Name: Late-stage radioligand binding assay to identify inhibitors of NADPH oxidase 1 (NOX1): PDSP screen Set 2 ..more
Tested Compound:
Depositor Specified Assays
Show more |
| AID | Name | Type | Probe | Comment |
|---|---|---|---|---|
| 1792 | Luminescence-based primary cell-based high throughput screening assay to identify inhibitors of NADPH oxidase 1 (Nox1): Maybridge Library | screening | Primary screen (NOX1 inhibitors in singlicate) | |
| 1796 | Summary of probe development efforts to identify inhibitors of NADPH oxidase 1 (Nox1) | summary | 2 | Summary (NOX1 inhibitors) |
| 1823 | Luminescence-based counterscreen for inhibitors of NADPH oxidase 1 (Nox1): biochemical high throughput screening assay to identify inhibitors of luminol (Maybridge Library) | screening | Counterscreen (luminal in singlicate) | |
| 2532 | Late stage results from the probe development effort to identify inhibitors of NOX1: HEK/293 IC50 | confirmatory | Dose response (NOX1 inhibitors, HEK/293 cells) | |
| 2538 | Luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1) | confirmatory | Dose response screen (NOX1 inhibitors) | |
| 2539 | Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: Family selectivity | screening | Counterscreen (NOX2, NOX3, NOX4 inhibitors) | |
| 2541 | Luminescence-based cell-based assay to identify inhibitors of NADPH oxidase 1 (NOX1) | screening | Confirmation screen (NOX1 inhibitors) | |
| 2545 | Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: HEK/293 percent inhibition | screening | Confirmation screen (NOX1 inhibitors, HEK/293 cells) | |
| 2556 | Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: Xanthine Oxidase | confirmatory | Counterscreen (Xanthine oxidase inhibitors) | |
| 2664 | Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Synthesized analogs | confirmatory | Primary screen (NOX1 inhibitors, synthesized analogs) | |
| 2752 | Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Synthesized analogs 2 | confirmatory | Primary screen (NOX1 inhibitors, synthesized analogs set 2) | |
| 2773 | Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Synthesized analogs 3 | confirmatory | Primary screen (NOX1 inhibitors, synthesized analogs set 3) | |
| 2808 | Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Purchased analogs | confirmatory | Primary screen (NOX1 inhibitors, purchased analogs) | |
| 2819 | Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Purchased analogs 2 | confirmatory | Primary screen (NOX1 inhibitors, purchased analogs set 2) | |
| 434974 | Late-stage radioligand binding assay to identify inhibitors of NADPH oxidase 1 (NOX1): PDSP screen | screening | Psychoactive Drug Screening Program primary screen (NOX1 inhibitors) | |
| 434953 | Late-stage radioligand binding dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): PDSP screen Ki | screening | Psychoactive Drug Screening Program secondary screen (NOX1 inhibitors) | |
| 434992 | Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Cytotoxicity assay | other | Late stage dose response (Cytotoxicity) | |
| 434993 | Late-stage microscopic assay to identify inhibitors of NADPH oxidase 1 (NOX1): Inhibition of invadopodia formation | other | Late stage microscopic assay (Invadopodia inhibition) | |
| 434997 | Luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Cherry picks 2 | confirmatory | Dose response (NOX1 inhibitors, cherry picks 2) | |
| 435002 | Late stage results from the probe development effort to identify inhibitors of NOX1: HEK/293 IC50 Set 2 | confirmatory | Late stage dose response (NOX1 inhibitors, HEK/293 cells set 2) | |
| 435009 | Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: Xanthine Oxidase Set 2 | confirmatory | Late stage dose response (NOX1 inhibitors, xanthine oxidase set 2) | |
| 435013 | Late stage results from the probe development effort to identify inhibitors of (NADPH oxidase 1) NOX1: Family selectivity: Set 2 | confirmatory | Late stage dose response (NOX1 inhibitors, family selectivity set 2) | |
| 463255 | Late-stage luminescence-based cell-based dose response assay to identify inhibitors of NADPH oxidase 1 (NOX1): Cytotoxicity assay 2 | confirmatory | Late stage dose response counterscreen (Cytotoxicity assay 2) | |
| 488778 | Late-stage microscopic assay to identify inhibitors of NADPH oxidase 1 (NOX1): Inhibition of extracellular matrix degradation | other | Late stage (ECM degradation inhibitors) |
Description:
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_4X%INH_PDSP SCREEN_SET 2
Name: Late-stage radioligand binding assay to identify inhibitors of NADPH oxidase 1 (NOX1): PDSP screen 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, 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.
