Fluorescence-based cell-based primary high throughput screening assay to identify inhibitors of the interaction of nucleotide-binding oligomerization domain containing 2 (NOD2) and the receptor-interacting serine-threonine kinase 2 (RIPK2)
Name: Fluorescence-based cell-based primary high throughput screening assay to identify inhibitors of the interaction of nucleotide-binding oligomerization domain containing 2 (NOD2) and the receptor-interacting serine-threonine kinase 2 (RIPK2). ..more
BioActive Compounds: 1383
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Peter S. Tobias, The Scripps Research Institute (TSRI)
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: R21 AI090446
Grant Proposal PI: Peter S. Tobias, The Scripps Research Institute (TSRI)
External Assay ID: NOD2-RIPK2_INH_BLA_1536_1X%INH PRUN
Name: Fluorescence-based cell-based primary high throughput screening assay to identify inhibitors of the interaction of nucleotide-binding oligomerization domain containing 2 (NOD2) and the receptor-interacting serine-threonine kinase 2 (RIPK2).
The function of members of the cytosolic NOD-like receptor (NLR) family has been the object of much research in the last several years focused on determining their triggers and the signaling pathways that they control. NLRs are involved in initiating pathogenic inflammatory responses to a wide variety of pathogens as well as mediating several important diseases of chronic sterile inflammation (1-2). The nucleotide-binding oligomerization domain containing protein (NOD) subfamily is characterized by proteins with a caspase-activating and recruitment domain (CARD). NOD1 and NOD2 both sense peptidoglycan, a heterogeneous polymer found in the cell walls of bacteria, but they detect distinct entities within the peptidoglycan polymer: whereas NOD2 detects muramyl dipeptide (MDP), which is a motif common to Gram-positive and Gram-negative bacterial peptidoglycan (3-4), NOD1 specifically detects diaminopimelic acid (DAP)-type peptidoglycan, which is found almost exclusively in Gram-negative bacteria (5-6). Through their N-terminal CARD domains, NOD1 and NOD2 couple peptidoglycan recognition to the NFkB pathway through a homophilic interaction with the adaptor kinase RIPK2 (7). This pathway leads to the activation of inflammatory mediators that impact on both host defense and the regulation of the immune response (8).
NOD2 malfunction seems to be involved in Crohn's disease and Blau's syndrome (4, 9-12). Although there are inhibitors of several of the downstream effectors of NOD2 activation, such as IL-1ss, we are not aware of any potent and specific inhibitors, and especially no small molecule inhibitors, of NOD2 activation. Such inhibitors could be very important in novel therapeutic approaches to Crohn's disease and Blau's syndrome. The high throughput assay we have designed and tested has the potential to point to such small molecule inhibitors of NOD2. Further, it has the potential to establish proof-of-concept for the idea of designing small molecule inhibitors of other CARD-CARD interactions important in a large number of other NLR related diseases (12).
1. Geddes, K., Magalhaes, J. G., and Girardin, S. E. (2009) Unleashing the therapeutic potential of NOD-like receptors, Nat Rev Drug Discov 8, 465-479.
2. Magalhaes, J. G., Sorbara, M. T., Girardin, S. E., and Philpott, D. J. (2011) What is new with Nods?, Curr Opin Immunol 23, 29-34.
3. Girardin, S. E., Boneca, I. G., Viala, J., Chamaillard, M., Labigne, A., Thomas, G., Philpott, D. J., and Sansonetti, P. J. (2003) Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection, J Biol Chem 278, 8869-8872.
4. Inohara, N., Ogura, Y., Fontalba, A., Gutierrez, O., Pons, F., Crespo, J., Fukase, K., Inamura, S., Kusumoto, S., Hashimoto, M., Foster, S. J., Moran, A. P., Fernandez-Luna, J. L., and Nunez, G. (2003) Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn's disease, J Biol Chem 278, 5509-5512.
5. Chamaillard, M., Hashimoto, M., Horie, Y., Masumoto, J., Qiu, S., Saab, L., Ogura, Y., Kawasaki, A., Fukase, K., Kusumoto, S., Valvano, M. A., Foster, S. J., Mak, T. W., Nunez, G., and Inohara, N. (2003) An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid, Nat Immunol 4, 702-707.
6. Girardin, S. E., Boneca, I. G., Carneiro, L. A., Antignac, A., Jehanno, M., Viala, J., Tedin, K., Taha, M. K., Labigne, A., Zahringer, U., Coyle, A. J., DiStefano, P. S., Bertin, J., Sansonetti, P. J., and Philpott, D. J. (2003) Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan, Science 300, 1584-1587.
7. Krieg, A., Correa, R. G., Garrison, J. B., Le Negrate, G., Welsh, K., Huang, Z., Knoefel, W. T., and Reed, J. C. (2009) XIAP mediates NOD signaling via interaction with RIPK2, Proc Natl Acad Sci U S A 106, 14524-14529.
8. Bertrand, M. J., Doiron, K., Labbe, K., Korneluk, R. G., Barker, P. A., and Saleh, M. (2009) Cellular inhibitors of apoptosis cIAP1 and cIAP2 are required for innate immunity signaling by the pattern recognition receptors NOD1 and NOD2, Immunity 30, 789-801.
