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_SET 2

Name: Late-stage radioligand binding dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): PDSP screen Ki Set 2.

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, powders, 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.

Receptors

Data Table(All)

Show moreShow less

PID§		Name	Substance		Panel Targets	Description	Additional Information
			Active	Inactive
1		Serotonin receptor 5ht2b	1		5-hydroxytryptamine (serotonin) receptor 2B [Homo sapiens] [gi:119591355]		Taxonomy id: 9606 Gene id: 3357
2		Adrenergic receptor Alpha2A		1	alpha-2A adrenergic receptor [Homo sapiens] [gi:194353970]		Taxonomy id: 9606 Gene id: 150
3		Adrenergic receptor Alpha2B		1	Adrenergic, alpha-2B-, receptor [Homo sapiens] [gi:187953265]		Taxonomy id: 9606 Gene id: 151
4		Adrenergic receptor Alpha2C		1	adrenergic, alpha-2C-, receptor [Homo sapiens] [gi:166706749]		Taxonomy id: 9606 Gene id: 152
5		Dopamine receptor D3		1	Dopamine receptor D3 [Homo sapiens] [gi:118764187]		Taxonomy id: 9606 Gene id: 1814
6		Opioid receptor KOR		1	kappa opioid receptor [Homo sapiens] [gi:39986053]		Taxonomy id: 9606 Gene id: 4986
7		Muscarinic receptor M1		1	Cholinergic receptor, muscarinic 1 [gi:74754970]		Taxonomy id: 9606 Gene id: 1128
8		Muscarinic receptor M2		1	cholinergic receptor, muscarinic 2 [Homo sapiens] [gi:119604271]		Taxonomy id: 9606 Gene id: 1129

---

§ Panel component ID.

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 SID 57287864 in selected assays from the "Late-stage radioligand binding assay to identify inhibitors of NADPH oxidase 1 (NOX1): PDSP screen Set 2" (AID 504381).

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 uL 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 uL 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 -log(IC50) ) )

Where:

Bottom is defined as the residual radioligand binding measured in the presence of 10 muM reference compound (i.e., non-specific binding).
Top is defined as 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 is defined as the assay radioligand concentration.
KD is defined as the affinity constant of the radioligand for the target receptor.

PubChem Activity Outcome and Score:

A Ki of less than or equal to 10,000 is considered active, and a Ki of greater than 10,000 is considered inactive.

List of Reagents:

Reagents were provided by the NIMH Psychoactive Drug Screening Program.

Grant Number: 1 R03 MH083264-01A1