronic acid (FLBA), 3.1 × 104 M-1s-1 for 7-(p-aminophenyl)-coumarin (APC), 1.6 × 104 M-1s-1 for 3'-(p-aminophenyl)-fluorescein (APF), and 1 × 107 M-1s-1 for 4-thiomorpholino-7-nitrobenz-2-oxa-1,3-diazole (NBD-TM). The high reaction rate constant of NBD-TM with HOCl makes this probe the most reliable tool to monitor HOCl formation in the presence of compounds showing HOCl-scavenging activity.

Water-soluble cationic boronate probe based on coumarin imidazolium scaffold: Synthesis, characterization, and application to cellular peroxynitrite detection.

Peroxynitrite (ONOO-) has been implicated in numerous pathologies associated with an inflammatory component, but its selective and sensitive detection in biological settings remains a challenge. Here, the development of a new water-soluble and cationic boronate probe based on a coumarin-imidazolium scaffold (CI-Bz-BA) for the fluorescent detection of ONOO- in cells is reported. The chemical reactivity of the CI-Bz-BA probe toward selected oxidants known to react with the boronate moiety was characterized, and the suitability of the probe for the direct detection of ONOO- in cell-free and cellular system is reported. Oxidation of the probe results in the formation of the primary hydroxybenzyl product (CI-Bz-OH), followed by the spontaneous elimination of the quinone methide moiety to produce the secondary phenol (CI-OH), which is accompanied by a red shift in the fluorescence emission band from 405 nm to 481 nm. CI-Bz-BA reacts with ONOO- stoichiometrically with a rate constant of ∼1 × 106 M-1s-1 to form, in addition to the major phenolic product CI-OH, the minor nitrated product CI-Bz-NO2, which is not formed by other oxidants tested or via myeloperoxidase-catalyzed oxidation/nitration. Both CI-OH and CI-Bz-NO2 products were also formed in the presence of cogenerated fluxes of nitric oxide and superoxide radical anion produced during decomposition of a SIN-1 donor. Using RAW 264.7 cells, we demonstrate the ability of the probe to report endogenously produced ONOO-via fluorescence measurements, including plate reader real time monitoring and two-photon fluorescence imaging. Liquid chromatography/mass spectrometry analyses of cell extracts and media confirmed the formation of both CI-OH and CI-Bz-NO2 in macrophages activated to produce ONOO-. We propose the use of a combination of real-time monitoring of probe oxidation using fluorimetry and fluorescence microscopy with liquid chromatography/mass spectrometry-based product identification for rigorous detection and quantitative analyses of ONOO- in biological systems.

Novel Boronate Probe Based on 3-Benzothiazol-2-yl-7-hydroxy-chromen-2-one for the Detection of Peroxynitrite and Hypochlorite.

Derivatives of coumarin, containing oxidant-sensitive boronate group, were recently developed for fluorescent detection of inflammatory oxidants. Here, we report the synthesis and the characterization of 3-(2-benzothiazolyl)-7-coumarin boronic acid pinacol ester (BC-BE) as a fluorescent probe for the detection of peroxynitrite (ONOO-), with high stability and a fast response time. The BC-BE probe hydrolyzes in phosphate buffer to 3-(2-benzothiazolyl)-7-coumarin boronic acid (BC-BA) which is stable in the solution even after a prolonged incubation time (24 h). BC-BA is slowly oxidized by H2O2 to form the phenolic product, 3-benzothiazol-2-yl-7-hydroxy-chromen-2-one (BC-OH). On the other hand, the BC-BA probe reacts rapidly with ONOO-. The ability of the BC-BA probe to detect ONOO- was measured using both authentic ONOO- and the system co-generating steady-state fluxes of O2•- and •NO. BC-BA is oxidized by ONOO- to BC-OH. However, in this reaction 3-benzothiazol-2-yl-chromen-2-one (BC-H) is formed in the minor pathway, as a peroxynitrite-specific product. BC-OH is also formed in the reaction of BC-BA with HOCl, and subsequent reaction of BC-OH with HOCl leads to the formation of a chlorinated phenolic product, which could be used as a specific product for HOCl. We conclude that BC-BA shows potential as an improved fluorescent probe for the detection of peroxynitrite and hypochlorite in biological settings. Complementation of the fluorescence measurements by HPLC-based identification of oxidant-specific products will help to identify the oxidants detected.

Identification of Peroxynitrite by Profiling Oxidation and Nitration Products from Mitochondria-Targeted Arylboronic Acid.

