The modulation of immune response activity is one of the major goals in the development of novel therapeutics for auto-immune and inflammatory diseases. The innate system resides at the intersection of the pathways of microbial recognition, inflammation, and cell death, thereby offering various therapeutic targets. In this context, NOD1 and NOD2 are of particular interest, since they recognize distinct structures derived from bacterial peptidoglycans and directly activate NF-kB, a central regulator of immune response, inflammation, and apoptosis. ..more
Target
Depositor Specified Assays
Show more |
| AID | Name | Type | Comment |
|---|---|---|---|
| 1578 | uHTS luminescence assay for the identification of compounds that inhibit NOD1 | confirmatory | uHTS luminescence assay for the identification of compounds that inhibit NOD1 |
| 1849 | uHTS Fluorescence assay for the identification of cytotoxic compounds among compounds active in NOD1 cell inhibition assay | confirmatory | Cytotox counter screen |
| 1852 | HTS assay for identification of inhibitors of TNF-a-specific NF-kB induction | other | Multiple cellular stimuli acting through various pathways lead to NF-kB induction. This assay uses tumor necrosis factor alpha (TNF-a), a canonical NF-kB inducer, and is designed for identification of hits specific to TNF-a-modulated pathways. |
| 2245 | SAR analysis of tumor necrosis factor alpha (TNF-alpha) induced IL-8 secretion in MCF-7/NOD1 cells. | confirmatory | This assay uses tumor necrosis factor alpha (TNFa), a canonical NF-kB inducer, and is designed for identification of hits specific to TNFa-modulated pathways |
| 2250 | SAR analysis of GM-Tri-DAP induced IL-8 secretion in MCF-7/NOD1 cells | confirmatory | SAR analysis of hit analogs |
| 2255 | SAR analysis of NF-kB dependent luciferase using Doxorucibin as an inducer | confirmatory | SAR analysis of hit analogs |
| 2260 | SAR analysis of muramyl dipeptide (MDP) induced IL-8 secretion in MCF-7/NOD2 cells. | confirmatory | SAR analysis of hit analogs |
| 2261 | SAR analysis of NF-kB dependent luciferase using PMA/Ionomycin as an inducer | confirmatory | SAR analysis of hit analogs |
| 2264 | SAR analysis of NF-kB dependent luciferase using DAP as an inducer | confirmatory | SAR analysis of hit analogs |
| 2333 | SAR analysis of compounds that inhibit NOD1 revised | confirmatory | SAR analysis of hit analogs |
| 2335 | SAR analysis of compounds that are cytotoxic to HEK293 revised | confirmatory | Cytotox counter screen |
| 2337 | SAR analysis of inhibitors of TNFa specific NF-kB induction revised | confirmatory | SAR analysis of hit analogs |
| 2466 | SAR analysis of compounds that inhibit NOD1 - Set 2 | confirmatory | SAR analysis of hit analogs |
| 2469 | SAR analysis of compounds that are cytotoxic to HEK293 - Set 2 | confirmatory | Cytotox counter screen |
| 2483 | SAR analysis of inhibitors of TNFa specific NF-kB induction - Set 2 | confirmatory | SAR analysis of hit analogs |
| 2485 | HTS dose response assay for identification of inhibitors of TNFa-specific NF-kB induction | confirmatory | Dose response confirmation of primary screen hits |
| 2503 | SAR analysis of Muramyl dipeptide (MDP) induced IL-8 secretion in MCF-7/NOD2 cells - Set 2 | confirmatory | SAR analysis of hit analogs |
| 2504 | SAR analysis of Tumor necrosis factor alpha (TNF-alpha) induced IL-8 secretion in MCF-7/NOD1 cells - Set 2 | confirmatory | SAR analysis of hit analogs |
| 2505 | SAR analysis of GM-Tri-DAP induced IL-8 secretion in MCF-7/NOD1 cells - Set 2 | confirmatory | SAR analysis of hit analogs |
| 2789 | SAR analysis of NF-kappaB dependent luciferase using Doxorucibin as an inducer - Set 2 | confirmatory | SAR analysis of hit analogs |
| 2792 | SAR analysis of NF-kappaB dependent luciferase using PMA/Ionomycin as an inducer - 2 | confirmatory | SAR analysis of hit analogs |
