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Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.

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Probe Report for NOX1 Inhibitors

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Received: ; Last Update: September 2, 2010.

NADPH oxidase 1 (NOX1) is highly expressed in colon epithelial cells, where it generates reactive oxygen species (ROS) to interact with normal and pathogenic bacteria. Excessive reactive ROS production is associated with damage to the intestinal mucosa, particularly in mucosal lesions of inflammatory bowel disease (IBD). Studies have shown that NOX1 levels are increased in human prostate cancer, and might also play a role in angiogenesis, cell growth, and tumor pathogenesis. The identification of potent, selective inhibitors of NOX1 may lead to potential therapeutic candidates for excess cell proliferation, cancer, and IBD. This project demonstrated that the molecular probe ML090 (CID-616479) is neither a hydrogen peroxide scavenger, nor a general cell toxin on the time scale of cellular NOX inhibition assays. The specificity of the probe for NOX1 over NOX2, 3 and 4 in a 293 assay system suggests that a target specific to the NOX1 system is the molecular target. ML090 should serve as a useful probe for cellular systems where inhibition of NOX1, and not other members of the NOX family, is desired. This compound provides a significant improvement over the previously existing non-selective NOX inhibitor, diphenylene iodium.

Assigned Assay Grant #: 1 R03 MH083264-01A1

Screening Center Name & PI: Scripps Research Institute Molecular Screening Center, H. Rosen, P. Hodder

Chemistry Center Name & PI: Scripps Research Institute Molecular Screening Center, Scripps Research Institute Molecular Screening Center, H. Rosen, W. Roush

Assay Submitter & Institution: Gary Bokoch, TSRI

PubChem Summary Bioassay Identifier (AID): AID-1796

Probe Structure & Characteristics

CID-616479, 5,11-Dihydroquinoxalino[2,3-b]quinoxaline, has been tested in 10 primary screening assays reported in Pubchem and is reported inactive in all of these. In this report, we describe a series of experiments that demonstrate that CID-616479 selectively inhibits NOX1 and is a significant improvement over diphenyl iodium—the existing, nonselective NOX inhibitor.

Image ml090fu1

NOX1 Inhibitor Probe

SID-26535836

CID-616479

ML090

CID/MLSIDTarget NameTarget IC50 micromolarAnti-targetsAnti-target IC50SelectivitySecondary Assay: 293-NOX1 IC50 [AID]
CID-616479/ML090SID-26535836NOX10.09NOX2, NOX3, and NOX4>10 μM>1000.36 micromolar

Recommendations for the scientific use of this probe

This compound is useful for cell-based assays in which it is desirable to specifically block NOX1 activity, including models of cancer cell proliferation and inflammation. Inhibition of NOX blocks reactive oxygen species generation. The current probe, diphenylene iodium, is widely used in research, for example there are 293 references found on searching Pubmed for “DPI and ROS”. This probe is the first selective inhibitor of NOX1 and provides a significant improvement over the widely used, nonspecific compound diphenylene iodium.

1. Scientific Rationale for Project

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 potent, selective inhibitors of NOX1 may lead to potential candidates for excess cell proliferation, cancer, and IBD. The known NOX inhibitors are of low micromolar potencies and are non-selective(10).

2. Project Description

a. The main goal of this project is to find cell based -selective inhibitors of NOX1.

The following goals were listed in the CPDP:

  1. Probes should not induce cell death (page 29, R03 application).
  2. Probes should exhibit saturable inhibitor activity (page 29).
  3. Probes should exhibit inhibitory activity against NOX-1 selectively (page 29), or against other NOX proteins in general (page 32);
  4. Probes should block the NOX1-derived ROS-dependent production of pro-inflammatory cytokines (page 38).

b. Assay implementation and screening

i. PubChem Bioassay Name(s), AID(s) Assay-Type (Primary, DR Counterscreen, Secondary)

