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BioAssay: AID 463255

Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Cytotoxicity assay 2

Name: Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Cytotoxicity assay 2. ..more
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 Tested Compounds
 Tested Compounds
All(1)
 
 
Inactive(1)
 
 
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 Tested Substances
All(1)
 
 
Inactive(1)
 
 
 Related BioAssays
 Related BioAssays
AID: 463255
Data Source: The Scripps Research Institute Molecular Screening Center (HT29CYTOX_INH_LUMI_384_4XCC50_Cytotox_2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-09-23

Data Table ( Complete ):           View All Data
Tested Compound:
Related Experiments
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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 inhibitors in singlicate)
1796Summary of probe development efforts to identify inhibitors of NADPH oxidase 1 (Nox1)Summary2 depositor-specified cross reference: Summary AID (NOX1 inhibitors)
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 inhibitors in singlicate)
2532Late stage results from the probe development effort to identify inhibitors of NOX1: HEK/293 IC50Confirmatory depositor-specified cross reference: Dose response (NOX1 inhibitors, HEK/293 cells in triplicate)
2538Luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1)Confirmatory depositor-specified cross reference: Dose response screen (NOX1 inhibitors in triplicate)
2539Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: Family selectivityScreening depositor-specified cross reference: Counterscreen (NOX2, NOX3, NOX4 inhibitors in triplicate)
2541Luminescence-based cell-based assay to identify inhibitors of NADPH oxidase 1 (NOX1)Screening depositor-specified cross reference: Confirmation screen (NOX1 inhibitors in triplicate)
2545Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: HEK/293 percent inhibitionScreening depositor-specified cross reference: Confirmation screen (NOX1 inhibitors, HEK/293 cells in singlicate)
2556Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: Xanthine OxidaseConfirmatory depositor-specified cross reference: Dose response (Xanthine oxidase inhibitors in triplicate)
2664Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Synthesized analogsConfirmatory depositor-specified cross reference: Dose response (NOX1 inhibitors, synthesized analogs in triplicate)
2752Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Synthesized analogs 2Confirmatory depositor-specified cross reference: Dose response (NOX1 inhibitors, synthesized analogs set 2 in triplicate)
2773Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Synthesized analogs 3Confirmatory depositor-specified cross reference: Dose response (NOX1 inhibitors, synthesized analogs set 3 in triplicate)
2808Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Purchased analogsConfirmatory depositor-specified cross reference: Dose response (NOX1 inhibitors, purchased analogs in triplicate)
2819Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Purchased analogs 2Confirmatory depositor-specified cross reference: Dose response (NOX1 inhibitors, purchased analogs set 2 in triplicate)
434953Late-stage radioligand binding dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): PDSP screen KiScreening depositor-specified cross reference: Psychoactive Drug Screening Program secondary screen (NOX1 inhibitors)
434974Late-stage radioligand binding assay to identify inhibitors of NADPH oxidase 1 (NOX1): PDSP screenScreening depositor-specified cross reference: Psychoactive Drug Screening Program primary screen (NOX1 inhibitors in quadruplicate)
434992Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Cytotoxicity assayOther depositor-specified cross reference: Counterscreen (Cytotoxicity in triplicate)
434993Late-stage microscopic assay to identify inhibitors of NADPH oxidase 1 (NOX1): Inhibition of invadopodia formationOther depositor-specified cross reference: Late-stage MOA assay (inhibition of invadopodia formation)
434997Luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Cherry picks 2Confirmatory depositor-specified cross reference: Dose response (NOX1 inhibitors in triplicate)
435002Late stage results from the probe development effort to identify inhibitors of NOX1: HEK/293 IC50 Set 2Confirmatory depositor-specified cross reference: Dose response (NOX1 inhibitors, HEK/293 cells in triplicate)
435009Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: Xanthine Oxidase Set 2Confirmatory depositor-specified cross reference: Counterscreen (Xanthine oxidase inhibitors in triplicate)
435013Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: Family selectivity: Set 2Confirmatory depositor-specified cross reference: Counterscreen (NOX2, NOX3, NOX4 inhibitors in triplicate)
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
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: HT29CYTOX_INH_LUMI_384_4XCC50_Cytotox_2

Name: Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Cytotoxicity assay 2.