The development of boronic probes enabled reliable detection and quantitative analysis of hydrogen peroxide , other nucleophilic hydroperoxides, hypochlorite , and peroxynitrite . The major product, in which boronate moiety of the probe is replaced by the hydroxyl group, is, however, common for all those oxidants. Here, we describe how ortho-isomer of mitochondria-targeted phenylboronic acid can be used to detect and differentiate peroxynitrite-dependent and independent probe oxidation. This method highlights detection and quantification of both the major, phenolic product and the minor, peroxynitrite-specific cyclic and nitrated products of probe oxidation.

Two-photon fluorescent probe for cellular peroxynitrite: Fluorescence detection, imaging, and identification of peroxynitrite-specific products.

A new naphthalene-based boronate probe, NAB-BE, for the fluorescence-based detection of inflammatory oxidants, including peroxynitrite, hypochlorous acid, and hydrogen peroxide, is reported. The chemical reactivity and fluorescence properties of the probe and the products are described. The major, phenolic oxidation product, NAB-OH, is formed in case of all three oxidants tested. This product shows green fluorescence, with a maximum at 512 nm, and can be excited either at 340 nm or in the near infrared region (745 nm) for two-photon fluorescence imaging. Peroxynitrite is the fastest of the oxidants tested and, in addition to the phenolic product, leads to the formation of a nitrated product, NAB-NO2, which can serve as a fingerprint for peroxynitrite. The probe was applied to detect peroxynitrite in activated macrophages using fluorimetry and two-photon fluorescence microscopy, and both NAB-OH and NAB-NO2 products were detected in cell extracts by liquid chromatography-mass spectrometry. The combined use of fluorometric high-throughput analyses, fluorescence imaging, and liquid chromatography-mass spectrometry-based product identification and quantitation is proposed for most comprehensive and rigorous characterization of oxidants in biological systems.

Boronate-Based Probes for Biological Oxidants: A Novel Class of Molecular Tools for Redox Biology.

Boronate-based molecular probes are emerging as one of the most effective tools for detection and quantitation of peroxynitrite and hydroperoxides. This review discusses the chemical reactivity of boronate compounds in the context of their use for detection of biological oxidants, and presents examples of the practical use of those probes in selected chemical, enzymatic, and biological systems. The particular reactivity of boronates toward nucleophilic oxidants makes them a distinct class of probes for redox biology studies. We focus on the recent progress in the design and application of boronate-based probes in redox studies and perspectives for further developments.

Increased formation of reactive oxygen species during tumor growth: Ex vivo low-temperature EPR and in vivo bioluminescence analyses.

Previous studies have shown that reactive oxygen species (ROS) such as superoxide or hydrogen peroxide generated at low levels can exert a tumor-promoting role via a redox-signaling mechanism. Reports also suggest that both tumorigenesis and tumor growth are associated with enhanced ROS formation. However, whether ROS levels or ROS-derived oxidative marker levels increase during tumor growth remains unknown. In this study, in vivo bioluminescence imaging with a boronate-based pro-luciferin probe was used to assess ROS formation. Additionally, probe-free cryogenic electron paramagnetic resonance was used to quantify a characteristic aconitase [3Fe4S]+ center that arises in the tumor tissue of mouse xenografts from the reaction of the native [4Fe4S]2+ cluster with superoxide. Results indicated that tumor growth is accompanied by increased ROS formation, and revealed differences in oxidant formation in the inner and outer sections of tumor tissue, respectively, demonstrating redox heterogeneity. Studies using luciferin and pro-luciferin probes enabled the assessment of tumor size, ROS formation, and bioenergetic status (e.g., ATP) in luciferase-transfected mice tumor xenografts. Probe-free ex vivo low-temperature electron paramagnetic resonance can also be translated to clinical studies.

Detection and Characterization of Reactive Oxygen and Nitrogen Species in Biological Systems by Monitoring Species-Specific Products.

SIGNIFICANCE: Since the discovery of the superoxide dismutase enzyme, the generation and fate of short-lived oxidizing, nitrosating, nitrating, and halogenating species in biological systems has been of great interest. Despite the significance of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in numerous diseases and intracellular signaling, the rigorous detection of ROS and RNS has remained a challenge. Recent Advances: Chemical characterization of the reactions of selected ROS and RNS with electron paramagnetic resonance (EPR) spin traps and fluorescent probes led to the establishment of species-specific products, which can be used for specific detection of several forms of ROS and RNS in cell-free systems and in cultured cells in vitro and in animals in vivo. Profiling oxidation products from the ROS and RNS probes provides a rigorous method for detection of those species in biological systems. CRITICAL ISSUES: Formation and detection of species-specific products from the probes enables accurate characterization of the oxidative environment in cells. Measurement of the total signal (fluorescence, chemiluminescence, etc.) intensity does not allow for identification of the ROS/RNS formed. It is critical to identify the products formed by using chromatographic or other rigorous techniques. Product analyses should be accompanied by monitoring of the intracellular probe level, another factor controlling the yield of the product(s) formed. FUTURE DIRECTIONS: More work is required to characterize the chemical reactivity of the ROS/RNS probes, and to develop new probes/detection approaches enabling real-time, selective monitoring of the specific products formed from the probes. Antioxid. Redox Signal. 28, 1416-1432.

