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BioAssay: AID 434953

Late-stage radioligand binding dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): PDSP screen Ki

Name: Late-stage radioligand binding dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): PDSP screen Ki ..more
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 Tested Compounds
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Unspecified(1)
 
 
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Unspecified(1)
 
 
AID: 434953
Data Source: The Scripps Research Institute Molecular Screening Center (NOX1_INH_RAD_96_Ki_PDSP Screen)
BioAssay Type: Panel, Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-05-27
Hold-until Date: 2010-08-18
Modify Date: 2010-08-19

Data Table ( Complete ):           View All Data
Target
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)
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)
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)
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)
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)
2541Luminescence-based cell-based assay to identify inhibitors of NADPH oxidase 1 (NOX1)Screening depositor-specified cross reference: Confirmation screen (NOX1 inhibitors)
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)
2556Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: Xanthine OxidaseConfirmatory depositor-specified cross reference: Counterscreen (Xanthine oxidase inhibitors)
2664Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Synthesized analogsConfirmatory depositor-specified cross reference: 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 depositor-specified cross reference: 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 depositor-specified cross reference: Primary screen (NOX1 inhibitors, synthesized analogs set 3)
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
2808Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Purchased analogsConfirmatory same project related to Summary assay
2819Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Purchased analogs 2Confirmatory same project related to Summary assay
434974Late-stage radioligand binding assay to identify inhibitors of NADPH oxidase 1 (NOX1): PDSP screenScreening 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_RAD_96_Ki_PDSP Screen

Name: Late-stage radioligand binding dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): PDSP screen Ki

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, 96, inhibitor, inhibition, Ki, late stage, Psychoactive Drug Screening Program, PDSP, radioligand, radioligand binding assay, receptor, transporter, ion channel, NIMH, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Center Network, MLPCN.
Panel Information
panel - panel
    Data Table(All)Show more
PID§NameSubstancePanel TargetsDescriptionAdditional Information
ActiveInactive
1Serotonin receptor 5ht2b15-hydroxytryptamine (serotonin) receptor 2B [Homo sapiens] [gi:119591355]
Taxonomy id: 9606
Gene id: 3357
2Serotonin receptor 5ht2c15-hydroxytryptamine (serotonin) receptor 2C, isoform CRA_b [Homo sapiens] [gi:119623029]
Taxonomy id: 9606
Gene id: 3358
3Serotonin receptor 5ht415-hydroxytryptamine (serotonin) receptor 4 [Homo sapiens] [gi:238908255]
Taxonomy id: 9606
Gene id: 3360
4Adrenergic receptor Alpha2A1alpha-2A adrenergic receptor [Homo sapiens] [gi:194353970]
Taxonomy id: 9606
Gene id: 150
5Adrenergic receptor Alpha2B1Adrenergic, alpha-2B-, receptor [Homo sapiens] [gi:187953265]
Taxonomy id: 9606
Gene id: 151
6Adrenergic receptor Alpha2C1adrenergic, alpha-2C-, receptor [Homo sapiens] [gi:166706749]
Taxonomy id: 9606
Gene id: 152
7Dopamine receptor D31Dopamine receptor D3 [Homo sapiens] [gi:118764187]
Taxonomy id: 9606
Gene id: 1814
8Muscarinic receptor M31Cholinergic receptor, muscarinic 3 [Homo sapiens] [gi:111306506]
Taxonomy id: 9606
Gene id: 1131
9Muscarinic receptor M41Cholinergic receptor, muscarinic 4 [Homo sapiens] [gi:187953629]
Taxonomy id: 9606
Gene id: 1132
10Muscarinic receptor M51Cholinergic receptor, muscarinic 5 [Homo Sapiens] [gi:74759612]
Taxonomy id: 9606
Gene id: 1133
11mGlu5 RatBrain1glutamate receptor, ionotropic kainate 5 precursor [Rattus norvegicus] [gi:13994161]
Taxonomy id: 10116
Gene id: 24407
12Biogenic amine transporter NET1Net [Homo sapiens] [gi:531523]
Taxonomy id: 9606
Gene id: 2004
13Oxytocin receptor1Oxytocin receptor [Homo sapiens] [gi:187951685]
Taxonomy id: 9606
Gene id: 5021
14Biogenic amine transporter SERT1serotonin transporter [Homo sapiens] [gi:58700442]
Taxonomy id: 9606
Gene id: 6532
15Sigma receptor 11opioid receptor, sigma 1 [Homo sapiens] [gi:123231939]
Taxonomy id: 9606
Gene id: 10280