Table 2PubChem BioAssays

Assay Brief NameAssay NameAssay TypeTargetPowder SampleCompound Concentration
Maybridge 16K library: primary screen AID-1792Primary cellular high-throughput screening assay to measure NOX1 inhibitionPrimary Assay (1X% INH)NOX1No7 μM
Maybridge 16K library: counterscreen screen Summary AID-1796Primary biochemical high-throughput screening assay to measure H202- luminol inhibitionPrimary Assay (1X% INH)H2O2No7 μM
Maybridge 16K Confirmation screen Summary AID-1796Confirmation cellular high- throughput screening assay to measure NOX1 inhibitionConfirmation Assay (triplicate) CounterscreenNOX1No7 and 3.3 μM
Cytoxicity Summary AID-1796Cellular CytotoxicityAssay (triplicate) ConfirmationCellular ATPNo7 μM
NOX1 293 confirmation Summary AID-1796Confirmation screening cellular assay to measure NOX 1 inhibitionAssay (3X% INH)NOX1Yes100 μM
NOX1 293 dose response Summary AID-1796Dose response cellular assay to measure NOX 1 inhibitionDose Response AssayNOX1Yes5-point, 1:4 dilution starting at 33 μM
NOX2 293 counterscreen Summary AID-1796Counterscreening cellular assay to measure NOX 2 inhibitionCell-based Low Throughput AssayNOX2Yes10 μM
NOX 3 293 counterscreen Summary AID-1796Counterscreening cellular assay to measure NOX 3 inhibitionCell-based Low Throughput AssayNOX3Yes10 μM
NOX 4 293 Summary AID-1796Counterscreening cellular assay to measure NOX 4 inhibitionCell-based Low Throughput AssayNOX4Yes10 μM
Xanthine Oxidase Summary AID-1796Counterscreen for inhibitors of Xanthine OxidaseBiochemical AssayXanthine OxidaseYesDose response

ii. Assay Rationale and Description

Table 3Assay Rationale and Description

AIDAssay RationaleAssay DescriptionZ′S:B
NOX1 HT29 Primary HTS- MaybridgeLibrary AID-1792The purpose of this assay is to measure the ability of compounds to inhibit NOX1 activity.This cell-based assay utilizes a luminol chemiluminescence assay to monitor intracellular reactive oxygen species (ROS) in the transformed colonic epithelial cell line HT29. In this assay, cells are incubated with compounds for 1 hour, and then luminol, and horseradish peroxidase (HRP) are added. Interaction between the luminol and cell-generated ROS yields an unstable endoperoxide and emission of photons that can be detected by a luminometer. As designed, compounds that inhibit NOX1 activity will reduce cellular ROS production, leading to reduced probe-ROS interactions and reduced well luminescence. Compounds were tested in singlicate at a concentration of 7 μM.0.48 ± 0.0818.5 ± 4.4
Maybridge 16K library: counterscreen screen AID-1823The purpose of this assay to eliminate H202 scavenger compounds that would behave as false positives in the primary screening assayThis cell-free assay utilizes a luminol probe-based chemiluminescence assay. In this assay, hydrogen peroxide and test compound are incubated for 1 hour and then luminol and horseradish peroxidase (HRP) are added. Interaction between the luminol and ROS yields an unstable endoperoxide and emission of photons that can be detected by a luminometer. As designed, compounds that scavenge hydrogen peroxide will reduce well luminescence. Compounds were tested in singlicate at a concentration of 7 μM.0.930
Cytotoxicity Assay (Please see AID-1796)The purpose of this assay to eliminate cytotoxic compounds that would behave as false positives in the primary screening assayIn this assay, cells were incubated for 1 hour with test compound. Cell viability was measure with the Cell Titre Glo reagent that measures cellular ATP.NANA
NOX1 HT29 confirmation AID-1796The purpose of this assay is to confirm the ability of compounds to inhibit NOX1 activity.Same as the Primary screen except that the compounds were tested in triplicate at 6.7 and 2.2 micromolarNANA
NOX 1-HEK confirmatory assays AID-1796The purpose of this assay is to confirm the ability of compounds to inhibit NOX1 activity in the NOX1 -293 transfection formatPrior to the transfection, 90% confluent 293 cells are seeded into 6-well plates and incubated overnight. The NOX1 components pRK5-Myc-NOX1, -NOXO1, and -NOXA1and were cotransfected with pRK5-myc-Rac1CA- Q61L the 293 cells in 6 well plates. A total of two micrograms total DNA, combined with four microliters lipofectamine and 100 ul OptiMem was added per well. After 16 hours the media was removed and replaced with 2 ml fresh media containing either the test or control compounds or vehicle control. Following 2 hours treatment at 37 degrees Celsius the cells were trypsinized and the cells from each well were resuspended into 1 mL HBSS. Sixty microliters of the cell suspension was dispensed in wells of a 96 well plate and 60 microliters of HBSS was added assay followed by 80 microlitres of a mixture of luminol and HRP. Chemiluminescence was measured for 30 minutes. Compounds were tested in singlicate at a concentration of 100 μM.NANA
NOX 1 HEK DR confirmation assay AID-1796The purpose of this assay is to determine the potency of compounds to inhibit NOX1 activity in the NOX -293 transfection format.This assay is the same as the NOX1-HEK confirmatory except that the cells were treated with a 10 point dose response of test compound.NANA
NOX -2, -3 and -4 HEK selectivity assays AID-1796The purpose of this assay is to evaluate the ability of compounds to inhibit NOX-2, -3 or -4 activity in the NOX -293 transfection formatIn these selectivity assays the cells are prepared in the same manner as the NOX1-HEK transfection assay except that the cells are transfected with the appropriate expression vectors for each NOX subtype. NOX2: pRK5-Myc- NOX2, pRK5-p67phox, pRK5-p47phox and pRK5-myc-Rac1CA-Q61L. NOX-3: pRK5-Myc-NOX3, pRK5-NOXO1, pRK5-NOXA1and pRK5- myc-Rac1CA-Q61L. NOX-4: pRK5-Myc-NOX4 and pRK5-p22phox. NOX activity was determined by chemiluninescence as for the NOX1 293 assay. Compounds were tested in tripicate at a concentration of 10 μM.NANA
Xanthine Oxidase AID-1796The purpose of this assay is to evaluate the ability of compounds to inhibit ROS production by another cellular sources such as xanthine oxidaseIn this assay, 50 microliters of a solution of 0.25 U/ml of xanthine oxidase in HBSS (0.5 mg/mL) was dispensed to all wells of a 96 well plate followed by 1 microliters of 1 mM DPI, DMSO or test compounds. The plates were incubated for 5 minutes at room temperature and then 50 microliters of 2 mM hypoxanthine was dispensed to all wells followed by 80 microliters of HRP/luminol mix and reading luminesenceNANA