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_4X%INH_PDSP SCREEN_SET 2
Name: Late-stage radioligand binding assay to identify inhibitors of NADPH oxidase 1 (NOX1): PDSP screen 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, 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.
Panel Information
Receptors
§ Panel component ID.
| Data Table(All) | Show more |
| PID§ | Name | Substance | Panel Targets | Description | Additional Information | ||
|---|---|---|---|---|---|---|---|
| Active | Inactive | ||||||
| 1 | Serotonin receptor 5ht1a | 1 | 5-hydroxytryptamine (serotonin) receptor 1A [Homo sapiens] [gi:119571762] | Taxonomy id: 9606 Gene id: 3350 | |||
| 2 | Serotonin receptor 5ht1b | 1 | 5-hydroxytryptamine (serotonin) receptor 1B [Homo sapiens] [gi:119569107] | Taxonomy id: 9606 Gene id: 3351 | |||
| 3 | Serotonin receptor 5ht1d | 1 | 5-hydroxytryptamine (serotonin) receptor 1D [Homo sapiens] [gi:119615443] | Taxonomy id: 9606 Gene id: 3352 | |||
| 4 | Serotonin receptor 5ht1e | 1 | 5-hydroxytryptamine (serotonin) receptor 1E [Homo sapiens] [gi:10635353] | Taxonomy id: 9606 Gene id: 3354 | |||
| 5 | Serotonin receptor 5ht2a | 1 | 5-hydroxytryptamine (serotonin) receptor 2A [Homo sapiens] [gi:119629175] | Taxonomy id: 9606 Gene id: 3356 | |||
| 6 | Serotonin receptor 5ht2b | 1 | 5-hydroxytryptamine (serotonin) receptor 2B [Homo sapiens] [gi:119591355] | Taxonomy id: 9606 Gene id: 3357 | |||
| 7 | Serotonin receptor 5ht2c | 1 | 5-hydroxytryptamine (serotonin) receptor 2C, isoform CRA_b [Homo sapiens] [gi:119623029] | Taxonomy id: 9606 Gene id: 3358 | |||
| 8 | Serotonin receptor 5ht3 | 1 | 5-hydroxytryptamine (serotonin) receptor 3A, isoform CRA_c [Homo sapiens] [gi:119587644] | Taxonomy id: 9606 Gene id: 3359 | |||
| 9 | Serotonin receptor 5ht5a | 1 | 5-hydroxytryptamine (serotonin) receptor 5A [Homo sapiens] [gi:119624931] | Taxonomy id: 9606 Gene id: 3361 | |||
| 10 | Serotonin receptor 5ht6 | 1 | 5-hydroxytryptamine (serotonin) receptor 6 [Homo sapiens] [gi:119615302] | Taxonomy id: 9606 Gene id: 3362 | |||
| 11 | Serotonin receptor 5ht7 | 1 | 5-hydroxytryptamine (serotonin) receptor 7 (adenylate cyclase-coupled), isoform CRA_c [Homo sapiens] [gi:119570505] | Taxonomy id: 9606 Gene id: 3363 | |||
| 12 | Adrenergic receptor Alpha1A | 1 | Adrenergic, alpha-1A-, receptor [Homo sapiens] [gi:66267329] | Taxonomy id: 9606 Gene id: 148 | |||
| 13 | Adrenergic receptor Alpha1B | 1 | Adrenergic, alpha-1B-, receptor [Homo sapiens] [gi:187953275] | Taxonomy id: 9606 Gene id: 147 | |||
| 14 | Adrenergic receptor Alpha1D | 1 | adrenergic, alpha-1D-, receptor [Homo sapiens] [gi:119630860] | Taxonomy id: 9606 Gene id: 146 | |||
| 15 | Adrenergic receptor Beta1 | 1 | adrenergic, beta-1-, receptor [Homo sapiens] [gi:166706747] | Taxonomy id: 9606 Gene id: 153 | |||
| 16 | Adrenergic receptor Beta2 | 1 | beta 2 adrenergic receptor [Homo sapiens] [gi:22658465] | Taxonomy id: 9606 Gene id: 154 | |||
| 17 | Adrenergic receptor Beta3 | 1 | adrenergic, beta-3-, receptor [Homo sapiens] [gi:68248548] | Taxonomy id: 9606 Gene id: 155 | |||
| 18 | BZP Rat Brain Site | 1 | Taxonomy id: 10116 | ||||
