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 ..
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)
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.
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.
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.
Categorized Comment - additional comments and annotations
Assay Cell Type: HEK293
From MLP Probe Report:
Probe count: 2
MLP Probe ML# for probe 1: ML090
PubChem Substance ID (SID) for probe 1: 26535836
PubChem Compound ID (CID) for probe 1: 616479
Probe type for probe 1: Inhibitor
IC50/EC50 (nM) for probe 1: 90
Target for probe 1: Nox1 (gi: 83699673)
Anti-target for probe 1: Nox2, Nox3, Nox4
Fold selectivity for probe 1: >100
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK47342/ (ID: 2358652)
Grant number for probe 1: MH083264-01
MLP Probe ML# for probe 2: ML171
PubChem Substance ID (SID) for probe 2: 57287864
PubChem Compound ID (CID) for probe 2: 81131
Probe type for probe 2: Inhibitor
IC50/EC50 (nM) for probe 2: <156
Target for probe 2: NOX1 (gi: 83699673)
Disease relevance for probe 2: Cancer
Anti-target for probe 2: NOX2, NOX3, NOX4, xanthine oxidase
Fold selectivity for probe 2: >20
NCBI Book chapter link for probe 2: http://www.ncbi.nlm.nih.gov/books/NBK98925/ (ID: 2961912)
Grant number for probe 2: MH083264-01A1
PubMed Publication ID (PMID) for probe 2: 20715845
NCBI Book chapter title for probe 1: Probe Report for NOX1 Inhibitors
NCBI Book chapter title for probe 2: Optimization and Characterization of an Inhibitor for NADPH Oxidase 1 (NOX-1)
Data Table (Concise)