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 hypochlorous 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 over-expressing 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. & Sumimoto H. Molecular mechanism for activation of superoxide-producing NADPH oxidases. Molecules and cells 2003 Dec 31;16(3):271-7
2. Cheng G., Cao Z., Xu X., van Meir EG. & Lambeth JD. Homologs of gp91phox: cloning and tissue expression of Nox3, Nox4, and Nox5. Gene 2001 May 16;269(1-2):131-40
3. Suh YA., Arnold RS., Lassegue B., Shi J., Xu X., Sorescu D., Chung AB., Griendling KK. & Lambeth JD. Cell transformation by the superoxide-generating oxidase Mox1. Nature 1999 Sep 2;401(6748):79-82
4. Szanto I., Rubbia-Brandt L., Kiss P., Steger K., Banfi B., Kovari E., Herrmann F., Hadengue A. & Krause KH. Expression of NOX1, a superoxide-generating NADPH oxidase, in colon cancer and inflammatory bowel disease. The Journal of pathology 2005 Oct;207(2):164-76
5. Rokutan K., Kawahara T., Kuwano Y., Tominaga K., Nishida K. & Teshima-Kondo S. Nox enzymes and oxidative stress in the immunopathology of the gastrointestinal tract. Seminars in immunopathology 2008 Jul;30(3):315-27
6. Lim SD., Sun C., Lambeth JD., Marshall F., Amin M., Chung L., Petros JA. & Arnold RS. Increased Nox1 and hydrogen peroxide in prostate cancer. The Prostate 2005 Feb 1;62(2):200-7
7. Arnold RS., Shi J., Murad E., Whalen AM., Sun CQ., Polavarapu R., Parthasarathy S., Petros JA. & Lambeth JD. Hydrogen peroxide mediates the cell growth and transformation caused by the mitogenic oxidase Nox1. Proceedings of the National Academy of Sciences of the United States of America 2001 May 8;98(10):5550-5
8. Ushio-Fukai M. & Nakamura Y. Reactive oxygen species and angiogenesis: NADPH oxidase as target for cancer therapy. Cancer letters 2008 Jul 18;266(1):37-52
9. Kobayashi S., Nojima Y., Shibuya M. & Maru Y. Nox1 regulates apoptosis and potentially stimulates branching morphogenesis in sinusoidal endothelial cells. Experimental cell research 2004 Nov 1;300(2):455-62

Keywords:

