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 (O2̇̄), 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 O2̇̄ and nitric oxide synthase-derived nitric oxide.

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 (O 2 . ), hydrogen peroxide (H 2 O 2 ), 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 O 2 . and nitric oxide synthase-derived nitric oxide.
NADPH oxidase (Nox) 4 enzymes (Nox1-5 and Duox1/2) have been proposed as potential therapeutic targets in the treatment of a variety of inflammatory and fibrotic diseases, including cancer (1)(2)(3). Unlike other redox-active enzymes for which generation of reactive oxygen species (ROS) is an "accidental" by-product of their primary catalytic function, the only known function of Nox enzymes is generation of ROS (e.g. O 2 . and H 2 O 2 ) ( Fig. 1) (4,5). A major impediment to advancing Nox research is the paucity of selective inhibitors of Nox isoforms, including Nox1 and -2 (6). This is due in part to the lack of reliable and high throughput-compatible detection probes and assays that are specific for O 2 . and H 2 O 2 . With the recent discovery of new probes with well defined redox chemistry that form highly diagnostic marker products upon reaction with ROS/RNS both under in vitro and in vivo conditions and high throughput global profiling assays (Table 1) (7), we can now screen a small library of bioactive compounds. One of the objectives of this study is to identify small molecule inhibitors of the Nox2 isoform using the high throughput screening (HTS)/ROS-based assay(s) that largely eliminate false positives. Previously, we reported the power of our newly developed HTS/ROS assays in identifying true "hits" for Nox2 inhibition and eliminating false positives at the outset (8). Typically, the chemiluminescent probe, L-012, has been used in Nox assay (9). Comparison between L-012 assay and our HTS/ROS assay revealed that L-012 increased false positives by at least a factor of 4 and that this increase is due to inhibition of peroxidase enzyme used in the L-012/Nox assay (10). A related objective of this study is to also identify new small molecule inhibitors of RNS (e.g. peroxynitrite). Peroxynitrite (ONOO Ϫ ) is a potent oxidizing and nitrating species formed from a diffusion-controlled reaction between O 2 . and nitric oxide ( ⅐ NO) ( Fig. 1) (11,12) and has been implicated in various neurodegenerative and cardiovascular diseases (13)(14)(15). Although ongoing efforts focus on antinitration strategies mostly through direct scavenging of ONOO Ϫ and/or related species (16), a better approach is to suppress the sources of generation of O 2 . (Nox) and/or inhibition of nitric-oxide synthase, particularly inducible NOS ( Fig.  1) (17,18). In this study, we identified several candidate Nox2 inhibitors through HTS-based ROS assays from testing a library of Ͼ2,000 bioactive compounds at Broad Institute. Selected hits for Nox2 inhibition were further tested for inhibition of ONOO Ϫ formation in activated macrophages. Results suggest that the HTS/ROS strategy developed herein could be used to

Structures of ROS/RNS-specific probes, their reaction products and detection methods
identify Nox2 inhibitors that inhibit ONOO Ϫ formation. In this study we also discovered a new diagnostic marker product for specific detection and quantitation of peroxynitrite in biological systems. One of the objectives of this study is to also use these candidate inhibitors of Nox2 as potential inhibitors of ONOO Ϫ generated via Nox2 intermediacy.
For organic synthesis, THF was distilled under dry argon atmosphere in the presence of sodium and benzophenone. All reagents were used as received without further purification. The reactions were monitored by TLC on silica gel (Merck 60F254). Crude materials were purified by flash chromatography on Merck silica gel 60 (0.040 -0.063 mm). 31  HTS-compatible Cellular Models of Nox2-Human promyelocytic leukemia HL60 cells (Sigma) differentiated into neutrophil-like cells by all-trans-retinoic acid were used as the HTS-compatible source of Nox2 model system for screening Nox2 inhibitors (25,26). HL60 cells were incubated with alltrans-retinoic acid (1 M) for 4 -5 days for converting nondifferentiated cells into differentiated cells. Nox2 activation was achieved by treating differentiated HL60 (dHL60) cells with phorbol myristate acetate (PMA, 1 M) (8).