| 19 | Dopamine receptor D1 | 1 | Dopamine receptor D1 [Homo sapiens] [gi:67677841] | Taxonomy id: 9606 Gene id: 1812 | |||
| 20 | Dopamine receptor D2 | 1 | Dopamine receptor D2 [Homo sapiens] [gi:18203703] | Taxonomy id: 9606 Gene id: 1813 | |||
| 21 | Dopamine receptor D3 | 1 | Dopamine receptor D3 [Homo sapiens] [gi:118764187] | Taxonomy id: 9606 Gene id: 1814 | |||
| 22 | Dopamine receptor D4 | 1 | dopamine receptor D4 [Homo sapiens] [gi:166714265] | Taxonomy id: 9606 Gene id: 1815 | |||
| 23 | Dopamine receptor D5 | 1 | dopamine receptor D5 [Homo sapiens] [gi:119613085] | Taxonomy id: 9606 Gene id: 1816 | |||
| 24 | Biogenic amine transporter DAT | 1 | dopamine transporter [Homo sapiens] [gi:139522363] | Taxonomy id: 9606 Gene id: 6531 | |||
| 25 | Opioid receptor DOR | 1 | delta-type opioid receptor [Homo sapiens] [gi:63477962] | Taxonomy id: 9606 Gene id: 4985 | |||
| 26 | GABA receptor GABAa | 1 | gamma-aminobutyric acid receptor type A rho-1 subunit [Homo sapiens] [gi:182911] | Taxonomy id: 9606 Gene id: 2569 | |||
| 27 | Histamine receptor H1 | 1 | histamine H1 receptor [Homo sapiens] [gi:149158709] | Taxonomy id: 9606 Gene id: 3269 | |||
| 28 | Histamine receptor H2 | 1 | histamine receptor H2 [Homo sapiens] [gi:197692667] | Taxonomy id: 9606 Gene id: 3274 | |||
| 29 | Histamine receptor H3 | 1 | histamine H3 receptor [Homo sapiens] [gi:194018562] | Taxonomy id: 9606 Gene id: 11255 | |||
| 30 | Histamine receptor H4 | 1 | histamine H4 receptor [Homo sapiens] [gi:167887581] | Taxonomy id: 9606 Gene id: 59340 | |||
| 31 | Opioid receptor KOR | 1 | kappa opioid receptor [Homo sapiens] [gi:39986053] | Taxonomy id: 9606 Gene id: 4986 | |||
| 32 | Muscarinic receptor M1 | 1 | Cholinergic receptor, muscarinic 1 [gi:74754970] | Taxonomy id: 9606 Gene id: 1128 | |||
| 33 | Muscarinic receptor M2 | 1 | cholinergic receptor, muscarinic 2 [Homo sapiens] [gi:119604271] | Taxonomy id: 9606 Gene id: 1129 | |||
| 34 | Muscarinic receptor M3 | 1 | Cholinergic receptor, muscarinic 3 [Homo sapiens] [gi:111306506] | Taxonomy id: 9606 Gene id: 1131 | |||
| 35 | Muscarinic receptor M4 | 1 | Cholinergic receptor, muscarinic 4 [Homo sapiens] [gi:187953629] | Taxonomy id: 9606 Gene id: 1132 | |||
| 36 | Muscarinic receptor M5 | 1 | Cholinergic receptor, muscarinic 5 [gi:74759612] | Taxonomy id: 9606 Gene id: 1133 | |||
| 37 | Opioid receptor MOR | 1 | Mu opioid receptor [gi:74758917] | Taxonomy id: 9606 Gene id: 4988 | |||
| 38 | Biogenic amine transporter NET | 1 | Net [Homo sapiens] [gi:531523] | Taxonomy id: 9606 Gene id: 2004 | |||
| 39 | Biogenic amine transporter SERT | 1 | serotonin transporter [Homo sapiens] [gi:58700442] | Taxonomy id: 9606 Gene id: 6532 | |||
| 40 | Sigma receptor 1 | 1 | opioid receptor, sigma 1 [Homo sapiens] [gi:123231939] | Taxonomy id: 9606 Gene id: 10280 | |||
| 41 | Sigma receptor 2 | 1 | Taxonomy id: 9606 | ||||
§ Panel component ID.
Protocol
Assay Overview:
The purpose of this panel of radioligand binding assays performed by the NIMH Psychoactive Drug Screening Program (PDSP) is to identify a subset of potential receptors, transporters, ion channels, etc. for which the NOX1 inhibitor compound SID 57287864 displays affinity.