NA, Not Applicable

Table 4Reagents and Source

AssayReagent (Source)
Primary and confirmation screensHT29 cells (ATCC, part HTB-38)
DPI (Sigma, part D2926-10mg)
Luminol (Sigma, part 09253-5g)
HRP (EMD Bioscience, part 516531-5KU).
HBSS(Invitrogen, part 14025-092)
384-well plates (Corning, part 3704)
Hydrogen peroxide scavenger CounterscreenSame as above, except that HT29 cells and DPI are not used and Hydrogen Peroxide (Sigma, part 216763)
N-acetyl cysteine (EMD, part 106425)
Cytotoxicity AssayHT29 cells (ATCC, part HTB-38)
DPI (Sigma, part D2926-10mg)
Cell Titre Glo (Promega, part G7572)
Rotenone (Sigma Part
NOX 1-4 HEK selectivity assays293 cells (ATCC, part CRL-1573)
DPI (Sigma, part D2926-10mg)
Luminol (Sigma, part 09253-5g)
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 (Corning part Add Number
Xanthine Oxidase CounterscreenXanthine oxidase (Sigma, part X4376)
Hypoxanthine (Sigma, part H9377)

iii. Summary of Results

Analysis of the NOX1 primary screening campaign identified 131 hits from NOX1-primary screen. The full deck cell-free H2O2-based counterscreen assay eliminated 31 compounds because they demonstrated significant H2O2 scavenger activity. The available hit compounds (96) were cherry picked and run in the cell based confirmation assay for NOX1 activity. The compounds were tested in triplicate at 6.7 and 2.2 micromolar. These same cherry picked hits were then tested for cellular toxicity. None showed significant cellular toxicity after 1 hr of incubation, the same incubation time as for the primary and confirmatory assays. The results of the confirmatory assay were rank ordered and 44 compounds were selected and obtained as powders. The remaining experiments were conducted with these samples. The repurchased compounds were tested for their ability to inhibit ROS production in 293 cells transfected with all NOX1 components at 100 micromolar concentration. In this assay, only 11 compounds out of 44 tested were able to significantly block ROS generation. These 11 compounds were tested in dose-response starting at 33 micromolar in the NOX1-293 system. Only 7 compounds were able to significantly block ROS generation at this concentration. These 7 compounds were tested for their ability to block ROS generation by other NOX family members (NOX2, NOX3 and NOX4) in the 293 transfection system. The 7 compounds active in the 293 NOX1 system were also tested for their ability to block ROS generation mediated by xanthine oxidase, another cellular source of ROS. The most potent NOX1 inhibitor (in the 293 transfection assay) also demonstrated remarkable selectivity for NOX 1 over NOX2, 3 and 4. The data from the selectivity assays for CID-616479 are shown in the Summary Table below. CID-616479 represents an improvement over known NOX1 inhibitors (e.g. DPI) because of its selectivity for NOX1 over NOX2, NOX3 and NOX4.

c. Probe Optimization

i. SAR and Chemistry Strategy that led to the probe

The probe molecule is a primary screening hit.