| 2793 | SAR analysis of NF-kappaB dependent luciferase using DAP as an inducer - Set 2 | confirmatory | SAR analysis of hit analogs |
| 2798 | SAR analysis of compounds that inhibit NOD1 - Set 3 | confirmatory | SAR analysis of hit analogs |
| 2800 | SAR analysis of compounds that are cytotoxic to HEK293 - Set 3 | confirmatory | Cytotox counter screen |
| 2801 | SAR analysis of inhibitors of TNFa specific NF-kB induction - Set 3 | confirmatory | SAR analysis of hit analogs |
| 1566 | uHTS luminescence assay for the identification of compounds that inhibit NOD2 | confirmatory | |
| 2475 | SAR analysis of compounds that inhibit NOD2 - Set 2 | confirmatory | |
| 2799 | SAR analysis of compounds that inhibit NOD2 - Set 3 | confirmatory | |
| 2334 | SAR analysis of compounds that inhibit NOD2 revised | confirmatory |
Description:
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number: 1 R03 MH084844-01
Assay Provider: Dr. John C. Reed, Sanford-Burnham Medical Research Institute, San Diego, CA
The modulation of immune response activity is one of the major goals in the development of novel therapeutics for auto-immune and inflammatory diseases. The innate system resides at the intersection of the pathways of microbial recognition, inflammation, and cell death, thereby offering various therapeutic targets. In this context, NOD1 and NOD2 are of particular interest, since they recognize distinct structures derived from bacterial peptidoglycans and directly activate NF-kB, a central regulator of immune response, inflammation, and apoptosis.
Mutations in the NOD1 and NOD2 genes are associated with a number of human inflammatory disorders, including Crohn's disease (CD), Blau syndrome, early-onset sarcoidosis, and atopic diseases, which characteristically cause constitutive NF-kB activation. Chemical inhibitors of NOD1 and NOD2 would provide powerful research tools for elucidating the roles of these proteins in primary cultured cells from humans and in animal models.
The main objective of this study was to identify small molecule inhibitors of NF-kappaB activity induced by NOD1. This was achieved by generating and validating a cell-based, Luciferase reporter gene assay for use in high throughput screening (HTS) to identify small molecule inhibitors of NOD1-dependent NF-kappaB activation (AID 1578). Various downstream counter-screens and secondary assays were employed to further characterize the selectivity of the hits, setting the stage for subsequent structure activity relationship (SAR) studies which led to the optimization of the chemical probes.
The first probe molecule CID1088438(ML130) selectively (> 40-fold) inhibits NOD1 dependent activation of NF-kB pathways as ascertained through gamma-tri-DAP stimulated luciferase signaling in a NF-kB-linked reporter assay in HEK293T cells containing endogenous NOD1 levels (AID 2333) with sub-micromolar potency (0.52 microM IC50), while not inhibiting MDP stimulated (NOD2- dependent) signaling in reporter cell lines containing both low (AID 2334) and, in earlier iterations, over-expressed (AID 1566) NOD2 proteins. The probe molecule is selective over the non-NOD stimulated pathways (TNFalpha stimulation) of NF-kB in these reporter assays (AID 2337). The probe does not appear to be cytotoxic at the concentrations tested (AID 2335). Furthermore, the probe molecule appears to selectively inhibit a biologically relevant terminal effect of NOD1 (gamma-tri-DAP) dependent NF-kB activation (AID 2250), namely IL-8 secretion, but not NOD2 (MDP) dependent (AID 2260), nor TNFalpha-dependent (AID 2245) IL-8 secretion in MCF-7 cells, as determined by IL-8 ELISA assays of cell culture supernatants. Finally, selectivity assays suggest that the probe inhibits a NOD1 (gamma-tri-DAP) dependent pathway to NF-kB activation (AID 2264) and is inactive in both PMA-induced (AID 2261) and doxorubicin-induced (AID 2255) NF-kB activation models.