Protocol Summary:
Test and reference compounds are diluted to 5X final assay concentration (50 uM for a final assay concentration of 10 uM) in the appropriate radioligand binding buffer. Then, 50 uL aliquots of buffer (negative control), test compound, and reference compound (positive control) are added in quadruplicate to the wells of a 96-well plate, each of which contains 50 uL of 5X radioligand and 100 uL of buffer. Finally, receptor-containing, crude membrane fractions are resuspended in an appropriate volume of buffer and dispensed (50 mul per well) into the 96-well plate. Radioligand binding is allowed to equilibrate for 1.5 hours at room temperature, and then bound radioactivity is isolated by filtration onto 0.3% polyethyleneimine-treated, 96-well filter mats using a 96-well Filtermate harverster. The filter mats are dried, then scintillant is melted onto the filters and the radioactivity retained on the filters is counted in a Microbeta scintillation counter. As designed, membrane fractions to which substance SID 57287864 binds will bind less radioligand and decrease the radioactivity measured in the assay.
Raw data from the Microbeta counter are analyzed on the PDSP DB. Total bound radioactivity is estimated from quadruplicate wells containing no test or reference compound and adjusted to 100%; non-specifically bound radioactivity is assessed from quadruplicate wells containing 10 uM of a suitable reference compound and adjusted to 0%. The average bound radioactivity in the presence of the test compound is expressed on a percent scale. The percent inhibition of radioligand binding is calculated as follows:
%_Inhibition = 100% - %_radioactivity_bound
Inhibition of > 50% is considered significant. Negative inhibition represents a stimulation of binding.
The PDSP on-line data entry and analysis system calculates the variance of the quadruplicate determinations (for the total, non-specific, and test compound binding values) and variances greater than 20% are flagged for further inspection and assays are repeated if necessary. Additionally, % inhibition values that are greater than the total binding (i.e., 100%) by at least 20% are also flagged for inspection; such results could indicate allosteric modulation of radioligand binding.
List of Reagents:
Reagents were provided by the NIMH Psychoactive Drug Screening Program.
The purpose of this panel of radioligand binding assays performed by the NIMH Psychoactive Drug Screening Program (PDSP) is to identify a subset of potential receptors, transporters, ion channels, etc. for which the NOX1 inhibitor compound SID 57287864 displays affinity.
Protocol Summary:
Test and reference compounds are diluted to 5X final assay concentration (50 uM for a final assay concentration of 10 uM) in the appropriate radioligand binding buffer. Then, 50 uL aliquots of buffer (negative control), test compound, and reference compound (positive control) are added in quadruplicate to the wells of a 96-well plate, each of which contains 50 uL of 5X radioligand and 100 uL of buffer. Finally, receptor-containing, crude membrane fractions are resuspended in an appropriate volume of buffer and dispensed (50 mul per well) into the 96-well plate. Radioligand binding is allowed to equilibrate for 1.5 hours at room temperature, and then bound radioactivity is isolated by filtration onto 0.3% polyethyleneimine-treated, 96-well filter mats using a 96-well Filtermate harverster. The filter mats are dried, then scintillant is melted onto the filters and the radioactivity retained on the filters is counted in a Microbeta scintillation counter. As designed, membrane fractions to which substance SID 57287864 binds will bind less radioligand and decrease the radioactivity measured in the assay.
Raw data from the Microbeta counter are analyzed on the PDSP DB. Total bound radioactivity is estimated from quadruplicate wells containing no test or reference compound and adjusted to 100%; non-specifically bound radioactivity is assessed from quadruplicate wells containing 10 uM of a suitable reference compound and adjusted to 0%. The average bound radioactivity in the presence of the test compound is expressed on a percent scale. The percent inhibition of radioligand binding is calculated as follows:
%_Inhibition = 100% - %_radioactivity_bound
Inhibition of > 50% is considered significant. Negative inhibition represents a stimulation of binding.
The PDSP on-line data entry and analysis system calculates the variance of the quadruplicate determinations (for the total, non-specific, and test compound binding values) and variances greater than 20% are flagged for further inspection and assays are repeated if necessary. Additionally, % inhibition values that are greater than the total binding (i.e., 100%) by at least 20% are also flagged for inspection; such results could indicate allosteric modulation of radioligand binding.
List of Reagents:
Reagents were provided by the NIMH Psychoactive Drug Screening Program.
