LC-MS/MS

All analytical methods were in MRM mode where the parent ion was selected in Q1 of the mass spectrometer. The parent ion was fragmented and a characteristic fragment ion monitored in Q3. MRM mass spectroscopy methods are particularly sensitive because additional time is spent monitoring the desired ions and not sweeping a large mass range. Methods were rapidly set up using Automaton^® (Applied Biosystems), where the compounds were listed with their name and mass in an Excel datasheet. Compounds were submitted in a 96-well plate to the HPLC autosampler and slowly injected without a column present. A narrow range centered on the indicated mass was scanned to detect the parent ion. The software then evaluated a few pre-selected parameters to determine conditions that maximized the signal for the parent ion. The molecule was then fragmented in the collision cell of the mass spectrometer and fragments with m/z larger than 70 but smaller than the parent mass were determined. Three separate collision energies were evaluated to fragment the parent ion and the largest three ions were selected. Each of these three fragment ions was further optimized and the best fragment was chosen. The software then inserted the optimized masses and parameters into a template method and saved it with a unique name that indicated the individual compound being optimized. Spectra for the parent ion and the fragmentation pattern were saved and reviewable later.

Solubility

The solubility of compounds was tested in phosphate buffered saline, pH 7.4. Compounds were inverted for 24 hours in test tubes containing 1–2 mg of compound with 1 mL of PBS. The samples were centrifuged and analyzed by HPLC (Agilent 1100 with diode-array detector). Peak area was compared to a standard of known concentration. In cases when the concentration was too low for UV analysis or when the compound did not possess a good chromophore, LC-MS/MS analysis was used.

Stability

Demonstration of stability in PBS was conducted under conditions likely to be experienced in a laboratory setting. The compound was dissolved in 1 mL of PBS at a concentration of 10 μM, unless its maximum solubility was insufficient to achieve this concentration. Low solubility compounds were tested between ten and fifty percent of their solubility limit. The solution was immediately aliquoted into seven standard polypropylene microcentrifuge tubes which were stored at ambient temperature in a block microcentrifuge tube holder. Individual tubes were frozen at −80°C at 0, 1, 2, 4, 8, 24, and 48 hours. The frozen samples were thawed in a room temperature and an equal volume of acetonitrile was added prior to determination of concentration by LC-MS/MS.

Determination of glutathione reactivity

One μL of a 10 mM compound stock solution was added to 1 mL of a freshly prepared solution of 100 μM reduced glutathione. Final compound concentration was 10 μM unless solubility limited. The solution was allowed to incubate at 37°C for two hours prior to being directly analyzed for glutathione adduct formation. LC-MS/MS analysis of GSH adducts was performed on an API 4000 Q-TrapTM mass spectrometer equipped with a Turboionspray source (Applied Biosystems, Foster City, CA). Two methodologies were utilized—a negative precursor ion (PI) scan of m/z 272, corresponding to GSH fragmenting at the thioether bond, and a neutral loss scan of −129 AMU to detect GSH adducts. This triggered positive ion enhanced resolution and enhanced product ion scans [32,33].

Primary screen to identify NOX1 inhibitors (AIDs 1792, 2541, 2538, 2664, 2752, 2773, 2808, 2819, and 434997)

Assay Overview: The purpose of this cell-based assay was to identify compounds in the Maybridge collection that inhibit NOX1 activity. This chemiluminescence assay employs a luminol probe to monitor intracellular ROS in the HT29 transformed colonic epithelial cell line. HT29 cells express high endogenous levels of known NOX1 components and no other NOX family members. In this assay, the cells are incubated with test compounds, cell permeable luminol, and horseradish peroxidase. The interaction of luminol with NOX1-generated ROS/superoxide inside cells yields an unstable endoperoxide that generates light, leading to increased well luminescence. As designed, compounds that inhibit cellular NOX1 activity will reduce intracellular ROS and endoperoxide levels, leading to reduced luminol-ROS interactions, reduced endoperoxide production, reduced light emission, and reduced well luminescence. Test compounds were assayed in singlicate at a final nominal concentration of 6.7 μM (AID 1792), in triplicate at a final nominal concentration of 3.3 μM (AID 2541), in triplicate using a dilution series starting at a maximum concentration of 10 μM (AID 2538), in triplicate using a dilution series starting at a maximum concentration of 20 μM (AIDs 2808, 2819), in triplicate using a dilution series starting at a maximum concentration of 25 μM (AID 2773), or in triplicate using a dilution series starting at a maximum concentration of 50 μM (AIDs 2664, 2752).