late stage, late-stage AID, powder, NOX1, NADPH oxidase 1, cancer, inflammation, 384, inhibitor, inhibition, late stage, dose response, HT29, ROS, luciferase, luminescence, CellTiter Glo, cell viability, cytotoxicity, cell-based, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Center Network, MLPCN.
Protocol
Assay Overview:
The purpose of this assay is to determine cytotoxicity of a powder sample of a compound identified as active in a previous assay Late stage results from the probe development effort to identify inhibitors of NOX1: HEK/293 IC50 Set 2 (AID 435002). In this assay, HT29 cells are incubated with test compound, 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 7-point 1:3 dilution series starting at a nominal test concentration of 20 uM.
Protocol Summary:
HT29 cells were grown in Dulbecco's Modified Eagle's Media supplemented with 10% v/v fetal bovine serum, 2 mM L-Glutamine, and 100 U/mL penicillin and streptomycin. Prior to the start of the assay, 2E5 HT29 cells in a 20 uL volume of HBSS were dispensed into wells of a 384-well tissue culture-treated microtiter plates. Test compound was diluted 1:100 in growth medium (100 uM final concentration) and then serially diluted 1:3 in growth medium. The assay was started immediately by dispensing 5 uL of test compound, media alone, or rotenone as a positive control (150 uM final concentration) to the appropriate wells. The plates were then incubated for 2 hours at 37 C. Plate was then equilibrated at room temperature for 30 minutes. The assay was stopped by dispensing 25 uL of CellTiter-Glo reagent to each well, followed by incubation in the dark at room temperature for 10 minutes. Well luminescence was measured on the ViewLux plate reader.
PubChem Activity Outcome and Score:
Compounds with a CC50 value of <= 10 uM were considered active (cytotoxic). Compounds with a CC50 > 10 uM were considered inactive (nontoxic).
Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score range for inactive compounds is 0-0. There are no active compounds.
List of Reagents:
HT29 cells (provided by Assay Provider)
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)
Rotenone (Sigma R8875)
CellTiter Glo (Promega, part S-G7570)
DMSO (Sigma 472301)
150 mm tissue culture dishes (Corning, part 430599)
384-well plates (Corning, 3570)
Comment
This assay was performed with compound ordered as a powder. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that non-specifically modulate luciferase activity, and compounds that quench or emit luminescence within the well.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Type: Toxicity
From ChEMBL:
Assay Format: Cell-based
Assay Type: Functional
Assay Cell Type: HT-29
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1QualifierActivity Qualifier identifies if the resultant data CC50 came from a fitted curve or was determined manually to be less than or greater than its listed CC50 concentration.String
2CC50*The value for concentration at which 50% of surviving cells are observed; CC50 shown in micromolar.FloatμM
3Inhibition at 20 uM [1] (20μM**)Value of % inhibition of growth at 20 uM inhibitor concentration; replicate one.Float%
4Inhibition at 20 uM [2] (20μM**)Value of % inhibition of growth at 20 uM inhibitor concentration; replicate two.Float%
5Inhibition at 20 uM [3] (20μM**)Value of % inhibition of growth at 20 uM inhibitor concentration; replicate three.Float%
6Inhibition at 20 uM [4] (20μM**)Value of % inhibition of growth at 20 uM inhibitor concentration; replicate four.Float%
7Inhibition at 6.7 uM [1] (6.7μM**)Value of % inhibition of growth at 6.7 uM inhibitor concentration; replicate one.Float%
8Inhibition at 6.7 uM [2] (6.7μM**)Value of % inhibition of growth at 6.7 uM inhibitor concentration; replicate two.Float%
9Inhibition at 6.7 uM [3] (6.7μM**)Value of % inhibition of growth at 6.7 uM inhibitor concentration; replicate three.Float%
10Inhibition at 6.7 uM [4] (6.7μM**)Value of % inhibition of growth at 6.7 uM inhibitor concentration; replicate four.Float%
11Inhibition at 2.2 uM [1] (2.2μM**)Value of % inhibition of growth at 2.2 uM inhibitor concentration; replicate one.Float%
12Inhibition at 2.2 uM [2] (2.2μM**)Value of % inhibition of growth at 2.2 uM inhibitor concentration; replicate two.Float%
13Inhibition at 2.2 uM [3] (2.2μM**)Value of % inhibition of growth at 2.2 uM inhibitor concentration; replicate three.Float%
14Inhibition at 2.2 uM [4] (2.2μM**)Value of % inhibition of growth at 2.2 uM inhibitor concentration; replicate four.Float%
15Inhibition at 0.74 uM [1] (0.74μM**)Value of % inhibition of growth at 0.74 uM inhibitor concentration; replicate one.Float%
16Inhibition at 0.74 uM [2] (0.74μM**)Value of % inhibition of growth at 0.74 uM inhibitor concentration; replicate two.Float%
17Inhibition at 0.74 uM [3] (0.74μM**)Value of % inhibition of growth at 0.74 uM inhibitor concentration; replicate three.Float%
18Inhibition at 0.74 uM [4] (0.74μM**)Value of % inhibition of growth at 0.74 uM inhibitor concentration; replicate four.Float%
19Inhibition at 0.25 uM [1] (0.25μM**)Value of % inhibition of growth at 0.25 uM inhibitor concentration; replicate one.Float%
20Inhibition at 0.25 uM [2] (0.25μM**)Value of % inhibition of growth at 0.25 uM inhibitor concentration; replicate two.Float%
21Inhibition at 0.25 uM [3] (0.25μM**)Value of % inhibition of growth at 0.25 uM inhibitor concentration; replicate three.Float%
22Inhibition at 0.25 uM [4] (0.25μM**)Value of % inhibition of growth at 0.25 uM inhibitor concentration; replicate four.Float%
23Inhibition at 0.082 uM [1] (0.082μM**)Value of % inhibition of growth at 0.082 uM inhibitor concentration; replicate one.Float%
24Inhibition at 0.082 uM [2] (0.082μM**)Value of % inhibition of growth at 0.082 uM inhibitor concentration; replicate two.Float%
25Inhibition at 0.082 uM [3] (0.082μM**)Value of % inhibition of growth at 0.082 uM inhibitor concentration; replicate three.Float%
26Inhibition at 0.082 uM [4] (0.082μM**)Value of % inhibition of growth at 0.082 uM inhibitor concentration; replicate four.Float%
27Inhibition at 0.027 uM [1] (0.027μM**)Value of % inhibition of growth at 0.027 uM inhibitor concentration; replicate one.Float%
28Inhibition at 0.027 uM [2] (0.027μM**)Value of % inhibition of growth at 0.027 uM inhibitor concentration; replicate two.Float%
29Inhibition at 0.027 uM [3] (0.027μM**)Value of % inhibition of growth at 0.027 uM inhibitor concentration; replicate three.Float%
30Inhibition at 0.027 uM [4] (0.027μM**)Value of % inhibition of growth at 0.027 uM inhibitor concentration; replicate four.Float%

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

Data Table (Concise)
Data Table ( Complete ):     View All Data
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