High Throughput Screening of the Small Library of Bioactive Compounds at Broad Institute-To test the inhibitory effects of the compounds included in the library of the bioactive compounds, we preincubated dHL60 cells with the potential inhibitors for 30 min, followed by addition of PMA and the appropriate probe, as shown in Table 2. After 90 min of incubation at 37°C in a CO 2 -free incubator, the extent of oxidation of the probes was measured with PerkinElmer Life Sciences Envision plate reader (PerkinElmer Life Sciences) using the following excitation/emission filter sets: 485/590 nm, 355/460 nm, and 531/595 nm, for hydropropidine ϩ DNA, coumarin boronic acid, and Amplex Red ϩ HRP, respectively.
Signal Optimization, HTS Statistics, and ZЈ Values-We used the ZЈ factor method as a measure of the assay quality or performance. As positive control (controlϩ signal), dHL60 cells were incubated with PMA in the presence of DMSO. The negative control (controlϪ signal) included cells in the presence of phenylarsine oxide (1 M) used as Nox2 inhibitor. The dynamic range was established by the difference between averaged maximal (controlϩ) and minimal (controlϪ) signals. The ZЈ factor was calculated using Equation 1 (27), where controlϩ and controlϪ correspond to PMA-stimulated cells in the presence of DMSO only or 1 M phenylarsine oxide, respectively, and S.D. values are the corresponding standard deviations. The calculated ZЈ values determined at Broad Institute in 384-well plates for all three HTS assays (hydropropidine ϩ DNA, coumarin boronic acid, and Amplex Red ϩ HRP) were 0.45, 0.64, and 0.79, respectively. Assuming the inhibitor identification threshold of 3ϫ S.D. deviation of the neutral (negative) control (DMSO), we can determine positive hits with a 42, 28, and 15% inhibition for hydropropidine ϩ DNA, coumarin boronic acid, and Amplex Red ϩ HRP, respectively.
Oxygen Consumption Experiments-Nox activity was determined by measuring rates of oxygen consumption in PMAactivated differentiated HL60 cells using a Seahorse XF96 extracellular flux analyzer (8,28). Cell suspensions were prepared in phenol red-free RPMI 1640 medium (without bicarbonate) and aliquoted (80 l per well) into 96-well plates to obtain a final cell count of 2 ϫ 10 4 cells per well. After spinning down the cells, additional RPMI 1640 medium (100 l per well) was added. Oxygen measurements were initiated, and at the specified time points, inhibitors of Nox were added, followed by injection of PMA. Alternatively, cells were pre-incubated with Nox2 inhibitors and transferred into measurement plates, and the response to PMA was tested. We used rotenone (1 M) and antimycin (10 M) to dissect out the contribution of mitochondrial respiration to the total oxygen consumption rate. This also enables one to monitor the effects of Nox2 inhibitors on mitochondrial respiration (8).
The NMR parameters of compound 6 are as follows: 31 3.09 (3H, s). 2Ј-Diphenylphosphino-2-methoxymethylbiphenyl 6 (380 mg, 0.88 mmol) was dissolved in 10 ml of HBr in glacial acetic acid and heated under reflux for 3 h. The solvent was removed, and the cyclo-o-MitoPh was recrystallized from ethyl acetate/ethanol (290 mg, 93%). The parameters for cyclo-o-MitoPh are as follows: 31  HPLC Analyses of the Specific Products Formed from Oxidation of Probes-HPLC-based analyses of the products of oxidation of HE and CBA probes were carried out using Agilent 1100 system equipped with absorption and fluorescence detectors, as described elsewhere (7,8). Rapid simultaneous monitoring of superoxide and hydrogen peroxide was carried out as reported previously (8), but the Supelco Ascentis Express phenyl-hexyl column (5 cm ϫ 4.6 mm, 2.7 m) was used. The compounds were eluted isocratically using mobile phase consisting of water (65%), acetonitrile (35%), and trifluoroacetic acid (0.1%) at a flow rate of 2 ml/min. The column temperature was set at 30°C. Under these conditions the following probes and products were monitored: HE (0.30 min), 2-OH-E ϩ (0.60 min), E ϩ (0.67 min), E ϩ -E ϩ (1.30 min), CBA (0.35 min), and COH (0.42 min). This method was also used in the rapid quantitative analyses of 2-OH-E ϩ or COH formation from HE or CBA in RAW 264.7 cells generating O 2 . or ONOO Ϫ . 2 and its oxidation/nitration products were performed as described recently (21,32). The method was modified for newly characterized products, including cyclo-o-MitoPh, and deuterated analogs were used as internal standards for quantitative analyses.