The second probe molecule CID5310346(ML146) selectively (> 8-fold) inhibits NOD1 dependent activation of NF-kB pathways as ascertained through gamma-tri-DAP stimulated luciferase signaling in a NF-kB-linked reporter assay in HEK293T cells containing endogenous NOD1 levels with submicromolar potency (1.54 uM IC50), while not inhibiting MDP stimulated (NOD2-dependent) signaling in both reporter cell lines containing both low (AID 2334) and, in earlier iterations, over-expressed (AID 1566) NOD2 proteins. The probe molecule is also selective over the non-NOD stimulated pathways (TNFalpha stimulation) of NF-kB in these reporter assays (AID 2337). While this probe is not as potent or selective as our first probe, CID1088438, it does meet probe criteria and represents a second bonafide scaffold for a NOD1 selective probe. The probe does not appear to be cytotoxic at the concentrations tested (AID 2335). Furthermore, the probe molecule appears to selectively inhibit a biologically relevant terminal effect of NOD1 (gamma-tri-DAP) dependent NF-kB activation (AID 2505), namely IL-8 secretion, but not NOD2 (MDP) dependent (AID 2503), nor TNFalpha-dependent (AID 2504) IL-8 secretion in MCF-7 cells, as determined by IL-8 ELISA assays of cell culture supernatants. Finally, selectivity assays suggest that the probe inhibits a NOD1 (gamma-tri-DAP) dependent pathway to NF-kB activation (AID 2793) and is inactive in both PMA-induced (AID 2792) and doxorubicin-induced (AID 2789) NF-kB activation models.
References
1)Strober W, Murray PJ, Kitani A, Watanabe T. Nat Rev Immunol. 2006 Jan;6(1):9-20. Review. Signalling pathways and molecular interactions of NOD1 and NOD2.
2. da Silva Correia J, Miranda Y, Austin-Brown N, Hsu J, Mathison J, Xiang R, Zhou H, Li Q, Han J, Ulevitch RJ. Proc Natl Acad Sci U S A. 2006 Feb 7;103(6):1840-5. Epub 2006 Jan 30. Nod1-dependent control of tumor growth.
3. Joosten LA, Heinhuis B, Abdollahi-Roodsaz S, Ferwerda G, Lebourhis L, Philpott DJ, Nahori MA, Popa C, Morre SA, van der Meer JW, Girardin SE, Netea MG, van den Berg WB. Proc Natl Acad Sci U S A. 2008 Jul 1;105(26):9017-22. Epub 2008 Jun 23. Differential function of the NACHT-LRR (NLR) members Nod1 and Nod2 in arthritis.
4. Shaw MH, Reimer T, Kim YG, Nunez G. Curr Opin Immunol. 2008 Aug;20(4):377-82. Epub 2008 Jul 2. Review. NOD-like receptors (NLRs): bona fide intracellular microbial sensors.
5. Kim YG, Park JH, Shaw MH, Franchi L, Inohara N, Nunez G. Immunity. 2008 Feb;28(2):246-57. Epub 2008 Feb 7. The cytosolic sensors Nod1 and Nod2 are critical for bacterial recognition and host defense after exposure to Toll-like receptor ligands.
6. Rescigno M, Nieuwenhuis EE. Curr Opin Gastroenterol. 2007 Jan;23(1):21-6. Review. The role of altered microbial signaling via mutant NODs in intestinal inflammation.