Result Definitions
| Show more |
| TID | Name | Description | PID§ | Panel Targets | Histogram | Type | Unit | |
|---|---|---|---|---|---|---|---|---|
| Outcome | The BioAssay activity outcome | Outcome | ||||||
| 1 | Serotonin 5ht1a (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 1 | 5-hydroxytryptamine (serotonin) receptor 1A [Homo sapiens] |  | Float | % | |
| 2 | Serotonin 5ht1a (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 1 | | Outcome | |||
| 3 | Serotonin 5ht1b (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 2 | 5-hydroxytryptamine (serotonin) receptor 1B [Homo sapiens] | | Float | % | |
| 4 | Serotonin 5ht1b (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 2 | | Outcome | |||
| 5 | Serotonin 5ht1d (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 3 | 5-hydroxytryptamine (serotonin) receptor 1D [Homo sapiens] | | Float | % | |
| 6 | Serotonin 5ht1d (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 3 | | Outcome | |||
| 7 | Serotonin 5ht1e (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 4 | 5-hydroxytryptamine (serotonin) receptor 1E [Homo sapiens] | | Float | % | |
| 8 | Serotonin 5ht1e (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 4 | | Outcome | |||
| 9 | Serotonin 5ht2a (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 5 | 5-hydroxytryptamine (serotonin) receptor 2A [Homo sapiens] | | Float | % | |
| 10 | Serotonin 5ht2a (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 5 | | Outcome | |||
| 11 | Serotonin 5ht2b (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 6 | 5-hydroxytryptamine (serotonin) receptor 2B [Homo sapiens] | | Float | % | |
| 12 | Serotonin 5ht2b (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 6 | | Outcome | |||
| 13 | Serotonin 5ht2c (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 7 | 5-hydroxytryptamine (serotonin) receptor 2C, isoform CRA_b [Homo sapiens] | | Float | % | |
| 14 | Serotonin 5ht2c (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 7 | | Outcome | |||
| 15 | Serotonin 5ht3 (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 8 | 5-hydroxytryptamine (serotonin) receptor 3A, isoform CRA_c [Homo sapiens] | | Float | % | |
| 16 | Serotonin 5ht3 (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 8 | | Outcome | |||
| 17 | Serotonin 5ht5a (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 9 | 5-hydroxytryptamine (serotonin) receptor 5A [Homo sapiens] | | Float | % | |
| 18 | Serotonin 5ht5a (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 9 | | Outcome | |||
| 19 | Serotonin 5ht6 (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 10 | 5-hydroxytryptamine (serotonin) receptor 6 [Homo sapiens] | | Float | % | |
| 20 | Serotonin 5ht6 (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 10 | | Outcome | |||
| 21 | Serotonin 5ht7 (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 11 | 5-hydroxytryptamine (serotonin) receptor 7 (adenylate cyclase-coupled), isoform CRA_c [Homo sapiens] | | Float | % | |
| 22 | Serotonin 5ht7 (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 11 | | Outcome | |||
| 23 | Adrenergic Alpha1A (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 12 | Adrenergic, alpha-1A-, receptor [Homo sapiens] | | Float | % | |
| 24 | Adrenergic Alpha1A (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 12 | | Outcome | |||
| 25 | Adrenergic Alpha1B (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 13 | Adrenergic, alpha-1B-, receptor [Homo sapiens] | | Float | % | |
| 26 | Adrenergic Alpha1B (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 13 | | Outcome | |||
| 27 | Adrenergic Alpha1D (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 14 | adrenergic, alpha-1D-, receptor [Homo sapiens] | | Float | % | |
| 28 | Adrenergic Alpha1D (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 14 | | Outcome | |||
| 29 | Adrenergic Beta1 (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 15 | adrenergic, beta-1-, receptor [Homo sapiens] | | Float | % | |
| 30 | Adrenergic Beta1 (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 15 | | Outcome | |||
| 31 | Adrenergic Beta2 (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 16 | beta 2 adrenergic receptor [Homo sapiens] | | Float | % | |
| 32 | Adrenergic Beta2 (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 16 | | Outcome | |||
| 33 | Adrenergic Beta3 (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 17 | adrenergic, beta-3-, receptor [Homo sapiens] | | Float | % | |
| 34 | Adrenergic Beta3 (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 17 | | Outcome | |||
| 35 | BZP Rat Brain Site (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 18 | | Float | % | ||
| 36 | BZP Rat Brain Site (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 18 | | Outcome | |||
| 37 | Dopamine D1 (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 19 | Dopamine receptor D1 [Homo sapiens] | | Float | % | |
| 38 | Dopamine D1 (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 19 | | Outcome | |||
| 39 | Dopamine D2 (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 20 | Dopamine receptor D2 [Homo sapiens] | | Float | % | |