Protocol Summary: HT29 cells were routinely cultured in 150 mm dishes at 37 °C and 95% relative humidity (RH). The growth media consisted of Dulbecco’s Modified Eagle’s Media supplemented with 10% v/v fetal bovine serum, 2 mM L-Glutamine, and 100U/mL penicillin and streptomycin. Prior to the start of the assay, cells were suspended to a concentration of 2.25 million cells/mL in Hank’s Balanced Salt Solution. 30 μL of cell suspension (67,500 cells) were dispensed into each well of 384-well tissue culture-treated microtiter plates and the plates were centrifuged briefly. The assay was started by dispensing 100 nL of test compound in DMSO, diphenylene iodonium (DPI) in DMSO, or DMSO alone (0.33% final concentration) to the appropriate wells. The plates were incubated for 1 hour at 37 °C, 5% CO₂ and 95% RH, followed by the addition of 20 μL of a solution containing 400U/mL horseradish peroxidase and 50 mM luminol mix to all wells. The plates were centrifuged briefly and incubated for 10 minutes at 37 °C (5% CO₂, 95% RH). Well luminescence was measured on the EnVision plate reader (Perkin Elmer). Assay Cutoff: 1) compounds that inhibited NOX1 ≥ 75% [(AID 1792); 2) compounds with ≥ 50% inhibitory activity (AID 2541); 3) compounds with an IC₅₀ < 20 μM (AIDs 2538, 2664, 2752, 2773); and 4) compounds with an IC₅₀ ≤ 17 μM (AIDs 2808, 2819) were declared active.

Maybridge library uHTS counterscreen to identify inhibitors of luminol (AID 1823)

Assay Overview: The purpose of this biochemical assay was to identify compounds in the Maybridge collection that directly inhibit luminescence generated by luminol in the peroxide reaction. This assay served as a counterscreen for a previous set of experiments entitled, “Luminescence-based primary cell-based high throughput screening assay to identify inhibitors of NADPH oxidase 1 (NOX1): Maybridge Library” (PubChem AID 1792). In this assay, compounds are incubated with hydrogen peroxide (H₂O₂), horseradish peroxidase (HRP), and luminol. The interaction between luminol and H₂O₂ yields unstable peroxide radicals that generate light that can be detected by a luminometer. As designed, compounds that inhibit this reaction will reduce luminol interactions with H₂O₂ and production of the unstable peroxide, leading to reduced light emission and well luminescence. Test compounds were assayed in singlicate at a final nominal concentration of 6.7 μM.

Protocol Summary: Prior to the start of the assay, H₂O₂ was diluted to a concentration of 88 μM in Hank’s Balanced Salt Solution. 30 μL of the diluted H₂O₂ were dispensed into each well of 384-well tissue culture-treated microtiter plates and the plates were centrifuged briefly. The assay was started by dispensing 100 nL of test compound in DMSO, N-acetyl cysteine (NAC; 2.5 mM final concentration) in DMSO, or DMSO alone (0.33% final concentration) to the appropriate wells. The plates were incubated for 30 minutes at 25 °C, followed by the addition of 20 μL of a solution containing 400U/mL horseradish peroxidase and 50 mM luminol mix to all wells. The plates were centrifuged briefly and incubated for 10 minutes at 25 °C. Well luminescence was measured on the EnVision plate reader (Perkin Elmer). Assay Cutoff: compounds that inhibited luminol ≥ 25% were declared active.

Dose response assay to identify inhibitors of NOX1 in a human embryonic kidney (HEK) 293 transfection format (AIDs 2532, 2545, and 435002)

Assay Overview: The purpose of this cell-based assay is to determine the potency of selected compounds identified as active in previous assays in a HEK293 transfection format. This chemiluminescence assay employs a luminol probe to monitor intracellular ROS in HEK293 cells. In this assay, HEK293 cells seeded into 6-well plates were cotransfected with expression vectors for NOX1, NOXA1, NOXO1 and constitutive active RAC1. After 16 hours, test or control compounds were added, followed 2 hours later by luminol and horseradish peroxidase. NOX activity was determined by chemiluminescence as described above (AID 1792). Test compounds were assayed in triplicate using a 4-point 1:3 dilution series starting at a maximum concentration of 10 μM.