Recent Developments in the Probes and Assays for Measurement of the Activity of NADPH Oxidases.

NADPH oxidases are a family of enzymes capable of transferring electrons from NADPH to molecular oxygen. A major function of NADPH oxidases is the activation of molecular oxygen into reactive oxygen species. Increased activity of NADPH oxidases has been implicated in various pathologies, including cardiovascular disease, neurological dysfunction, and cancer. Thus, NADPH oxidases have been identified as a viable target for the development of novel therapeutics exhibiting inhibitory effects on NADPH oxidases. Here, we describe the development of new assays for measuring the activity of NADPH oxidases enabling the high-throughput screening for NADPH oxidase inhibitors.

Recent developments in detection of superoxide radical anion and hydrogen peroxide: Opportunities, challenges, and implications in redox signaling.

In this review, some of the recent developments in probes and assay techniques specific for superoxide (O2-) and hydrogen peroxide (H2O2) are discussed. Over the last decade, significant progress has been made in O2- and H2O2 detection due to syntheses of new redox probes, better understanding of their chemistry, and development of specific and sensitive assays. For superoxide detection, hydroethidine (HE) is the most suitable probe, as the product, 2-hydroxyethidium, is specific for O2-. In addition, HE-derived dimeric products are specific for one-electron oxidants. As red-fluorescent ethidium is always formed from HE intracellularly, chromatographic techniques are required for detecting 2-hydroxyethidium. HE analogs, Mito-SOX and hydropropidine, exhibit the same reaction chemistry with O2- and one-electron oxidants. Thus, mitochondrial superoxide can be unequivocally detected using HPLC-based methods and not by fluorescence microscopy. Aromatic boronate-based probes react quantitatively with H2O2, forming a phenolic product. However, peroxynitrite and hypochlorite react more rapidly with boronates, forming the same product. Using ROS-specific probes and HPLC assays, it is possible to screen chemical libraries to discover specific inhibitors of NADPH oxidases. We hope that rigorous detection of O2- and H2O2 in different cellular compartments will improve our understanding of their role in redox signaling.

On the use of peroxy-caged luciferin (PCL-1) probe for bioluminescent detection of inflammatory oxidants in vitro and in vivo - Identification of reaction intermediates and oxidant-specific minor products.

Peroxy-caged luciferin (PCL-1) probe was first used to image hydrogen peroxide in living systems (Van de Bittner et al., 2010 [9]). Recently this probe was shown to react with peroxynitrite more potently than with hydrogen peroxide (Sieracki et al., 2013 [11]) and was suggested to be a more suitable probe for detecting peroxynitrite under in vivo conditions. In this work, we investigated in detail the products formed from the reaction between PCL-1 and hydrogen peroxide, hypochlorite, and peroxynitrite. HPLC analysis showed that hydrogen peroxide reacts slowly with PCL-1, forming luciferin as the only product. Hypochlorite reaction with PCL-1 yielded significantly less luciferin, as hypochlorite oxidized luciferin to form a chlorinated luciferin. Reaction between PCL-1 and peroxynitrite consists of a major and minor pathway. The major pathway results in luciferin and the minor pathway produces a radical-mediated nitrated luciferin. Radical intermediate was characterized by spin trapping. We conclude that monitoring of chlorinated and nitrated products in addition to bioluminescence in vivo will help identify the nature of oxidant responsible for bioluminescence derived from PCL-1.

Mitigation of NADPH Oxidase 2 Activity as a Strategy to Inhibit Peroxynitrite Formation.

Using high throughput screening-compatible assays for superoxide and hydrogen peroxide, we identified potential inhibitors of the NADPH oxidase (Nox2) isoform from a small library of bioactive compounds. By using multiple probes (hydroethidine, hydropropidine, Amplex Red, and coumarin boronate) with well defined redox chemistry that form highly diagnostic marker products upon reaction with superoxide (O2 (̇̄)), hydrogen peroxide (H2O2), and peroxynitrite (ONOO(-)), the number of false positives was greatly decreased. Selected hits for Nox2 were further screened for their ability to inhibit ONOO(-)formation in activated macrophages. A new diagnostic marker product for ONOO(-)is reported. We conclude that the newly developed high throughput screening/reactive oxygen species assays could also be used to identify potential inhibitors of ONOO(-)formed from Nox2-derived O2 (̇̄)and nitric oxide synthase-derived nitric oxide.