LC-MS/MS Analyses of o-MitoPhB(OH) 2 -derived Products-Analyses of o-MitoPhB(OH)
Dose-Response Analyses-For determination of the apparent IC 50 values, the dose-response fitting application was used, as implemented in OriginPro 9.1.0 program (OriginLab Corp.). Equation 2 used for the fitting is as follows: where y is the measured quantity (proportional to Nox2 enzyme activity); A 1 and A 2 correspond to bottom and top asymptotes, p to the Hill slope, and c is the concentration of the compound.

Overview of HTS/ROS Assays Used in the Identification of
Nox Inhibitors-To reliably identify inhibitors of Nox isoforms, we developed the following primary and secondary assays for HTS/ROS analysis (Fig. 2), as described recently (8). Hydropro-pidine (HPr ϩ ) was used as a primary probe for extracellular O 2 . measurement (19). HPr ϩ reacts with O 2 . (k ϭ 1.2 ϫ 10 4 M Ϫ1 s Ϫ1 ) to form a specific and diagnostic marker product, 2-hydroxypropidium (2-OH-Pr 2ϩ ) (19). 2-OH-Pr 2ϩ fluorescence quantum yield is enhanced Ͼ10-fold in the presence of added DNA. Coumarin-7-boronic acid (CBA) was used as another primary assay probe for measuring H 2 O 2 . CBA reacts with as compared with its catalase-insensitive reaction with ONOO Ϫ (k ϭ 1.1 ϫ 10 6 M Ϫ1 s Ϫ1 ), to form a highly fluorescent product, COH (33). Over the duration of the reaction between CBA and H 2 O 2 , there was significantly less spontaneous decomposition of CBA as compared with other boronate probes (33,34). The addition of PMA (activator of PKC signaling pathway) to differentiated HL60 cells (overexpressing Nox2) in the presence of HPr ϩ and DNA causes a linear increase in 2-OH-Pr 2ϩ fluorescence (O 2 . reaction) that was inhibited by SOD and not by catalase (Fig. 2). Concomitantly, there is an increase in COH fluorescence (H 2 O 2 reaction with CBA) that is inhibited by catalase and not by SOD (Fig. 2).
In the secondary assay, we used the probe hydroethidine (HE) and detected 2-hydroxyethidium (2-OH-E ϩ ) (specific product of HE reaction with O 2 . ) using ultra-HPLC (Fig. 2). For measuring H 2 O 2 , the probe Amplex Red was used in the presence of HRP to monitor resorufin (formed from Amplex Red oxidation with H 2 O 2 /HRP) using a plate reader. The addition of PMA to differentiated HL60 cells in the presence of Amplex Red and HRP causes an increase in the fluorescence intensity of resorufin that is abrogated by catalase but not by SOD (Fig. 2). Under these conditions, O 2 . is detected by measuring 2-OH-E ϩ (by HPLC) that was inhibited by SOD and not by catalase (Fig.  2). These assays provide the foundation for rigorous measurements of O 2 . and H 2 O 2 using three different probes for high throughput screening studies (8).
Screening the Library of Bioactive Compounds, Identification of Potential Nox2 Inhibitors-We applied the ROS/HTS assays, as discussed above, to screen a subset library of bioactive drugs as Nox2 inhibitors at Broad Institute Probe Development Center. First, we fulfilled the HTS assay "readiness" criteria for the primary and secondary assay probes (HPr ϩ , CBA, and Amplex Red).