Protocol Summary: HEK293 cells were maintained in DMEM containing 10% heat-inactivated fetal bovine serum, 2 mM glutamine, and antibiotics (100 U/ml penicillin and 100 g/ml streptomycin) at 37°C in 5% CO₂. 500,000 HEK293 cells were seeded into 6-well plates in media without antibiotics the day before transfection. Cells were transfected with pRK5-Myc-NOX1, -NOXO1, -NOXA1, and pRK5-myc-Rac1CAQ61L using Lipofectamine 2000. 16 hours after transfection, 50,000 HEK293 cells in 30 μL HBSS were dispensed into each well of a white 384-well plate. The assay was started by dispensing 50 nL of test compound in DMSO, diphenylene iodonium (DPI) in DMSO, or DMSO alone (0.33% final concentration) to the appropriate wells. The plates were incubated for 1 hour at 37 °C, 5% CO₂ and 95% RH, followed by the addition of 20 μL of a mixture of 1 mM luminol plus 8 units of HRP in HBSS and luminescence quantified using a 384-well plate luminometer (EnVision). Chemiluminescence was measured for 30 minutes. Assay Cutoff: compounds with an IC₅₀ ≤ 10 μM were declared active.

Dose response assay to identify inhibitors of NOX2, NOX3, or NOX4 in a human embryonic kidney (HEK)/293 transfection format (AIDs 2539 and 435013)

Assay Overview: The purpose of this family selectivity cell-based assay was to evaluate the ability of compounds identified as active in previous assays to inhibit NOX2, NOX3, or NOX4 activity in a HEK293 transfection format. This chemiluminescence assay employs a luminol probe to monitor intracellular ROS in HEK293 cells. HEK293 cells seeded into 6-well plates were cotransfected with the appropriate expression vectors for each NOX subtype. NOX activity was determined by chemiluminescence as described above (AID 1792).

Protocol Summary: HEK293 cells were maintained as described above (AID 435002). HEK293 cells seeded into 6-well plates were cotransfected with the appropriate expression vectors for each NOX subtype. For the NOX2 assay (Assay 1), cells were transfected with pRK5-Myc- NOX2, pRK5-p67phox, pRK5-p47phox and pRK5-myc-Rac1CA-Q61L. For the NOX-3 assay (Assay 2), cells were transfected with pRK5-Myc-NOX3, pRK5-NOXO1, pRK5-NOXA1and pRK5-myc- Rac1CA-Q61L. For the NOX-4 assay (Assay 3), cells were transfected with pRK5-Myc-NOX4 and pRK5-p22phox. After 16 hours, cells were dispensed into wells of a 384-well plate, test or control compounds were added, and NOX activity was determined by chemiluminescence as described above (AID 435002). Assay Cutoff: compounds with an IC₅₀ ≤ 10 μM were declared active.

Dose response assay to identify inhibitors of xanthine oxidase (AIDs 2556 and 435009)

Assay Overview: The purpose of this cell-free assay was to evaluate the ability of compounds identified as active in previous assays to inhibit ROS production by another cellular source, xanthine oxidase. This chemiluminescence assay employs a luminol probe to monitor ROS production. In this assay, xanthine oxidase was dispensed to all wells of a 96-well plate followed by test or control compounds. Hypoxanthine was dispensed to all wells, followed by the addition of horseradish peroxide and luminal and reading luminescence. The interaction of luminol with xanthine oxidase-generated ROS/superoxide inside cells yields an unstable endoperoxide that generates light, leading to increased well luminescence. As designed, compounds that inhibit cellular xanthine oxidase activity will reduce intracellular ROS and endoperoxide levels, leading to reduced luminol-ROS interactions, reduced endoperoxide production, reduced light emission, and reduced well luminescence. Test compounds were assayed in triplicate using a 7-point dilution series starting at a maximum concentration of 10 μM.

Protocol Summary: 25 μL of freshly-prepared 0.25 U/ml xanthine oxidase were dispensed into a 384-well plate (white Corning) by FlexDrop. 50 nL of DPI, DMSO or library compounds were dispensed by Biomek FX into each individual well. Upon 10 minutes of incubation at room temperature, 20 μL of 1 mM luminol plus 8 units of HRP in HBSS was added and luminescence was quantified using a 384-well plate luminometer (EnVision). Assay Cutoff: Compounds with an IC₅₀ ≤ 10 μM were declared active.

Dose response cytotoxicity assay (AID 434992 and 463255)

Assay Overview: The purpose of this assay was to eliminate cytotoxic compounds. In this assay, HT29 cells were incubated with test compounds, followed by determination of cell viability. The assay utilizes the CellTiter-Glo luminescent reagent to measure intracellular ATP in viable cells. Luciferase present in the reagent catalyzes the oxidation of beetle luciferin to oxyluciferin and light in the presence of cellular ATP. Well luminescence is directly proportional to ATP levels and cell viability. As designed, compounds that reduce cell viability will reduce ATP levels, luciferin oxidation and light production, resulting in decreased well luminescence. Compounds were tested in triplicate in a 10-point 1:3 dilution series starting at a nominal test concentration of 40 μM.