§ Panel component ID.
Protocol
Assay Overview:
The purpose of this panel of radioligand binding assays performed by the NIMH Psychoactive Drug Screening Program (PDSP) is to determine Ki values for the NOX1 inhibitor compound CID 717880 in selected assays from the "Late-stage radioligand binding assay to identify inhibitors of NADPH oxidase 1 (NOX1): PDSP screen".
Protocol Summary:
A solution of the compound to be tested is prepared as a 1 mg/ml stock in buffer or DMSO according to its solubility. A similar stock of a reference compound (positive control) is also prepared. Eleven dilutions (5X assay concentration) of the test and reference compounds are 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 uM, 5 uM, 50 uM (thus, the corresponding assay concentrations span from 10 pM to 10 uM and include semilog points in the range where high-to-moderate affinity ligands compete with radioligand for binding sites).
Radioligand is 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). Then, duplicate 50 μl aliquots of the test and reference compound dilutions are added. Finally, 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.
Raw data (dpm) representing total radioligand binding (i.e., specific + non-specific binding) are plotted as a function of the logarithm of the molar concentration of the competitor (i.e., test or reference compound). Non-linear regression of the normalized (i.e., percent radioligand binding compared to that observed in the absence of test or reference compound) raw data is performed in Prism 4.0 (GraphPad Software) using the built-in three parameter logistic model describing ligand competition binding to radioligand-labeled sites:
y = bottom + ( ( top - bottom ) / ( 1 + 10x - logIC50 ) )
where bottom equals the residual radioligand binding measured in the presence of 10 μM reference compound (i.e., non-specific binding) and top equals the total radioligand binding observed in the absence of competitor. The log IC50 (i.e., the log of the ligand concentration that reduces radioligand binding by 50%) is thus estimated from the data and used to obtain the Ki by applying the Cheng-Prusoff approximation:
Ki = IC50 / ( 1 + [ligand] / KD )
where [ligand] equals the assay radioligand concentration and KD equals the affinity constant of the radioligand for the target receptor.
A Ki of ≤ 10,000 is considered active, and a Ki of > 10,000 is considered inactive.
PubChem Activity Score and Outcome:
List of Reagents:
Reagents were provided by the NIMH Psychoactive Drug Screening Program.
Result Definitions
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TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1Serotonin 5ht2b (Qualifier)Activity Qualifier identifies if the resultant data Ki came from a fitted curve or was determined manually to be less than or greater than its listed Ki concentration.15-hydroxytryptamine (serotonin) receptor 2B [Homo sapiens]String
2Serotonin 5ht2b (Ki)*Value of Ki (nM)1FloatnM
3Serotonin 5ht2b (Outcome)The Assay outcome, one of Active, Inactive or Not Tested (NT).1Outcome
4Serotonin 5ht2c (Qualifier)Activity Qualifier identifies if the resultant data Ki came from a fitted curve or was determined manually to be less than or greater than its listed Ki concentration.25-hydroxytryptamine (serotonin) receptor 2C, isoform CRA_b [Homo sapiens]String
5Serotonin 5ht2c (Ki)*Value of Ki (nM)2FloatnM
6Serotonin 5ht2c (Outcome)The Assay outcome, one of Active, Inactive or Not Tested (NT).2Outcome
7Serotonin 5ht4 (Qualifier)Activity Qualifier identifies if the resultant data Ki came from a fitted curve or was determined manually to be less than or greater than its listed Ki concentration.35-hydroxytryptamine (serotonin) receptor 4 [Homo sapiens]String
8Serotonin 5ht4 (Ki)*Value of Ki (nM)3FloatnM
9Serotonin 5ht4 (Outcome)The Assay outcome, one of Active, Inactive or Not Tested (NT).3Outcome
10Adrenergic Alpha2A (Qualifier)Activity Qualifier identifies if the resultant data Ki came from a fitted curve or was determined manually to be less than or greater than its listed Ki concentration.4alpha-2A adrenergic receptor [Homo sapiens]String
11Adrenergic Alpha2A (Ki)*Value of Ki (nM)4FloatnM
12Adrenergic Alpha2A (Outcome)The Assay outcome, one of Active, Inactive or Not Tested (NT).4Outcome