TABLE 2
Step-by-step protocol applied to screen the library of bioactive compounds at The Broad Institute The list of 2,029 compounds, together with the results of screening of their normalized effects on Nox2 activity using HPr ϩ , CBA, and Amplex Red probes, are shown in supplemental Table 1. The stepwise protocol used in the HTS/ROS studies at Broad Institute is shown in Table 2. The effects of 2,029 compounds on O 2 . formation by monitoring the formation of 2-OH-Pr 2ϩ in PMA-activated differentiated HL60 cells are shown in Fig. 3. 2-OH-Pr 2ϩ formation in the presence of DMSO and phenylarsine oxide (1 M) was normalized to 0% (no effect) and Ϫ100% (complete inhibition) (Fig. 3a). We set a 50% inhibition (Fig. 3a, green dashed line) as a threshold criterion for a "hit." Blue dots in Fig. 3a indicate compounds inhib-iting 2-OH-Pr 2ϩ formation by Ͼ50%, and red dots indicate compounds exhibiting Ͻ50% inhibition (Fig. 3a). Fig. 3b shows the plate-to-plate comparison showing the reproducibility of data for each compound, and the pie chart (inset in Fig. 3b) shows 63 out of 2,029 compounds exhibit Ͼ50% inhibition of 2-OH-Pr 2ϩ formation (blue chart with positive Ͼ50% inhibition in both plates). Fig. 3, c and d, shows similar results for COH formation from CBA. One hundred fifty four compounds from the screened library inhibited COH formation by Ͼ50% in both plates (Fig. 3d, inset). Corresponding results obtained using the Amplex Red probe measuring H 2 O 2 are shown in Fig.  3, e and f. The three-dimensional hit correlation plot for all three assay probes, HPr ϩ , CBA, and Amplex Red, is shown in Fig. 4a. Of the 2,029 compounds tested, 49 compounds (2.4%, blue pie chart in Fig. 4b) were identified as hits with all three probes (Table 3), using 50% inhibition threshold. The red color in Fig. 4b corresponds to negative result for all three probes (Ϫ/Ϫ/Ϫ); magenta is used for compounds that scored a positive result for a single probe (ϩ/Ϫ/Ϫ); green is used for compounds that scored positively with two probes (ϩ/ϩ/Ϫ); and blue is used for positive results for all three probes (ϩ/ϩ/ϩ). The list of "positive hits" shown in blue in Fig. 4b, together with the average extent of inhibition at ϳ30 M concentration is shown (Table  3). This list contains Food and Drug Administration-approved drugs that include dopamine receptor blockers and drugs used in the treatment of schizophrenia and antimalarial and antifungal agents. Some of these drugs have been previously used or identified as inhibitors of Nox isoforms (35,36). Interestingly, the compound, mitoxantrone, identified as the most potent inhibitor of Nox2 activity in dHL60 cells (Table 2) strongly inhibited pancreatic ductal adenocarcinoma survival (37). The positive hits were further evaluated to filter out pan assay interference compounds (38,39). Of the 49 hits, nine were tested positive when screened against the library of the pan assay interference compounds, as defined in Ref. 38 and shown in Table 3.
Confirmatory Assays, Dose Response, and Oxygen Consumption Measurements-We confirmed the inhibitory effect of compounds selected from positive hits (Table 3) on Nox2 activ-ity by determining their dose response. We screened over 20 commercially available compounds selected from Table 3 for the confirmatory assays. The apparent IC 50 values for inhibition of Nox2 activity in the dHL60 model determined with a fluorescence plate reader using the CBA-based assay are shown in Table 4. Next, the effect of selected compounds on Nox2 activity was tested by HPLC-based simultaneous monitoring of O 2 . and H 2 O 2 (Fig. 5, a and b), by measuring oxygen consumption stimulated by Nox2 activation using the 96-well platebased extracellular flux analyzer (Seahorse XF96, Fig. 5, c and d) and by monitoring the inhibitory effect on spin-trapped superoxide adduct using DEPMPO (data not shown). Fig. 5a shows the results of the confirmatory assays for selected compounds, sulconazole, mefloquine, cetylpyridinium cation, and DPI, on formation of 2-hydroxyethidium, the specific marker product for O 2 . . Similarly, Fig. 5b shows the effect of these compounds production also compare favorably with those measured using Nox2-mediated oxygen consumption. We next verified that at these concentrations sulconazole, mefloquine, and cetylpyridinium cations inhibited formation of the DEPMPO-superoxide adduct. All three compounds completely blocked formation of DEPMPO-⅐ OOH adduct, when used at 100 M for sulconazole and mefloquine or 10 M for cetylpyridinium and DPI cations (data not shown). Inhibition of Nox2 in Activated RAW 264.7 Macrophages-Following the experiments on dHL60 cells, we tested whether the selected hits can also inhibit Nox2 activity in a different cellular model for Nox2 