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BioAssay: AID 2532

Late stage results from the probe development effort to identify inhibitors of NOX1: HEK/293 IC50

Name: Late stage results from the probe development effort to identify inhibitors of NOX1: HEK/293 IC50 ..more
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 Tested Compounds
 Tested Compounds
All(6)
 
 
Active(6)
 
 
 Tested Substances
 Tested Substances
All(6)
 
 
Active(6)
 
 
AID: 2532
Data Source: The Scripps Research Institute Molecular Screening Center (NOX1_INH_LUMI_96_3XIC50_HEK/293)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-03-12
Hold-until Date: 2010-08-19
Modify Date: 2010-08-19

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 6
Depositor Specified Assays
Show more
AIDNameTypeProbeComment
1792Luminescence-based primary cell-based high throughput screening assay to identify inhibitors of NADPH oxidase 1 (Nox1): Maybridge Libraryscreening Primary Screen (NOX1)
1796Summary of probe development efforts to identify inhibitors of NADPH oxidase 1 (Nox1)summary2 Summary AID (NOX1)
1823Luminescence-based counterscreen for inhibitors of NADPH oxidase 1 (Nox1): biochemical high throughput screening assay to identify inhibitors of luminol (Maybridge Library)screening Counterscreen (luminal)
2538Luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1)confirmatory Dose response screen (NOX1 inhibitors)
2539Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: Family selectivityscreening Counterscreen (NOX2, NOX3, NOX4 inhibitors)
2541Luminescence-based cell-based assay to identify inhibitors of NADPH oxidase 1 (NOX1)screening Confirmation screen (NOX1 inhibitors)
2545Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: HEK/293 percent inhibitionscreening Confirmation screen (NOX1 inhibitors, HEK/293 cells)
2556Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: Xanthine Oxidaseconfirmatory Counterscreen (Xanthine Oxidase inhibitors)
2664Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Synthesized analogsconfirmatory Primary screen (NOX1 inhibitors, synthesized analogs)
2752Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Synthesized analogs 2confirmatory Primary screen (NOX1 inhibitors, synthesized analogs set 2)
2773Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Synthesized analogs 3confirmatory
2808Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Purchased analogsconfirmatory
2819Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Purchased analogs 2confirmatory
434953Late-stage radioligand binding dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): PDSP screen Kiscreening
434974Late-stage radioligand binding assay to identify inhibitors of NADPH oxidase 1 (NOX1): PDSP screenscreening
434992Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Cytotoxicity assayother
434993Late-stage microscopic assay to identify inhibitors of NADPH oxidase 1 (NOX1): Inhibition of invadopodia formationother
434997Luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Cherry picks 2confirmatory
435002Late stage results from the probe development effort to identify inhibitors of NOX1: HEK/293 IC50 Set 2confirmatory
435009Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: Xanthine Oxidase Set 2confirmatory
435013Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: Family selectivity: Set 2confirmatory
463255Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Cytotoxicity assay 2confirmatory
488778Late-stage microscopic assay to identify inhibitors of NADPH oxidase 1 (NOX1): Inhibition of extracellular matrix degradationother
504381Late-stage radioligand binding assay to identify inhibitors of NADPH oxidase 1 (NOX1): PDSP screen Set 2other
504410Late-stage radioligand binding dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): PDSP screen Ki Set 2confirmatory
Description:
Data Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Gary Bokoch, TSRI
Network: Molecular Libraries Probe Production Center Network (MLPCN)
Grant Proposal Number: 1 R03 MH083264-01A1
Grant Proposal PI: Gary Bokoch, TSRI
External Assay ID: NOX1_INH_LUMI_96_3XIC50_HEK/293

Name: Late stage results from the probe development effort to identify inhibitors of NOX1: HEK/293 IC50

Description:

Host defense mechanisms are diverse and include receptor-initiated signaling pathways, antibody and cytokine production, and the generation of reactive oxygen species (ROS) such as hydroxyl radical and hypochlorus acid to kill microorganisms (1). In activated phagocytic cells, the membrane integrated protein gp91phox serves as the catalytic cytochrome b subunit of the respiratory burst oxidase used to generate superoxide in an NADPH-dependent manner for host defense (2). Generation of ROS has also been identified in non-phagocytic cells (3). One important enzyme involved in ROS production in non-leukocyte tissues is NADPH oxidase 1 (NOX1), a homolog of gp91phox. NOX1 is highly expressed in colon epithelial cells where it can generate ROS to interact with normal and pathogenic bacteria (3-5). However, excess ROS production is associated with damage to the intestinal mucosa, particularly in mucosal lesions of inflammatory bowel disease (IBD) (4). Studies showing that NOX1 levels are increased in human prostate cancer (6) and that cells overexpressing NOX1 have a transformed appearance, exhibit anchorage-independent growth, and induce vascularized tumor formation in athymic mice (3, 7), suggest that NOX1 may also play a role in angiogenesis, cell growth, and tumor pathogenesis (8, 9). The identification of inhibitors of NOX1 may lead to potential candidates for excess cell proliferation, cancer, and IBD.