7. Rosenstiel P, Hellmig S, Hampe J, Ott S, Till A, Fischbach W, Sahly H, Lucius R, Folsch UR, Philpott D, Schreiber S. Cell Microbiol. 2006 Jul;8(7):1188-98. Influence of polymorphisms in the NOD1/CARD4 and NOD2/CARD15 genes on the clinical outcome of Helicobacter pylori infection.
8. McGovern DP, Hysi P, Ahmad T, van Heel DA, Moffatt MF, Carey A, Cookson WO, Jewell DP. Hum Mol Genet. 2005 May 15;14(10):1245-50. Epub 2005 Mar 24. Association between a complex insertion/deletion polymorphism in NOD1 (CARD4) and susceptibility to inflammatory bowel disease.
9. Opitz B, Puschel A, Schmeck B, Hocke AC, Rosseau S, Hammerschmidt S, Schumann RR, Suttorp N, Hippenstiel S. J Biol Chem. 2004 Aug 27;279(35):36426-32. Epub 2004 Jun 23. Nucleotide-binding oligomerization domain proteins are innate immune receptors for internalized Streptococcus pneumoniae.
10. Le Bourhis L, Benko S, Girardin SE. Biochem Soc Trans. 2007 Dec;35(Pt 6):1479-84. Review. Nod1 and Nod2 in innate immunity and human inflammatory disorders.
11. Maeda S, Hsu LC, Liu H, Bankston LA, Iimura M, Kagnoff MF, Eckmann L, Karin M. Science. 2005 Feb 4;307(5710):734-8. Erratum in: Science. 2005 Apr 29;308(5722):633. Nod2 mutation in Crohn's disease potentiates NF-kappaB activity and IL-1beta processing.
12. Li J, Moran T, Swanson E, Julian C, Harris J, Bonen DK, Hedl M, Nicolae DL, Abraham C, Cho JH. Hum Mol Genet. 2004 Aug 15;13(16):1715-25. Epub 2004 Jun 15. Regulation of IL-8 and IL-1beta expression in Crohn's disease associated NOD2/CARD15 mutations.
13. Brideau C, Gunter B, Pikounis B, Liaw A. J Biomol Screen. 2003 Dec;8(6):634-47 Improved statistical methods for hit selection in high-throughput screening
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number: 1 R03 MH084844-01
Assay Provider: Dr. John C. Reed, Sanford-Burnham Medical Research Institute, San Diego, CA
The modulation of immune response activity is one of the major goals in the development of novel therapeutics for auto-immune and inflammatory diseases. The innate system resides at the intersection of the pathways of microbial recognition, inflammation, and cell death, thereby offering various therapeutic targets. In this context, NOD1 and NOD2 are of particular interest, since they recognize distinct structures derived from bacterial peptidoglycans and directly activate NF-kB, a central regulator of immune response, inflammation, and apoptosis.
Mutations in the NOD1 and NOD2 genes are associated with a number of human inflammatory disorders, including Crohn's disease (CD), Blau syndrome, early-onset sarcoidosis, and atopic diseases, which characteristically cause constitutive NF-kB activation. Chemical inhibitors of NOD1 and NOD2 would provide powerful research tools for elucidating the roles of these proteins in primary cultured cells from humans and in animal models.
The main objective of this study was to identify small molecule inhibitors of NF-kappaB activity induced by NOD1. This was achieved by generating and validating a cell-based, Luciferase reporter gene assay for use in high throughput screening (HTS) to identify small molecule inhibitors of NOD1-dependent NF-kappaB activation (AID 1578). Various downstream counter-screens and secondary assays were employed to further characterize the selectivity of the hits, setting the stage for subsequent structure activity relationship (SAR) studies which led to the optimization of the chemical probes.