activity, namely PMA-stimulated RAW 264.7 cells. Activation of RAW 264.7 cells in the presence of the hydroethidine probe led to a significant increase in the HPLC peak of 2-hydroxyethidium (monitored with a fluorescence detector) (Fig. 6a), as reported earlier (7). We measured the yield of 2-OH-E ϩ upon PMA-induced Nox2 activation in the presence of several compounds identified as apparent Nox2 inhibitors in dHL60 cells, with DPI used as a positive control for Nox2 inhibition. At these concentrations, all of the compounds tested decreased the intensity of the peak of 2-OH-E ϩ (Fig. 6a, fluorescence mode), confirming the ability of these compounds to inhibit Nox2 activity in intact cells. Quantitative analysis of the HPLC data (Fig. 6b) based on the use of authentic standards for 2-OH-E ϩ , E ϩ , and E ϩ -E ϩ shows that the compounds inhibit the yield of 2-OH-E ϩ , while having no effect on ethidium (E ϩ ), the nonspecific oxidation product (with the exception of benzethonium cation), and variable effects on the one-electron oxidation dimeric product (diethidium, E ϩ -E ϩ ). These results confirm that the HTS hits for Nox2 identified using the differ-entiated HL60 cells also inhibit O 2 . formation from Nox2 in RAW 264.7 cells. Do Nox2 Inhibitors Also Inhibit Generation of Peroxynitrite?-Next, we tested whether the compounds listed in Table 4 that inhibit Nox2 activity in PMA-stimulated RAW 264.7 cells could also inhibit generation of peroxynitrite formed from O 2 . and ⅐ NO reaction in the same cellular model. RAW macrophages were stimulated to co-generate both ⅐ NO and O 2 . via activation of inducible NOS and Nox2 (using LPS, interferon-␥, and phorbol ester), as reported earlier (7). CBA was used to monitor ONOO Ϫ in cell-free systems and from activated macrophages by monitoring COH (catalase-insensitive) generation (7,20,33). Results show that several but not all Nox2 inhibitors identified from the screen of the bioactive library using the dHL60 model inhibited ONOO Ϫ -dependent COH formed from CBA. Of note, the compounds inhibiting ONOO Ϫ formation in RAW 264.7 cells also inhibited Nox2 activity in these cells, as demonstrated in Fig. 6. Fig. 7b shows the dosedependent inhibition of ONOO Ϫ formed from activated macrophages in the presence of selected hits for Nox2 inhibition (e.g. DPI, sulconazole, mefloquine, and cetylpyridinium  (Fig. 7a) and the dHL60 Nox2 model (Table 4). These results suggest that the HTS/ROS assay with CBA can be used to identify novel inhibitors of ONOO Ϫ formation as well.

Specific Detection of a Cyclized Product during Peroxynitrite Reaction with Ortho-substituted Mitophenylboronic Acid-
Recently, we developed a new probe, o-MitoPhB(OH) 2 , that rapidly reacts with ONOO Ϫ forming a minor product, o-MitoPhNO 2 , that is very specific for ONOO Ϫ (Fig. 8a) and is unaffected by glutathione or other biologically relevant reductants (21,22). As this product is not fluorescent, we used HPLC with absorption detection for quantitative analyses, and we confirmed it is formed in RAW 264.7 cells in activated macrophages (22). However, using an LC-MS/MS method, we found that this product (i.e. o-MitoPhNO 2 ) was detected in a relatively low yield (21). Quantitative analysis of the LC-MS data revealed an additional minor product (m/z ϭ 351) formed during the reaction between o-MitoPhB(OH) 2 and ONOO Ϫ . The same product was detected in extracts from RAW 264.7 cells, when activated to produce ONOO Ϫ (see below). The structure of this product was assigned to cyclo-o-MitoPh (Fig. 8a, shown in red) formed by radical-induced intramolecular cyclization mechanism (Fig. 8a, minor radical pathway). This was supported by the observation that during the reaction of ONOO Ϫ with d 15o-MitoPhB(OH) 2 (with all protons on triphenylphosphonium moiety substituted by deuterium), the product with m/z ϭ 365 atomic mass units is formed, indicating the loss of one deuterium atom. The identity of this product was further verified by independent synthesis of cyclo-o-MitoPh (Fig. 9a), and the structure was confirmed by mass spectrometry, NMR, and

Nox2 Inhibition as Anti-nitration Strategy