Protocol Summary: Prior to the start of the assay 50,000 HT29 cells in 20 μL HBSS were dispensed into each well of 384-well tissue culture-treated microtiter plates. The assay was started by immediately dispensing 50 nL of test compound in DMSO, DMSO alone, or rotenone as a positive control (150 μM final concentration) to the appropriate wells. The plates were then incubated for 1 hour at 37 °C. The assay was stopped by dispensing 20 μL of CellTiter-Glo reagent to each well, followed by incubation at room temperature for 15 minutes. Well luminescence was measured on the ViewLux plate reader. Assay Cutoff: compounds with a CC₅₀ ≤ 10 μM were declared active.

Microscopic assay to identify inhibitors of invadopodia formation (AID 434993)

Assay Overview: The purpose of this assay was to determine whether a compound that inhibits NOX1 can block invadopodia formation in DLD1 human colon cancer cells. In this assay, cells were transfected with tyrosine kinase c-Src, which is required for the formation of functional invadopodia. Cells were then treated with test or control compounds, then stained with invadopodia markers phalloidin or cortactin, and then prepared for confocal and epifluorescence microscopy. As designed, a compound that inhibits NOX1 will inhibit invadopodia formation and result in fewer phalloidin- and contactin-positive structures.

Protocol Summary: Human DLD1 cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated fetal bovine serum, 2 mM glutamine, and antibiotics (100 units/ml penicillin and 100 g/ml streptomycin) at 37 °C in 5% CO₂. DLD1 cells were plated on glass coverslips and after 24 hours cells were transfected with active SrcYF or empty vector (mock) in the presence of Lipofectamine 2000. 48 hours after transfection, cells were treated for 1 hour with 10 μM test compound, DMSO, or 10 μM DPI. Afterwards, cells were fixed in 4% paraformaldehyde at room temperature for 10 minutes. Successively, cells were permeabilized in 0.5% Triton for 10 minutes, then blocked in 2% BSA in PBS for 45 minutes at room temperature. Cells were then immunolabeled with appropriate primary and Alexa-Fluor 568-conjugated secondary antibodies. F-actin was detected by using Alexa-Fluor 568-conjugated phalloidin. Cells were mounted on slides with Mowiol mounting medium (Calbiochem) according to the manufacturer’s instructions. Epifluorescence images of fixed cells were acquired on an inverted microscope (Eclipse TE 2000-U, Nikon) equipped with an electronically controlled shutter, filter wheels, and a 14-bit cooled CCD camera (Cool SNAP HQ, Photometrics) controlled by MetaMorph software (Universal Imaging Corp.) by using a 60x/1.4 NA Plan Apo DIC or a 40x/1.4 NA Plan Apo Ph3 objective lens (Nikon). Confocal images were acquired on a spinning disk confocal microscope system, equipped with a CoolSnapHQ camera and 100x/1.4 NA Plan Apo or a 60x/1.4 NA Plan Apo objective lens (Nikon). Invadopodia structures in typical fields were counted. Data were collected from three independent experiments. Assay Cutoff: compounds that exhibited a SEM of p < 0.05 compared to the DMSO control were declared active.

Microscopic assay to identify inhibitors of extracellular matrix (ECM) degradation (AID 488778)

Assay Overview: The purpose of this assay was to determine whether a NOX1 inhibitor compound that was shown to block invadopodia formation in DLD1 human colon cancer cells in a previous assay, “Late-stage microscopic assay to identify inhibitors of NADPH oxidase 1 (NOX1): Inhibition of invadopodia formation” (AID 434993), can block the ability of DLD1 cells to degrade ECM. This assay also tests whether NOX1 overexpression restores the capacity of the DLD1 cells to degrade ECM in the presence of NOX1 inhibitor compound. In this assay, cells are transfected with empty vector, tyrosine kinase c-Src (SrcYF), which is required for the formation of functional invadopodia, or SrcYF plus a NOX1 expression plasmid. After transfection, cells are plated on fluorescently labeled gelatin-coated coverslips. Cells are treated with test compound or control, then stained with invadopodia marker phalloidin (that stains actin), and then prepared for epifluorescence microscopy. As designed, a compound that inhibits NOX1 will inhibit the NOX1-mediated formation of functional invadopodia and reduce the ability of c-Src-transfected DLD1 cells to degrade the ECM