The first probe molecule CID1088438(ML130) selectively (> 40-fold) inhibits NOD1 dependent activation of NF-kB pathways as ascertained through gamma-tri-DAP stimulated luciferase signaling in a NF-kB-linked reporter assay in HEK293T cells containing endogenous NOD1 levels (AID 2333) with sub-micromolar potency (0.52 microM IC50), while not inhibiting MDP stimulated (NOD2- dependent) signaling in reporter cell lines containing both low (AID 2334) and, in earlier iterations, over-expressed (AID 1566) NOD2 proteins. The probe molecule is selective over the non-NOD stimulated pathways (TNFalpha stimulation) of NF-kB in these reporter assays (AID 2337). The probe does not appear to be cytotoxic at the concentrations tested (AID 2335). Furthermore, the probe molecule appears to selectively inhibit a biologically relevant terminal effect of NOD1 (gamma-tri-DAP) dependent NF-kB activation (AID 2250), namely IL-8 secretion, but not NOD2 (MDP) dependent (AID 2260), nor TNFalpha-dependent (AID 2245) IL-8 secretion in MCF-7 cells, as determined by IL-8 ELISA assays of cell culture supernatants. Finally, selectivity assays suggest that the probe inhibits a NOD1 (gamma-tri-DAP) dependent pathway to NF-kB activation (AID 2264) and is inactive in both PMA-induced (AID 2261) and doxorubicin-induced (AID 2255) NF-kB activation models.
The second probe molecule CID5310346(ML146) selectively (> 8-fold) inhibits NOD1 dependent activation of NF-kB pathways as ascertained through gamma-tri-DAP stimulated luciferase signaling in a NF-kB-linked reporter assay in HEK293T cells containing endogenous NOD1 levels with submicromolar potency (1.54 uM IC50), while not inhibiting MDP stimulated (NOD2-dependent) signaling in both reporter cell lines containing both low (AID 2334) and, in earlier iterations, over-expressed (AID 1566) NOD2 proteins. The probe molecule is also selective over the non-NOD stimulated pathways (TNFalpha stimulation) of NF-kB in these reporter assays (AID 2337). While this probe is not as potent or selective as our first probe, CID1088438, it does meet probe criteria and represents a second bonafide scaffold for a NOD1 selective probe. The probe does not appear to be cytotoxic at the concentrations tested (AID 2335). Furthermore, the probe molecule appears to selectively inhibit a biologically relevant terminal effect of NOD1 (gamma-tri-DAP) dependent NF-kB activation (AID 2505), namely IL-8 secretion, but not NOD2 (MDP) dependent (AID 2503), nor TNFalpha-dependent (AID 2504) IL-8 secretion in MCF-7 cells, as determined by IL-8 ELISA assays of cell culture supernatants. Finally, selectivity assays suggest that the probe inhibits a NOD1 (gamma-tri-DAP) dependent pathway to NF-kB activation (AID 2793) and is inactive in both PMA-induced (AID 2792) and doxorubicin-induced (AID 2789) NF-kB activation models.
References
1)Strober W, Murray PJ, Kitani A, Watanabe T. Nat Rev Immunol. 2006 Jan;6(1):9-20. Review. Signalling pathways and molecular interactions of NOD1 and NOD2.
2. da Silva Correia J, Miranda Y, Austin-Brown N, Hsu J, Mathison J, Xiang R, Zhou H, Li Q, Han J, Ulevitch RJ. Proc Natl Acad Sci U S A. 2006 Feb 7;103(6):1840-5. Epub 2006 Jan 30. Nod1-dependent control of tumor growth.
3. Joosten LA, Heinhuis B, Abdollahi-Roodsaz S, Ferwerda G, Lebourhis L, Philpott DJ, Nahori MA, Popa C, Morre SA, van der Meer JW, Girardin SE, Netea MG, van den Berg WB. Proc Natl Acad Sci U S A. 2008 Jul 1;105(26):9017-22. Epub 2008 Jun 23. Differential function of the NACHT-LRR (NLR) members Nod1 and Nod2 in arthritis.