x-ray crystallography (Fig. 9b). The LC-MS/MS parameters (retention time, m/z value, and MS/MS fragmentation pattern) of the minor product of o-MitoPhB(OH) 2 reaction with authentic ONOO Ϫ were identical to those of the authentic cyclo-o-MitoPh (Fig. 8, a and c). The specificity of this product was tested using other oxidants (H 2 O 2 and HOCl), which are known to oxidize boronic compounds and comparing the product identities and distribution (Fig. 8, b and c). Consistently with our previous research on the chemical reactivity of boronic compounds (20,32,40), the reaction between o-MitoPhB(OH) 2 . 8a) (22). In addition, a small amount of the protonated form of triphenylphosphonium oxide (TPPϭO, m/z ϭ 279) was also detected. With HOCl, the minor product observed (using excess of HOCl) is chlorinated phenol o-MitoPh(Cl)OH (Fig. 8a), showing up as a double peak attributed to two isomers. Interestingly, other oxidation products, including triphenylphosphine oxide and brominated phenolic product (o-MitoPh(Br)OH)were also formed (Fig. 8). Although triphenylphosphine oxide formation can be explained by a nucleophilic attack of ClO Ϫ on the phosphorus atom of the triphenylphosphonium moiety, the  MARCH 25, 2016 • VOLUME 291 • NUMBER 13 brominated phenolic product indicates formation of HOBr via oxidation of the bromide anion (present as a counterion of o-MitoPhB(OH) 2 probe) by HOCl (41,42). To test whether o-MitoPhB(OH) 2 can distinguish between peroxynitrite and myeloperoxidase (MPO)-mediated oxidation/nitration, we determined the products formed by MPO in the presence of hydrogen peroxide, with or without nitrite (Fig. 10a). The results indicate that both cyclo-o-MitoPh and o-MitoPhNO 2 products are specific for ONOO Ϫ , although MPO in the presence of nitrite leads to nitration of the phenolic product, formed from oxidation of o-MitoPhB(OH) 2 by H 2 O 2 . The quantitative analysis of the oxidation/nitration of o-MitoPhB(OH) 2 by ONOO Ϫ indicates that yields of the minor specific products, cyclo-o-MitoPh and o-MitoPhNO 2 , are 10.5 Ϯ 0.5 and 0.5 Ϯ 0.1%, respectively (Fig. 10b). In the case of MPO-mediated oxidation in the presence of sodium nitrite, the major product seems to be nitrated phenols (o-MitoPh(NO 2 )OH). The other product detected was triphenylphosphine oxide, formed by the MPO/H 2 O 2 system both in the absence and presence of nitrite (data not shown). Next, we investigated the formation of peroxynitrite-specific products in activated macrophages and the effect of selected Nox2 inhibitors on the reaction products of o-MitoPh-B(OH) 2 and ONOO Ϫ (Fig. 11). We have shown previously that stimulation of RAW 264.7 cells leads to formation of o-MitoPhOH and small amounts of o-MitoPhNO 2 (21,22). As shown in Fig. 11, the most exclusive minor product detected in cells was the cyclized product cyclo-o-MitoPh. In fact, the amount of o-MitoPhNO 2 formed in cells is less than 5% of the yield of cyclo-o-MitoPh (Fig. 11c). The analysis of the effect of inhibitors on the yield of the major phenolic oxidation product of o-MitoPhB(OH) 2 gives mixed results, with mefloquine actually increasing the yield of this product. This suggests the possibility of multiple pathways of the formation and/or metabolism of the phenolic product inside the cells. In contrast, results show that selected candidate Nox2 inhibitors, including cetylpyridinium, sulconazole, mefloquine, and DPI, inhibit formation of cyclo-o-MitoPh and o-MitoPhNO 2 , the diagnostic products for ONOO Ϫ (Fig. 11c). This is consistent with the results of measurements of extracellular ONOO Ϫ (Fig. 7b) and further demonstrates the added value of determining the specific minor product(s) for ONOO Ϫ reaction with o-MitoPhB(OH) 2 .

Discussion
Significance of Nox Inhibition-Nox enzyme-derived ROS/ RNS are now recognized to play a central role in both inflammatory and fibrotic diseases (1,2). The former includes both classical inflammatory conditions (e.g. arthritis and inflammatory bowel disease) and nonclassical conditions in which inflammation plays a central pathogenic role. For example, inflammatory lung diseases include acute respiratory distress syndrome, asthma, and chronic obstructive pulmonary disease, although other inflammatory conditions include Alzheimer and Parkinson diseases, ischemic stroke, and organ reperfusion injury during transplantation (43). Fibrotic diseases include liver fibrosis (e.g. following viral infection), idiopathic pulmonary fibrosis, and diabetic kidney disease. Nox2 has been implicated in inflammatory diseases and Nox4 in fibrotic diseases (1,2). Existing therapies for both classes of diseases include steroids and other anti-inflammatory approaches, but they have proven to be ineffective. Therefore, there is a need for developing novel anti-inflammatory and antifibrotic agents based on other molecular targets. Nox enzymes are especially promising

Nox2 Inhibition as Anti-nitration Strategy
in this regard due to mounting evidence in humans and in other experimental models (44). Peroxynitrite, the product of diffusion-controlled reaction of nitric oxide and superoxide, has been implicated in several inflammatory diseases (15), including neurodegenerative diseases and chemotherapy-induced nephrotoxicity (45). Recent data also suggest the involvement of ONOO Ϫ in traumatic brain injury-induced neurodegeneration via activation of calpain in neurons (46,47).
