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_Set 2
Name: Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: Family selectivity: Set 2
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.
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.
NOX1, NADPH oxidase 1, cancer, inflammation, 384, inhibitor, inhibition, late stage, HEK/293 cells, luminol, ROS, chemiluminescence, selectivity, family selectivity, NOX2, NOX3, NOX4, Scripps, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Center Network, MLPCN.
The purpose of this cell-based assay is to evaluate the ability of compounds identified as active in previous assays (the "Luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Cherry picks 2" (AID 434997) assay or the "Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Purchased analogs" (AID 2808) assay) to inhibit NOX-2, -3, or -4 activity in a HEK/293 transfection format. This chemiluminescence assay employs a luminol probe to monitor intracellular ROS in HEK/293 cells.
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 assayed in triplicate using a 10-point dilution series starting at a maximum concentration of 10 uM.
For each test compound, percent inhibition was plotted against compound concentration. The reported average IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value for each replicate, and taking the average value. Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.
PubChem Activity Outcome and Score:
Assay 1 (NOX2): The PubChem Activity Score range for active compounds is 100-1, and for inactive compounds 0-0.
Assay 2 (NOX3): The PubChem Activity Score range for active compounds is 100-73, and for inactive compounds 1-0.
Assay 3 (NOX4): The PubChem Activity Score range for active compounds is 100-1. There were no inactive compounds.
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
This assay was performed in the laboratory of the Assay Provider with compounds ordered as powders. The results of our probe development efforts can be found at http://mlpcn.florida.scripps.edu/index.php/probes/probe-reports.html.