References:

1. Takeya, R. and Sumimoto, H., Molecular mechanism for activation of superoxide-producing NADPH oxidases. Mol Cells, 2003. 16(3): p. 271-7.
2. Cheng, G., Cao, Z., Xu, X., van Meir, E.G., and Lambeth, J.D., Homologs of gp91phox: cloning and tissue expression of Nox3, Nox4, and Nox5. Gene, 2001. 269(1-2): p. 131-40.
3. Suh, Y.A., Arnold, R.S., Lassegue, B., Shi, J., Xu, X., Sorescu, D., Chung, A.B., Griendling, K.K., and Lambeth, J.D., Cell transformation by the superoxide-generating oxidase Mox1. Nature, 1999. 401(6748): p. 79-82.
4. Szanto, I., Rubbia-Brandt, L., Kiss, P., Steger, K., Banfi, B., Kovari, E., Herrmann, F., Hadengue, A., and Krause, K.H., Expression of NOX1, a superoxide-generating NADPH oxidase, in colon cancer and inflammatory bowel disease. J Pathol, 2005. 207(2): p. 164-76.
5. Rokutan, K., Kawahara, T., Kuwano, Y., Tominaga, K., Nishida, K., and Teshima-Kondo, S., Nox enzymes and oxidative stress in the immunopathology of the gastrointestinal tract. Semin Immunopathol, 2008. 30(3): p. 315-27.
6. Lim, S.D., Sun, C., Lambeth, J.D., Marshall, F., Amin, M., Chung, L., Petros, J.A., and Arnold, R.S., Increased Nox1 and hydrogen peroxide in prostate cancer. Prostate, 2005. 62(2): p. 200-7.
7. Arnold, R.S., Shi, J., Murad, E., Whalen, A.M., Sun, C.Q., Polavarapu, R., Parthasarathy, S., Petros, J.A., and Lambeth, J.D., Hydrogen peroxide mediates the cell growth and transformation caused by the mitogenic oxidase Nox1. Proc Natl Acad Sci U S A, 2001. 98(10): p. 5550-5.
8. Ushio-Fukai, M. and Nakamura, Y., Reactive oxygen species and angiogenesis: NADPH oxidase as target for cancer therapy. Cancer Lett, 2008. 266(1): p. 37-52.
9. Kobayashi, S., Nojima, Y., Shibuya, M., and Maru, Y., Nox1 regulates apoptosis and potentially stimulates branching morphogenesis in sinusoidal endothelial cells. Exp Cell Res, 2004. 300(2): p. 455-62.

Keywords:

NOX1, NADPH oxidase 1, cancer, inflammation, 384, inhibitor, inhibition, late stage, HEK/293 cells, luminol, ROS, chemiluminescence, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Center Network, MLPCN.
Protocol
Assay Overview:

The purpose of this cell-based assay is to determine the potency of compounds identified as active in the "Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: HEK/293 Percent inhibition" (AID 1792) assay. This chemiluminescence assay employs a luminol probe to monitor intracellular ROS in HEK/293 cells.

Protocol Summary:

In this assay, HEK/293 cells seeded into 6-well plates were cotransfected with pRK5-Myc-Nox1, -NoxO1, -NoxA1, and pRK5-myc-Rac1CAQ61L. After 16 hours, test or control compounds were added, followed 2 hours later by luminal 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 triplicate using a 4-point dilution series starting at a maximum concentration of 33 micromolar.

For each test compound, percent inhibition was plotted against compound concentration. The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value. Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.

Any compound with a percent activity value <50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.


PubChem Activity Outcome and Score:

Compounds with an IC50 of <10 micromolar were scored as "Active", and compounds with an IC50 of ≥10 micromolar were scored as "Inactive".

Activity score has been ranked by the potency, with the most potent compounds assigned the highest activity scores.

The PubChem Activity Score range for active compounds is 100-1, there are no inactive compounds.

List of Reagents:

HEK/293 cells
pRK5-Myc-Nox1 DNA
pRK5-Myc-NoxO1 DNA
pRK5-Myc-NoxA1 DNA
pRK5-myc-Rac1CAQ61L DNA
DMEM medium (GIBCO, part 25200)
Hank's Balanced Salt Solution (Invitrogen, part 14025-092)
100X Penicillin-Streptomycin mix (Invitrogen, part 15140)
Trypsin-EDTA solution (Invitrogen, part 25200-056)
Fetal Bovine Serum (Invitrogen, part 16140-071)
Luminol (Sigma, 09253-5g)
Horseradish peroxidase (EMD Bioscience, part 516531-5KU)
DPI (Sigma, D2926)
6-well plates (Corning, part 3516)
96-well plates
Comment
This assay was performed in the laboratory of the Assay Provider with compounds ordered as powders. Details of protocols, compound structures, and results from the original assays can be found in PubChem at the respective AIDs listed below. The results of our probe development efforts can be found at http://mlpcn.florida.scripps.edu/index.php/probes/probe-reports.html.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1QualifierActivity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentrationString
2IC50*The concentration at which 50 percent of the activity in the inhibition assay is observed; (IC50) shown in micromolar.FloatμM
3% Inhibition at 33 uM (33μM**)Value of % Inhibition at 33 uM activator concentration; average of triplicate measurement.Float%
4% Inhibition at 8.3 uM (8.3μM**)Value of % Inhibition at 8.3 uM activator concentration; average of triplicate measurement.Float%
5% Inhibition at 2.0 uM (2μM**)Value of % Inhibition at 2.0 uM activator concentration; average of triplicate measurement.Float%
6% Inhibition at 0.7 uM (0.7μM**)Value of % Inhibition at 0.7 uM activator concentration; average of triplicate measurement.Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH083264-01A1

Data Table (Concise)
Classification
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