It has been proposed that Nox2-derived ROS/RNS from myeloid-derived suppressor cells in the tumor microenvironment are potentially responsible for decreased T cell reactivity and immunosuppressive effects (48). In this regard, small molecular weight compounds that inhibit Nox2 activity could play a vital role in providing additional mechanistic insight on the immune system in tumor microenvironments. The role of Nox/ROS in biology is paradoxical (49) (50). Preliminary results show that several compounds identified as potential Nox2 inhibitors (Table 4) significantly inhibit proliferation of human pancreatic cancer cells (data not shown). It is likely that follow-up studies will greatly improve the potency and selectivity of hit compounds using medicinal chemistry. Although this study is restricted to the Nox2 isoform, future investigations will broaden the scope of work to include other Nox isoforms using the appropriate HTS-compatible cell lines. HTS/ROS/RNS Assay-ROS/RNS do not represent a single entity, but encompass a wide range of reactive species that exhibit oxidizing, nitrating, nitrosating, and halogenating properties (51,52). To better understand the pathophysiological consequences of ROS/RNS, it is crucial to identify and characterize the species that are specifically responsible for a given "biologic and toxicologic" effect and to inhibit the specific sources of ROS/RNS generation (53). Doxorubicin, one of the most widely used chemotherapeutics, causes myocardial inflammation that is attributed to Nox-induced ROS/RNS formation in inflammatory cells (54 -56). Genetic disruption of Nox2 mitigated doxorubicin-mediated contractile dysfunction, oxidative/nitrative stress, and inflammation (56). Thus, selective targeting of Nox2 may provide a novel therapeutic strategy for mitigation of ROS/RNS and cardiotoxic side effects, while maximizing its antitumor efficacy (56). To date, only a limited number of Nox isoform-selective inhibitors are available (57,58). This is due in part to serious limitations of the existing ROS/RNS assays as described below.
Previous approaches have used sensitive but nonselective and artifact-prone ROS/RNS probes (59) for detecting Noxderived oxidants, resulting in a high rate of false positives and potentially missing weaker but selective hits lost in the "noise." Many HTS-based H 2 O 2 assays to detect Nox inhibitors included the enzyme HRP. The lack of probes' selectivity for specific oxidant and the susceptibility of the HTS assays to peroxidase substrates and inhibitors led to the controversy over the Nox-inhibitory potency of the positive hits selected, including apocynin, VAS2870, and 2-acetylphenothiazine (36,60,61). In fact, one of the authors recently reported a new myeloperoxidase inhibitor, identified during the HTS campaign for Nox2 inhibitors, using the L-012 probe as a Nox2 activity reporter (62). Using the present HTS/ROS approach, it is conceivable that false positives will be decreased by Ͼ75%, with the assay specificity allowing identification of weaker inhibitors that can later be improved through medicinal chemistry. In this work, we describe the use of ROS/RNS-specific probes whose redox chemistry is better understood with respect to reaction kinetics, stoichiometry and reaction products (63). Selective monitoring of specific ROS in the HTS assay is important. For example, these HTS assays can be extended to other cell types to gain new insight regarding enzymatic sources of ROS generation and subsequently unravel the role of ROS in disease-related biological processes. It is conceivable that a similar HTS/ROS assay may be used to identify inhibitors of other oxidants (e.g. hypochlorous acid) and their sources of generation using other fluorescent probes (Table 1). In this study, we identified new, oxidant-specific, products of the reaction of o-MitoPhB(OH) 2 with ONOO Ϫ and HOCl (Fig.  8). In the presence of the excess oxidant, the major phenolic product undergoes nitration or chlorination reaction, respectively, leading to isomeric nitro-or chloro-derivatives of Mito-PhOH. Also, in the presence of HOBr, analogous products (i.e.  MitoPh(Br)OH) are formed. With excess ONOO Ϫ or HOCl, the triphenylphosphine oxide was also formed (Fig. 8). Interestingly, with peroxynitrite, the most exclusive minor product, cyclo-o-MitoPh, was formed even when the probe was present in excess of the oxidant. The lack of effect of biologically relevant reductants (GSH and NADH) and the low efficiency of trapping of the phenyl radical (22) may be explained by rapid intramolecular cyclization due to addition of the phenyl radical to the benzene ring of the triphenylphosphonium moiety (Fig.  8a). As this product is not formed with any other oxidant or nitrating agent tested, it can serve as a specific diagnostic product of ONOO Ϫ and should be used as the ultimate proof of peroxynitrite intermediacy.