3. Probe

a. Chemical name

6,11-dihydroquinoxalino[2,3-b]quinoxaline [ML090]

b. Chemical structure

Image ml090fu2

c. Structural Verification Information of probe SID

Maybridge states that the purity is greater than 90%. An LC-MS consistent with this purity was run on an aliquot of the sample for the MLSMR.

d. PubChem CID (corresponding to the SID)

CID-616479

e. Available from a vendor

Maybridge, part JFD 00196

f. Mode of action for biological activity

The generation of reactive oxygen species (ROS) by NOX enzymes requires multiple protein components. We have shown that the probe molecule is not a hydrogen peroxide scavenger, nor is a general cell toxin on the time scale of the cellular NOX inhibition assays. The specificity of the probe for NOX1 over NOX2, 3 and 4 in the 293 assay system suggest that a target specific to the NOX1 system is the molecular target. CID-616479 should be a useful probe of cellular systems where inhibition of NOX1 and not NOX 2, NOX3 or NOX4 is desired.

g. Detailed synthetic pathway for making probe

Not applicable, the probe is commercially available.

h. Summary of probe properties

Aqueous solubility, -3.42750688310091; ADMET BBB, 0.21; ADMET BBB level, 1; ADMET absorption level, 0; ADMET solubility, -5.215; ADMET solubility level, 2

i. Summary of known probe properties

Table 5Probe Properties

PubChem CIDCID-616479
PubChem SIDSID-26535836
IUPAC Name6,11-dihydroquinoxalino[2,3-b]quinoxaline
MLS002345386
MFC14H10N4
MW234.256
Formal Charge0
H Acceptor4
H Donor2
Atom Count18
Rotatable Bonds0
Rings4
Stereoatoms0
AlogP3.642
logD3.642
Polar surface area49.84
Aqueous solubilitya−3.42750688310091
ADMET BBBb0.21
ADMET BBB levelc1
ADMET absorption leveld0
ADMET solubilitye−5.215
ADMET solubility levelf2
VendorMaybridge
Vendor Catalog NumberJFD 00196
a

Aqueous solubility is expressed as logS, where S is solubility in mol/L. The method used is the multiple linear regression model based on Electrotopological State indices published in (11, 12).

b

ADMET_BBB: Log of Brain/Blood partition coefficient (LogBB). See (13) for details on this method.

c

ADMET_BBB_Level: Ranking of the LogBB values into one of the following levels (see (13, 14) for details): 0: Very High 1: High 2: Medium 3: Low 4: Undefined (molecule is outside the confidence area of the regression model).

d

ADMET Passive Intestinal Absorption properties. A ranking of the molecule into one of the following levels (see (13, 14) for details): 0: Good 1: Moderate 2: Poor 3: Very Poor

e

ADMET_Solubility: Log of the water solubility at 25 degrees, LogSw, in mol/L. See (11) for more information.

4. Appendices

a. Comparative data on (1) probes, (2) similar compound structures (establishing SAR) and (3) prior probes

Table 6Summary

Compound IDNOX1 IC50 (μM) HT29NOX1-293 IC50 (μM)NOX2-293 %INH at 10 μMNOX3-293 %INH at 10 μMNOX4-293 %INH at 10 μMXanthine Oxidase
CID-3101 Diphenylene iodium (DPI)0.17N.D. (100% inhibition at 10 μM)100100100N.D.1
CID-6164790.090.362.2−23−230.6

N.D. Not determined.

1

The IC50 is reported in the literature to be in the low micromolar range (15).

