Luminescence-based primary cell-based high throughput screening assay to identify inhibitors of NADPH oxidase 1 (Nox1): Maybridge Library
Name: Luminescence-based primary cell-based high throughput screening assay to identify inhibitors of NADPH oxidase 1 (Nox1): Maybridge Library ..more
BioActive Compounds: 130
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_LUMI_384_%INH
Name: Luminescence-based primary cell-based high throughput screening assay to identify inhibitors of NADPH oxidase 1 (Nox1): Maybridge Library
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, primary, primary screen, Maybridge, HTS, high throughput screen, 384, inhibitor, inhibition, HT29, luminol, ROS, chemiluminescence, 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 identify compounds in the Maybridge collection that inhibit Nox1 activity. This chemiluminescence assay employs a luminol probe to monitor intracellular ROS in the HT29 transformed colonic epithelial cell line. HT29 cells express high endogenous levels of known Nox1 components and no other Nox family members. In this assay, the cells are incubated with test compounds, cell permeable luminol, 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 singlicate at a final nominal concentration of 6.7 uM.
HT29 cells were routinely cultured in 150 mm dishes at 37 degrees C and 95% relative humidity (RH). The growth media consisted of Dulbecco's Modified Eagle's Media (DMEM) supplemented with 10% v/v fetal bovine serum, 2 mM L-Glutamine, and 100U/mL penicillin and streptomycin.
Prior to the start of the assay, cells were suspended to a concentration of 2.25 million cells/mL in Hank's Balanced Salt Solution. Next, 30 ul of cell suspension (67,500 cells) were dispensed into each well of 384-well tissue culture-treated microtiter plates and the plates were centrifuged briefly. The assay was started by dispensing 100 nL of test compound in DMSO, diphenylene iodonium (DPI) in DMSO, or DMSO alone (0.33% final concentration) to the appropriate wells. Next, the plates were incubated for 1 hour at 37 degrees C, 5% CO2
and 95% RH, followed by the addition of 20 ul of a solution containing 400U/mL horseradish peroxidase and 50 mM luminol mix to all wells. The plates were centrifuged briefly and incubated for 10 minutes at 37 degrees C (5% CO2, 95% RH). Well luminescence was measured on the EnVision plate reader (Perkin Elmer).
The percent inhibition for each compound was calculated as follows:
%Inhibition=[1-((Test_Compound-Median_High_Control)/(Median_Low_Control- Median__High_Control))] *100
Test_Compound is defined as wells containing cells in the presence of test compound.
Low_Control is defined as wells containing cells in the presence of DMSO.
High_Control is defined as wells containing cells in the presence of DPI.
For this Primary assay, test compounds that exhibited greater % inhibition than the assigned cutoff value for the screen were declared active.
The reported PubChem Activity Score has been normalized to 100% of the highest observed primary inhibition value. Negative % inhibition values are reported as activity score zero.
The inactive compounds of this assay have activity score range of 0 to 69 and active compounds range of activity score is 69 to 100.
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)
Luminol (Sigma, 09253-5g)
Horseradish peroxidase (EMD Bioscience, part 516531-5KU)
DPI (Sigma, D2926)
150 mm tissue culture dishes (Corning, part 430599)
384-well plates (Corning, 3704)
This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. In this assay, DPI had an IC50 of approximately 0.17 uM. 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. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)