13Adrenergic Alpha2B (Qualifier)Activity Qualifier identifies if the resultant data Ki came from a fitted curve or was determined manually to be less than or greater than its listed Ki concentration.5Adrenergic, alpha-2B-, receptor [Homo sapiens]String
14Adrenergic Alpha2B (Ki)*Value of Ki (nM)5FloatnM
15Adrenergic Alpha2B (Outcome)The Assay outcome, one of Active, Inactive or Not Tested (NT).5Outcome
16Adrenergic Alpha2C (Qualifier)Activity Qualifier identifies if the resultant data Ki came from a fitted curve or was determined manually to be less than or greater than its listed Ki concentration.6adrenergic, alpha-2C-, receptor [Homo sapiens]String
17Adrenergic Alpha2C (Ki)*Value of Ki (nM)6FloatnM
18Adrenergic Alpha2C (Outcome)The Assay outcome, one of Active, Inactive or Not Tested (NT).6Outcome
19Dopamine D3 (Qualifier)Activity Qualifier identifies if the resultant data Ki came from a fitted curve or was determined manually to be less than or greater than its listed Ki concentration.7Dopamine receptor D3 [Homo sapiens]String
20Dopamine D3 (Ki)*Value of Ki (nM)7FloatnM
21Dopamine D3 (Outcome)The Assay outcome, one of Active, Inactive or Not Tested (NT).7Outcome
22Muscarinic M3 (Ki)*Value of Ki (nM)8Cholinergic receptor, muscarinic 3 [Homo sapiens]FloatnM
23Muscarinic M3 (Qualifier)Activity Qualifier identifies if the resultant data Ki came from a fitted curve or was determined manually to be less than or greater than its listed Ki concentration.8String
24Muscarinic M3 (Outcome)The Assay outcome, one of Active, Inactive or Not Tested (NT).8Outcome
25Muscarinic M4 (Qualifier)Activity Qualifier identifies if the resultant data Ki came from a fitted curve or was determined manually to be less than or greater than its listed Ki concentration.9Cholinergic receptor, muscarinic 4 [Homo sapiens]String
26Muscarinic M4 (Ki)*Value of Ki (nM)9FloatnM
27Muscarinic M4 (Outcome)The Assay outcome, one of Active, Inactive or Not Tested (NT).9Outcome
28Muscarinic M5 (Qualifier)Activity Qualifier identifies if the resultant data Ki came from a fitted curve or was determined manually to be less than or greater than its listed Ki concentration.10Cholinergic receptor, muscarinic 5 [Homo Sapiens]String
29Muscarinic M5 (Ki)*Value of Ki (nM)10FloatnM
30Muscarinic M5 (Outcome)The Assay outcome, one of Active, Inactive or Not Tested (NT).10Outcome
31Glutamate mGluR5_RatBrain (Qualifier)Activity Qualifier identifies if the resultant data Ki came from a fitted curve or was determined manually to be less than or greater than its listed Ki concentration.11glutamate receptor, ionotropic kainate 5 precursor [Rattus norvegicus]String
32Glutamate mGluR5_RatBrain (Ki)*Value of Ki (nM)11FloatnM
33Glutamate mGluR5_RatBrain (Outcome)The Assay outcome, one of Active, Inactive or Not Tested (NT).11Outcome
34Biogenic amine transporter NET (Qualifier)Activity Qualifier identifies if the resultant data Ki came from a fitted curve or was determined manually to be less than or greater than its listed Ki concentration.12Net [Homo sapiens]String
35Biogenic amine transporter NET (Ki)*Value of Ki (nM)12FloatnM
36Biogenic amine transporter NET (Outcome)The Assay outcome, one of Active, Inactive or Not Tested (NT).12Outcome
37Oxytocin receptor (Qualifier)Activity Qualifier identifies if the resultant data Ki came from a fitted curve or was determined manually to be less than or greater than its listed Ki concentration.13Oxytocin receptor [Homo sapiens]String
38Oxytocin receptor (Ki)*Value of Ki (nM)13FloatnM
39Oxytocin receptor (Outcome)The Assay outcome, one of Active, Inactive or Not Tested (NT).13Outcome
40Biogenic amine transporter SERT (Ki)*Value of Ki (nM)14serotonin transporter [Homo sapiens]FloatnM
41Biogenic amine transporter SERT (Qualifier)Activity Qualifier identifies if the resultant data Ki came from a fitted curve or was determined manually to be less than or greater than its listed Ki concentration.14String
42Biogenic amine transporter SERT (Outcome)The Assay outcome, one of Active, Inactive or Not Tested (NT).14Outcome
43Sigma 1 (Qualifier)Activity Qualifier identifies if the resultant data Ki came from a fitted curve or was determined manually to be less than or greater than its listed Ki concentration.15opioid receptor, sigma 1 [Homo sapiens]String
44Sigma 1 (Ki)*Value of Ki (nM)15FloatnM
45Sigma 1 (Outcome)The Assay outcome, one of Active, Inactive or Not Tested (NT).15Outcome

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

Classification
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