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BioAssay: AID 1796

Summary of probe development efforts to identify inhibitors of NADPH oxidase 1 (Nox1)

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 more ..
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 Tested Compounds
 Tested Compounds
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Probe(2)
 
 
Active(2)
 
 
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 Tested Substances
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Probe(2)
 
 
Active(2)
 
 
AID: 1796
Data Source: The Scripps Research Institute Molecular Screening Center (NOX1_INH_LEADS_SUMMARY)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-06-02
Modify Date: 2011-04-13

Data Table ( Complete ):           Active    All
Target
Sequence: NADPH oxidase 1 [Homo sapiens]
Description ..   
Protein Family: NOX_Duox_like_FAD_NADP

Gene:NOX1     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: Chemical Probe: 2    Active: 2
Depositor Specified Assays
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AIDNameTypeComment
1792Luminescence-based primary cell-based high throughput screening assay to identify inhibitors of NADPH oxidase 1 (Nox1): Maybridge LibraryscreeningPrimary screen (NOX1 inhibitors in singlicate)
1823Luminescence-based counterscreen for inhibitors of NADPH oxidase 1 (Nox1): biochemical high throughput screening assay to identify inhibitors of luminol (Maybridge Library)screeningCounterscreen (luminal in singlicate)
2154Late stage results from the probe development effort to identify inhibitors of Nox1.screeningLate stage results (NOX1 inhibitors)
2532Late stage results from the probe development effort to identify inhibitors of NOX1: HEK/293 IC50confirmatoryDose response (NOX1 inhibitors, HEK/293 cells)
2538Luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1)confirmatoryDose response screen (NOX1 inhibitors)
2539Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: Family selectivityscreeningCounterscreen (NOX2, NOX3, NOX4 inhibitors)
2541Luminescence-based cell-based assay to identify inhibitors of NADPH oxidase 1 (NOX1)screeningConfirmation screen (NOX1 inhibitors)
2545Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: HEK/293 percent inhibitionscreeningConfirmation screen (NOX1 inhibitors, HEK/293 cells)
2556Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: Xanthine OxidaseconfirmatoryCounterscreen (Xanthine oxidase inhibitors)
2664Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Synthesized analogsconfirmatoryPrimary screen (NOX1 inhibitors, synthesized analogs)
2752Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Synthesized analogs 2confirmatoryPrimary 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 3confirmatoryPrimary screen (NOX1 inhibitors, synthesized analogs set 3)
2808Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Purchased analogsconfirmatoryPrimary screen (NOX1 inhibitors, purchased analogs)
2819Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Purchased analogs 2confirmatoryPrimary screen (NOX1 inhibitors, purchased analogs set 2)
434974Late-stage radioligand binding assay to identify inhibitors of NADPH oxidase 1 (NOX1): PDSP screenscreeningPsychoactive Drug Screening Program primary screen (NOX1 inhibitors)
434953Late-stage radioligand binding dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): PDSP screen KiscreeningPsychoactive Drug Screening Program secondary screen (NOX1 inhibitors)
434992Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Cytotoxicity assayotherLate stage dose response (Cytotoxicity)
434993Late-stage microscopic assay to identify inhibitors of NADPH oxidase 1 (NOX1): Inhibition of invadopodia formationotherLate stage microscopic assay (Invadopodia inhibition)
434997Luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Cherry picks 2confirmatoryDose response (NOX1 inhibitors, cherry picks 2)
435002Late stage results from the probe development effort to identify inhibitors of NOX1: HEK/293 IC50 Set 2confirmatoryLate stage dose response (NOX1 inhibitors, HEK/293 cells set 2)
435009Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: Xanthine Oxidase Set 2confirmatoryLate stage dose response (NOX1 inhibitors, xanthine oxidase set 2)
435013Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: Family selectivity: Set 2confirmatoryLate stage dose response (NOX1 inhibitors, family selectivity set 2)
463255Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Cytotoxicity assay 2confirmatoryLate stage dose response counterscreen (Cytotoxicity assay 2)
488778Late-stage microscopic assay to identify inhibitors of NADPH oxidase 1 (NOX1): Inhibition of extracellular matrix degradationotherLate stage (ECM degradation inhibitors)
504381Late-stage radioligand binding assay to identify inhibitors of NADPH oxidase 1 (NOX1): PDSP screen Set 2otherPsychoactive Drug Screening Program screen set 2 (NOX1 inhibitors)
504410Late-stage radioligand binding dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): PDSP screen Ki Set 2confirmatoryPsychoactive Drug Screening Program screen Ki set 2 (NOX1 inhibitors)
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Gary Bokoch, TSRI
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 MH083264-01A1
Grant Proposal PI: Gary Bokoch
External Assay ID: NOX1_INH_LEADS_SUMMARY

Name: Summary of probe development efforts to identify inhibitors of NADPH oxidase 1 (Nox1)

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 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.

Summary of Probe Development Effort:

This probe development effort is focused on the identification of NOX1 inhibitors. Following primary HTS of the Maybridge library to identify NOX1 inhibitors, luminol counterscreen, and confirmation of hit activity, a number of compounds of interest emerged. Titration assays to determine compound potency and selectivity, and confirmation of activity in the HEK/293 cell system were performed. Compound SID 26535836, a potent inhibitor of NOX1 with remarkable selectivity for NOX1 over NOX2, 3, and 4, was selected as a probe.

A number of other compounds of interest emerged from the HTS screening, including a phenothiazine. SAR analysis by purchase and by synthesis was undertaken on a number of related compounds. A potent and selective compound, SID 57287864, was chosen as a probe. In order to gain mechanistic insight into the inhibition of NOX1, assays were run to determine whether SID 57287864 could block the formation of functional invadopodia and inhibit extracellular matrix degradation in DLD1 human colon cancer cells transformed with the tyrosine kinase c-Src. Results of these assays showed that SID 57287864 significantly decreased invadopodia formation and the ability of these cells to degrade the extracellular matrix. SID 57287864 was submitted for screening to the NIMH's Psychoactive Drug Screening Program (PDSP) to identify potential off targets; the results indicated that SID 57287864 did not significantly bind most of the receptors tested.

All AIDs that contain results associated with this project can be found in the "Related Bioassays" section of this Summary AID. Two probe reports have been submitted. The results of our probe development efforts can be found at http://mlpcn.florida.scripps.edu/index.php/probes/probe-reports.html. A probe report for SID 26535836 can be found in the Molecular Libraries Bookshelf (PubMed Books) (http://www.ncbi.nlm.nih.gov/books) under ML090. A probe report for SID 57287864 can be found in the Molecular Libraries Bookshelf (PubMed Books) (http://www.ncbi.nlm.nih.gov/books) under ML171.

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:

Summary, NOX1, NADPH oxidase 1, cancer, inflammation, primary, primary screen, HTS, high throughput screen, 384, inhibitor, inhibition, HT29, luminol, ROS, chemiluminescence, Scripps, Maybridge, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Center Network, MLPCN.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1ML #Unique alphanumeric identifier assigned to a chemical probe molecule within the Molecular Libraries Probe Production Centers Network (MLPCN).String
Additional Information
Grant Number: 1 R03 MH083264-01A1

Data Table (Concise)
Classification
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