4. Shaw MH, Reimer T, Kim YG, Nunez G. Curr Opin Immunol. 2008 Aug;20(4):377-82. Epub 2008 Jul 2. Review. NOD-like receptors (NLRs): bona fide intracellular microbial sensors.
5. Kim YG, Park JH, Shaw MH, Franchi L, Inohara N, Nunez G. Immunity. 2008 Feb;28(2):246-57. Epub 2008 Feb 7. The cytosolic sensors Nod1 and Nod2 are critical for bacterial recognition and host defense after exposure to Toll-like receptor ligands.
6. Rescigno M, Nieuwenhuis EE. Curr Opin Gastroenterol. 2007 Jan;23(1):21-6. Review. The role of altered microbial signaling via mutant NODs in intestinal inflammation.
7. Rosenstiel P, Hellmig S, Hampe J, Ott S, Till A, Fischbach W, Sahly H, Lucius R, Folsch UR, Philpott D, Schreiber S. Cell Microbiol. 2006 Jul;8(7):1188-98. Influence of polymorphisms in the NOD1/CARD4 and NOD2/CARD15 genes on the clinical outcome of Helicobacter pylori infection.
8. McGovern DP, Hysi P, Ahmad T, van Heel DA, Moffatt MF, Carey A, Cookson WO, Jewell DP. Hum Mol Genet. 2005 May 15;14(10):1245-50. Epub 2005 Mar 24. Association between a complex insertion/deletion polymorphism in NOD1 (CARD4) and susceptibility to inflammatory bowel disease.
9. Opitz B, Puschel A, Schmeck B, Hocke AC, Rosseau S, Hammerschmidt S, Schumann RR, Suttorp N, Hippenstiel S. J Biol Chem. 2004 Aug 27;279(35):36426-32. Epub 2004 Jun 23. Nucleotide-binding oligomerization domain proteins are innate immune receptors for internalized Streptococcus pneumoniae.
10. Le Bourhis L, Benko S, Girardin SE. Biochem Soc Trans. 2007 Dec;35(Pt 6):1479-84. Review. Nod1 and Nod2 in innate immunity and human inflammatory disorders.
11. Maeda S, Hsu LC, Liu H, Bankston LA, Iimura M, Kagnoff MF, Eckmann L, Karin M. Science. 2005 Feb 4;307(5710):734-8. Erratum in: Science. 2005 Apr 29;308(5722):633. Nod2 mutation in Crohn's disease potentiates NF-kappaB activity and IL-1beta processing.
12. Li J, Moran T, Swanson E, Julian C, Harris J, Bonen DK, Hedl M, Nicolae DL, Abraham C, Cho JH. Hum Mol Genet. 2004 Aug 15;13(16):1715-25. Epub 2004 Jun 15. Regulation of IL-8 and IL-1beta expression in Crohn's disease associated NOD2/CARD15 mutations.
13. Brideau C, Gunter B, Pikounis B, Liaw A. J Biomol Screen. 2003 Dec;8(6):634-47 Improved statistical methods for hit selection in high-throughput screening
Protocol
Please see pertinent AIDs: 1578,1849, 1852, 2245, 2250, 2255, 2260, 2261, 2261, 2264, 2333, 2335, 2337,2466, 2469, 2483, 2485, 2503, 2504, 2505, 2789, 2792, 2793, 2798, 2799, 2800, 2801
Comment
Probe molecules are defined as the positives of this assay and assigned a score of 100. Compounds SID85248360 and SID 87225488 have been identified as probe molecules.
Result Definitions
| TID | Name | Description | Histogram | Type | Unit | |
|---|---|---|---|---|---|---|
| Outcome | The BioAssay activity outcome | Outcome | ||||
| Score | The BioAssay activity ranking score |  | Integer | |||
| 1 | ML# | ML# for the compound | String |
Additional Information
Grant Number: 1 R03 MH084844-01
Data Table (Concise)
Classification
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