Inhibitory Mechanism of Nox2 Positive Hits on ROS Formation-Screening of the library of over 2,000 bioactive compounds generated a relatively high amount of positive hits, with the lowest yield (3.1%) using the hydropropidine-based assay. This was expected, as the compounds tested show a variety of biological activities and do not predominantly target NADPH oxidase. However, screening of a large, chemically diverse library of compounds will produce a significantly lower yield of active compounds. The HTS compatibility of the three assays (HPr ϩ , CBA, and Amplex Red-based) and the use of rapid HPLC analyses in the 384-well plate format will enable rapid orthogonal screenings to filter out false positives. Additionally, the use of chemical structure-based filters to remove pan assay interference compounds should further decrease the amount of compounds selected for post-screening studies/hit optimization process.
HL60 cells differentiated into a neutrophil-like phenotype by DMSO or all-trans-retinoic acid exhibited high expression of Nox2 as confirmed by Western blotting of both membranebound gp91 phox and cytosolic p47 phox subunits (8). The differentiated cells were compared with nondifferentiated cells in their response to PMA, an activator of protein kinase C, leading to the phosphorylation of the p47 phox cytosolic subunit, which, in turn, binds to p22 phox membrane protein. After the assembly of all cytosolic and membrane components, NADPH is oxidized, and its electrons are transferred to oxygen, generating O 2 . . Small molecules can inhibit O 2 . and H 2 O 2 by targeting specific steps of Nox activation as follows: Nox expression, ligand receptor binding, trafficking of Nox components to cell membrane, activation and assembly of Nox complex, NADPH binding, and electron transfer from the active site of the enzyme (64). To identify target of inhibition of Nox2, it is essential to perform a comprehensive analysis of phosphorylation of proteins of the regulatory subunits and the effect of inhibitors on phosphorylation and translocation of the cytosolic subunits to the membrane. These aspects will be a part of our future research. Some of the Nox2 inhibitors (e.g. promazines) identified in this work have previously been shown to block Nox2 activity in cell-free assays (36). Of added significance is the fact that selected hits also affect basal mitochondrial respiration in dHL60 cells (Fig. 5, c and d). Whereas sulconazole inhibits PMA response (Nox2 activation) and basal respiration similarly, other compounds are more selective in their inhibition of Nox2 activity, with little or less pronounced inhibition of mitochondrial function at selected concentrations. Although the inhibition of basal mitochondrial respiration and Nox2 activity may be linked, the inhibition of mitochondrial respiration per se is unlikely to block Nox2 activity. In fact, we have previously shown that rotenone, an inhibitor of mitochondrial complex I, inhibits basal mitochondrial respiration in dHL60 cells, without affecting the Nox2 activity, as measured by the PMA-stimulated increase in the rate of oxygen consumption (8). It is also worth noting that DPI, a non-selective inhibitor of flavoenzymes, inhibits Nox2 activity at a significantly lower concentration (0.1 M) than required for inhibiting the basal mitochondrial respiration (Ϸ1 M) (Fig. 5d). The proposed method of high throughput screening of Nox inhibitors in intact cells will likely yield many positive hits that affect the Nox enzyme indirectly, as discussed above. The detailed mechanistic studies may provide new information on the pathways/factors controlling Nox enzyme activity and yield potentially new inhibitors for clinical trials, based on drug repurposing strategy.
Author Contributions-B. K. conceived and coordinated the study and wrote the paper. J. Z. designed, performed, analyzed, and interpreted the data, prepared the figures, and revised the paper critically.