5. Bibliography

1.
Takeya R, Sumimoto H. Molecular mechanism for activation of superoxide-producing NADPH oxidases. Mol Cells. 2003;16:271–277. [PubMed: 14744014]
2.
Cheng G, Cao Z, Xu X, van Meir EG, Lambeth JD. Homologs of gp91phox: cloning and tissue expression of Nox3, Nox4, and Nox5. Gene. 2001;269:131–140. [PubMed: 11376945]
3.
Suh YA, Arnold RS, Lassegue B, Shi J, Xu X, Sorescu D, Chung AB, Griendling KK, Lambeth JD. Cell transformation by the superoxide-generating oxidase Mox1. Nature. 1999;401:79–82. [PubMed: 10485709]
4.
Szanto I, Rubbia-Brandt L, Kiss P, Steger K, Banfi B, Kovari E, Herrmann F, Hadengue A, Krause KH. Expression of NOX1, a superoxide-generating NADPH oxidase, in colon cancer and inflammatory bowel disease. J Pathol. 2005;207:164–176. [PubMed: 16086438]
5.
Rokutan K, Kawahara T, Kuwano Y, Tominaga K, Nishida K, Teshima-Kondo S. Nox enzymes and oxidative stress in the immunopathology of the gastrointestinal tract. Semin Immunopathol. 2008;30:315–327. [PubMed: 18521607]
6.
Lim SD, Sun C, Lambeth JD, Marshall F, Amin M, Chung L, Petros JA, Arnold RS. Increased Nox1 and hydrogen peroxide in prostate cancer. Prostate. 2005;62:200–207. [PubMed: 15389790]
7.
Arnold RS, Shi J, Murad E, Whalen AM, Sun CQ, Polavarapu R, Parthasarathy S, Petros JA, Lambeth JD. Hydrogen peroxide mediates the cell growth and transformation caused by the mitogenic oxidase Nox1. Proc Natl Acad Sci U S A. 2001;98:5550–5555. [PMC free article: PMC33250] [PubMed: 11331784]
8.
Ushio-Fukai M, Nakamura Y. Reactive oxygen species and angiogenesis: NADPH oxidase as target for cancer therapy. Cancer Lett. 2008;266:37–52. [PMC free article: PMC2673114] [PubMed: 18406051]
9.
Kobayashi S, Nojima Y, Shibuya M, Maru Y. Nox1 regulates apoptosis and potentially stimulates branching morphogenesis in sinusoidal endothelial cells. Exp Cell Res. 2004;300:455–462. [PubMed: 15475009]
10.
Jaquet V, Scapozza L, Clark R, Krause KH, Lambeth JD. Small Molecule NOX Inhibitors: ROS-generating NADPH Oxidases as Therapeutic Targets. Antioxidants & redox signaling. 2009 [PubMed: 19309261]
11.
Cheng A, Merz KM Jr. Prediction of aqueous solubility of a diverse set of compounds using quantitative structure-property relationships. Journal of medicinal chemistry. 2003;46:3572–3580. [PubMed: 12904062]
12.
Tetko IV, Tanchuk VY, Kasheva TN, Villa AE. Estimation of aqueous solubility of chemical compounds using E-state indices. Journal of chemical information and computer sciences. 2001;41:1488–1493. [PubMed: 11749573]
13.
Egan WJ, Merz KM Jr, Baldwin JJ. Prediction of drug absorption using multivariate statistics. Journal of medicinal chemistry. 2000;43:3867–3877. [PubMed: 11052792]
14.
Egan WJ, Lauri G. Prediction of intestinal permeability. Advanced drug delivery reviews. 2002;54:273–289. [PubMed: 11922948]
15.
Aldieri E, Riganti C, Polimeni M, Gazzano E, Lussiana C, Campia I, Ghigo D. Classical inhibitors of NOX NAD(P)H oxidases are not specific. Current drug metabolism. 2008;9:686–696. [PubMed: 18855607]

Aqueous solubility is expressed as logS, where S is solubility in mol/L. The method used is the multiple linear regression model based on Electrotopological State indices published in (11, 12).

ADMET_BBB: Log of Brain/Blood partition coefficient (LogBB). See (13) for details on this method.

ADMET_BBB_Level: Ranking of the LogBB values into one of the following levels (see (13, 14) for details): 0: Very High 1: High 2: Medium 3: Low 4: Undefined (molecule is outside the confidence area of the regression model).

ADMET Passive Intestinal Absorption properties. A ranking of the molecule into one of the following levels (see (13, 14) for details): 0: Good 1: Moderate 2: Poor 3: Very Poor

ADMET_Solubility: Log of the water solubility at 25 degrees, LogSw, in mol/L. See (11) for more information.

The IC50 is reported in the literature to be in the low micromolar range (15).

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