Protocol Summary: Human DLD1 cells were maintained as described above (AID 434993). DLD1 cells were transfected with empty vector, vector with constitutive active Src (SrcYF), or SrcYF plus a NOX1 expresssion plasmid, in the presence of Lipofectamine 2000. 24 hrs after transfection, cells were trypsinized and plated on FITC-labeled gelatin-coated coverslips. After 2 hours, cells were treated with test compound, DPI (positive control), or DMSO (negative control) for 1.5 hours. 24 hours later, cells were fixed in 4% paraformaldehyde at room temperature for 10 minutes. Successively, cells were permeabilized in 0.5% Triton for 10 minutes and blocked in 2% BSA in PBS for 45 minutes at room temperature. Cells were then immunolabeled with Alexa-Fluor 568-conjugated phalloidin and visualized by epifluorescence microscopy (60X) as described above (AID 434993). Quantification from three independent biological experiments is reported for each condition. Assay Cutoff: compounds that exhibited a SEM of p < 0.05 compared to the DMSO control were declared active.

NIMH Psychoactive Drug Screening Program (PDSP) primary assay (AIDs 434974 and 504381)

Assay Overview: The purpose of this panel of radioligand binding assays performed by the NIMH PDSP was to identify a subset of potential receptors, transporters, or ion channels for which the test compound displays affinity. In this assay, test compounds are incubated with radioligand and receptor-containing crude membrane fractions. Bound radioactivity is isolated by filtration onto filter mats and counted. As designed, membrane fractions to which test compound bind will bind less radioligand and decrease the radioactivity measured in the assay.

Protocol Summary: Test and reference compounds were diluted to 5X final assay concentration (50 μM for a final assay concentration of 10 μM) in the appropriate radioligand binding buffer. 50 μL aliquots of buffer (negative control), test compound, and reference compound (positive control) were added in quadruplicate to the wells of a 96-well plate, each of which contained 50 μL 5X radioligand and 100 μL buffer. Receptor-containing, crude membrane fractions were resuspended in an appropriate volume of buffer and dispensed (50 μL per well) into the 96-well plate. Radioligand binding was allowed to equilibrate for 1.5 hours at room temperature, and then bound radioactivity was isolated by filtration onto 0.3% polyethyleneimine-treated, 96-well filter mats using a 96-well Filtermate harverster. The filter mats were dried, scintillant was melted onto the filters, and the radioactivity retained on the filters was counted in a Microbeta scintillation counter. Test compound was tested in quadruplicate. Assay Cutoff: inhibition of > 50% for a particular target was considered active.

NIMH Psychoactive Drug Screening Program (PDSP) secondary assay (AIDs 434953 and 504410)

Assay Overview: The purpose of this panel of radioligand binding assays performed by the NIMH PDSP was to determine Ki values for test compounds in selected assays from the PDSP primary assay (AID 434974). In this assay, test compounds are incubated with radioligand and receptor-containing crude membrane fractions. Bound radioactivity is isolated by filtration onto Whatman glass fiber filters and counted. As designed, membrane fractions to which test compound binds will bind less radioligand and decrease the radioactivity measured in the assay. Compounds were tested in duplicate in an 11-point dilution series starting at a nominal test concentration of 50 μM.

Protocol Summary: A solution of the compound to be tested was prepared as a 1 mg/ml stock in buffer or DMSO according to its solubility. A similar stock of a reference compound (positive control) was also prepared. Eleven dilutions (5X assay concentration) of the test and reference compounds were prepared by serial dilution: 0.05 nM, 0.5 nM, 1.5 nM, 5 nM, 15 nM, 50 nM, 150 nM, 500 nM, 1.5 μM, 5 μM, 50 μM (thus, the corresponding assay concentrations span from 10 pM to 10 μM and include semilog points in the range where high-to-moderate affinity ligands compete with radioligand for binding sites). Radioligand was dispensed into the wells of a 96-well plate. (Typically, the assay concentration of radioligand is a value between one half the KD and the KD of a particular radioligand at its target). Duplicate 50 μL aliquots of the test and reference compound dilutions are added. Then, crude membrane fractions of cells expressing recombinant receptor are dispensed into each well. The 250 μL reactions are incubated at room temperature and shielded from light (to prevent photolysis of light-sensitive ligands), then harvested by rapid filtration onto Whatman glass fiber filters. Filters are placed in scintillation tubes and allowed to dry overnight. The next day scintillation cocktail is added to each tube. The tubes are capped, labeled, and counted by liquid scintillation counting. Assay Cutoff: compounds with a Ki of ≤ 10 μM are considered active.

Table 1 gives an overview of the PubChem assays associated with the NOX1 inhibitor project and indicates which are associated with the current probe development effort for ML171.

Table 1

Overview of PubChem assays for NOX1 inhibitor project.