| 40 | Dopamine D2 (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 20 | | Outcome | |||
| 41 | Dopamine D3 (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 21 | Dopamine receptor D3 [Homo sapiens] | | Float | % | |
| 42 | Dopamine D3 (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 21 | | Outcome | |||
| 43 | Dopamine D4 (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 22 | dopamine receptor D4 [Homo sapiens] | | Float | % | |
| 44 | Dopamine D4 (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 22 | | Outcome | |||
| 45 | Dopamine D5 (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 23 | dopamine receptor D5 [Homo sapiens] | | Float | % | |
| 46 | Dopamine D5 (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 23 | | Outcome | |||
| 47 | Biogenic amine transporter DAT (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 24 | dopamine transporter [Homo sapiens] | | Float | % | |
| 48 | Biogenic amine transporter DAT (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 24 | | Outcome | |||
| 49 | Opioid DOR (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 25 | delta-type opioid receptor [Homo sapiens] | | Float | % | |
| 50 | Opioid DOR (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 25 | | Outcome | |||
| 51 | GABA GABAa (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 26 | gamma-aminobutyric acid receptor type A rho-1 subunit [Homo sapiens] | | Float | % | |
| 52 | GABA GABAa (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 26 | | Outcome | |||
| 53 | Histamine H1 (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 27 | histamine H1 receptor [Homo sapiens] | | Float | % | |
| 54 | Histamine H1 (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 27 | | Outcome | |||
| 55 | Histamine H2 (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 28 | histamine receptor H2 [Homo sapiens] | | Float | % | |
| 56 | Histamine H2 (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 28 | | Outcome | |||
| 57 | Histamine H3 (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 29 | histamine H3 receptor [Homo sapiens] | | Float | % | |
| 58 | Histamine H3 (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 29 | | Outcome | |||
| 59 | Histamine H4 (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 30 | histamine H4 receptor [Homo sapiens] | | Float | % | |
| 60 | Histamine H4 (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 30 | | Outcome | |||
| 61 | Opioid KOR (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 31 | kappa opioid receptor [Homo sapiens] | | Float | % | |
| 62 | Opioid KOR (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 31 | | Outcome | |||
| 63 | Muscarinic M1 (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 32 | Cholinergic receptor, muscarinic 1 | | Float | % | |
| 64 | Muscarinic M1 (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 32 | | Outcome | |||
| 65 | Muscarinic M2 (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 33 | cholinergic receptor, muscarinic 2 [Homo sapiens] | | Float | % | |
| 66 | Muscarinic M2 (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 33 | | Outcome | |||
| 67 | Muscarinic M3 (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 34 | Cholinergic receptor, muscarinic 3 [Homo sapiens] | | Float | % | |
| 68 | Muscarinic M3 (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 34 | | Outcome | |||
| 69 | Muscarinic M4 (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 35 | Cholinergic receptor, muscarinic 4 [Homo sapiens] | | Float | % | |
| 70 | Muscarinic M4 (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 35 | | Outcome | |||
| 71 | Muscarinic M5 (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 36 | Cholinergic receptor, muscarinic 5 | | Float | % | |
| 72 | Muscarinic M5 (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 36 | | Outcome | |||
| 73 | Opioid MOR (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 37 | Mu opioid receptor | | Float | % | |
| 74 | Opioid MOR (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 37 | | Outcome | |||
| 75 | Biogenic amine transporter NET (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 38 | Net [Homo sapiens] | | Float | % | |
| 76 | Biogenic amine transporter NET (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 38 | | Outcome | |||
| 77 | Biogenic amine transporter SERT (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 39 | serotonin transporter [Homo sapiens] | | Float | % | |
| 78 | Biogenic amine transporter SERT (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 39 | | Outcome | |||
| 79 | Sigma 1 (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 40 | opioid receptor, sigma 1 [Homo sapiens] | | Float | % | |
| 80 | Sigma 1 (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 40 | | Outcome | |||
| 81 | Sigma 2 (Inhibition) (10μM**) | Value of % Inhibition at 10 uM activator concentration; average of four measurements. | 41 | | Float | % | ||
| 82 | Sigma 2 (Outcome) | The Assay outcome, one of Active, Inactive or Not Tested. | 41 | | Outcome |
** Test Concentration. § Panel component ID.
Additional Information
Grant Number: 1 R03 MH083264-01A1
Classification
PageFrom: