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BioAssay: AID 2539

Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: Family selectivity

Name: Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: Family selectivity ..more
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 Tested Compounds
 Tested Compounds
All(7)
 
 
Active(5)
 
 
Inactive(2)
 
 
 Tested Substances
 Tested Substances
All(7)
 
 
Active(5)
 
 
Inactive(2)
 
 
AID: 2539
Data Source: The Scripps Research Institute Molecular Screening Center (NOX1_INH_LUMI_96_3X%INH_Selectivity)
BioAssay Type: Panel, Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-03-13
Hold-until Date: 2010-08-18
Modify Date: 2010-08-19

Data Table ( Complete ):           View Active Data    View All Data
Targets
BioActive Compounds: 5
Related Experiments
Show more
AIDNameTypeProbeComment
1792Luminescence-based primary cell-based high throughput screening assay to identify inhibitors of NADPH oxidase 1 (Nox1): Maybridge LibraryScreening depositor-specified cross reference: Primary Screen (NOX1)
1796Summary of probe development efforts to identify inhibitors of NADPH oxidase 1 (Nox1)Summary2 depositor-specified cross reference: 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 depositor-specified cross reference: Counterscreen (luminal)
2532Late stage results from the probe development effort to identify inhibitors of NOX1: HEK/293 IC50Confirmatory depositor-specified cross reference
2538Luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1)Confirmatory depositor-specified cross reference
2556Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: Xanthine OxidaseConfirmatory depositor-specified cross reference
2664Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Synthesized analogsConfirmatory depositor-specified cross reference
2752Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Synthesized analogs 2Confirmatory depositor-specified cross reference
2773Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Synthesized analogs 3Confirmatory depositor-specified cross reference
2808Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Purchased analogsConfirmatory depositor-specified cross reference
2819Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Purchased analogs 2Confirmatory depositor-specified cross reference
434953Late-stage radioligand binding dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): PDSP screen KiScreening depositor-specified cross reference
434974Late-stage radioligand binding assay to identify inhibitors of NADPH oxidase 1 (NOX1): PDSP screenScreening depositor-specified cross reference
434992Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Cytotoxicity assayOther depositor-specified cross reference
434993Late-stage microscopic assay to identify inhibitors of NADPH oxidase 1 (NOX1): Inhibition of invadopodia formationOther depositor-specified cross reference
434997Luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Cherry picks 2Confirmatory depositor-specified cross reference
435002Late stage results from the probe development effort to identify inhibitors of NOX1: HEK/293 IC50 Set 2Confirmatory depositor-specified cross reference
435009Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: Xanthine Oxidase Set 2Confirmatory depositor-specified cross reference
435013Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: Family selectivity: Set 2Confirmatory depositor-specified cross reference
463255Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Cytotoxicity assay 2Confirmatory depositor-specified cross reference
488778Late-stage microscopic assay to identify inhibitors of NADPH oxidase 1 (NOX1): Inhibition of extracellular matrix degradationOther depositor-specified cross reference
504381Late-stage radioligand binding assay to identify inhibitors of NADPH oxidase 1 (NOX1): PDSP screen Set 2Other depositor-specified cross reference
504410Late-stage radioligand binding dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): PDSP screen Ki Set 2Confirmatory depositor-specified cross reference
2154Late stage results from the probe development effort to identify inhibitors of Nox1.Screening same project related to Summary assay
2541Luminescence-based cell-based assay to identify inhibitors of NADPH oxidase 1 (NOX1)Screening same project related to Summary assay
2545Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: HEK/293 percent inhibitionScreening same project related to Summary assay
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_3X%INH_Selectivity

Name: Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: Family selectivity

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, selectivity, family selectivity, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Center Network, MLPCN.
Panel Information
NOX1 Family Selectivity Assays
PID§NameSubstancePanel TargetsDescriptionAdditional Information
ActiveInactive
1NOX2 Selectivity7Cytochrome b-245, beta polypeptide [Homo sapiens] [gi:21618561]
Taxonomy id: 9606
Gene id: 1536
2NOX3 Selectivity25NADPH oxidase 3 [Homo sapiens] [gi:119568074]
Taxonomy id: 9606
Gene id: 50508
3NOX4 Selectivity25NADPH oxidase 4 [Homo sapiens] [gi:25304044]
Taxonomy id: 9606
Gene id: 50507

§ Panel component ID.
Protocol
Assay Overview:

The purpose of this cell-based assay is to evaluate the ability of compounds to inhibit NOX-2, -3, or -4 activity in the NOX1-HEK/293 transfection format. This chemiluminescence assay employs a luminol probe to monitor intracellular ROS in HEK/293 cells.

Protocol Summary:

In the family selectivity assays, HEK/293 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, 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 tested in triplicate at a concentration of 10 micromolar.

Active Score:

Compounds with ≥50% inhibitory activity were scored as "Active", and compounds with < 50% inhibitory activity were scored as "Inactive".

List of Reagents:

293 cells (ATCC, part CRL-1573)
DPI (Sigma, part D2926-10 mg)
Luminol (Sigma, part 09253-5 g)
HRP (EMD Bioscience, part 516531-5KU).
Lipofectamine 2000 (Invitrogen 11688-019)
HBSS (Invitrogen, part 14025-092
OptiMem (Invitrogen, part 31985)
pRK5-Myc-Nox1 (Bokoch Lab)
pRK5-Myc-Nox2 (Bokoch Lab)
pRK5-NoxO1 (Bokoch Lab)
pRK5-NoxA1 (Bokoch Lab)
pRK5-myc-Rac1CA-Q61L (Bokoch Lab)
pRK5-p67phox(Bokoch Lab)
pRK5-p47phox(Bokoch Lab)
pRK5-p22phox(Bokoch Lab)
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.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: HEK293
Result Definitions
TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1NOX2 Selectivity (Inhibition) (10μM**)Normalized percent inhibition of the primary screen at a nominal compound concentration of 10 micromolar.1Cytochrome b-245, beta polypeptide [Homo sapiens]Float%
2NOX2 Selectivity (Outcome)The Assay outcome, one of Active, Inactive or Not Tested.1Outcome
3NOX3 Selectivity (Inhibition) (10μM**)Normalized percent inhibition of the primary screen at a nominal compound concentration of 10 micromolar.2NADPH oxidase 3 [Homo sapiens]Float%
4NOX3 Selectivity (Outcome)The Assay outcome, one of Active, Inactive or Not Tested.2Outcome
5NOX4 Selectivity (Inhibition) (10μM**)Normalized percent inhibition of the primary screen at a nominal compound concentration of 10 micromolar.3NADPH oxidase 4 [Homo sapiens]Float%
6NOX4 Selectivity (Outcome)The Assay outcome, one of Active, Inactive or Not Tested.3Outcome

** Test Concentration. § Panel component ID.
Additional Information
Grant Number: 1 R03 MH083264-01A1

Classification
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