9. Hugot, J. P., Chamaillard, M., Zouali, H., Lesage, S., Cezard, J. P., Belaiche, J., Almer, S., Tysk, C., O'Morain, C. A., Gassull, M., Binder, V., Finkel, Y., Cortot, A., Modigliani, R., Laurent-Puig, P., Gower-Rousseau, C., Macry, J., Colombel, J. F., Sahbatou, M., and Thomas, G. (2001) Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease, Nature 411, 599-603.
10. Miceli-Richard, C., Lesage, S., Rybojad, M., Prieur, A. M., Manouvrier-Hanu, S., Hafner, R., Chamaillard, M., Zouali, H., Thomas, G., and Hugot, J. P. (2001) CARD15 mutations in Blau syndrome, Nat Genet 29, 19-20.
11. Ogura, Y., Bonen, D. K., Inohara, N., Nicolae, D. L., Chen, F. F., Ramos, R., Britton, H., Moran, T., Karaliuskas, R., Duerr, R. H., Achkar, J. P., Brant, S. R., Bayless, T. M., Kirschner, B. S., Hanauer, S. B., Nunez, G., and Cho, J. H. (2001) A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease, Nature 411, 603-606.
12. Ting, J. P., Kastner, D. L., and Hoffman, H. M. (2006) CATERPILLERs, pyrin and hereditary immunological disorders, Nat Rev Immunol 6, 183-195.
13. Lee, H. K., Brown, S. J., Rosen, H., and Tobias, P. S. (2007) Application of beta-lactamase enzyme complementation to the high-throughput screening of toll-like receptor signaling inhibitors, Mol Pharmacol 72, 868-875.
14. Lee, H. K., Dunzendorfer, S., and Tobias, P. S. (2004) Cytoplasmic domain-mediated dimerizations of toll-like receptor 4 observed by beta-lactamase enzyme fragment complementation, J Biol Chem 279, 10564-10574.
singlicate, NOD2, nucleotide-binding oligomerization domain containing 2, CARD15, caspase activation and recruitment domain family member 15, RIP2, RIPK2, receptor-interacting serine-threonine kinase 2, Receptor interacting protein 2, NLR, nucleotide-binding domain leucine-rich repeat-containing protein, enzyme complementation, receptor, reporter assay, beta-lactamase, Bla, CHO cells, fluorescence, CCF4, inhibit, inhibitor, inhibition, protein-protein interaction, Crohn's disease, Blau's syndrome, primary, HTS, 1536, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
The purpose of this enzyme fragment complementation assay (13-14) is to identify compounds that inhibit RIPK2-NOD2 binding. This assay uses a CHO cell line that was generated to stably express two beta-lactamase (BLA) fragment fusion proteins: a chimera of RIPK2 fused to BLA fragment "a" and a chimera of NOD2 fused to BLA fragment "b". These cells have constitutive BLA activity due to the interaction of the RIPK2 and NOD2 components of the chimeras, which brings together BLA fragments "a" and "b" to reconstitute full length BLA. As designed, test compounds which interrupt RIPK2 and NOD2 interaction will disrupt BLA reconstitution and reduce BLA activity. BLA activity is detected by measuring fluorescence of the cleavable BLA substrate CCF4, which yields either blue fluorescence at 450 nm (FRET) or green fluorescence at 519 nm (no FRET). Test compounds will be screened for inhibition of constitutive RIPK2-NOD2 binding as measured by changes in the ratio of fluorescence emissions at 450 nm and 519 nm. Clavulanic acid, an inhibitor of BLA activity, was implemented as a non specific control. Compounds are tested in singlicate at a nominal test concentration of 4.98 uM.
The stably transfected NOD2 RIPK2 CHO cells were cultured in 500 sq cm triple flasks at 37 C and 95% relative humidity (RH). The growth media consisted of Dulbecco's Modified Eagle's Media (DMEM) containing 10% v/v fetal bovine serum, 0.1 mM NEAA, 1 mM Sodium Pyruvate, 25 mM HEPES, 5 mM L-Glutamine, 200 ug/mL geneticin (G418) and 1X Penicillin/Streptomycin.
Prior to assay, cells were suspended to a concentration of approximately 200,000 cells/ml in DMEM (Phenol Red Free), containing 10% v/v fetal bovine serum, 0.1 mM NEAA, 1mM Sodium Pyruvate, 25 mM Hepes, 5 mM L-Glutamine and 1X Penicillin/Streptomycin. To start the assay, 5 uL of assay media was dispensed into the first two columns of a 1536 well plate and 5 ul of cell suspension to the remaining wells (1,000 cells per well). Plates were centrifuged and then incubated at 37 C, 5% CO2 and 95% RH for 19 hours. Next, 25 nL of test compound in DMSO, Clavulanate (200 uM final concentration) or DMSO alone (0.5% final concentration) were added to the appropriate wells and incubated for 30 minutes at 37 C, 5% CO2 and 95% RH.
Then, 1 uL of the fluorogenic CCF4-AM substrate, prepared according to manufacturer's protocol (LiveBLAzer, Invitrogen), was added to each well. After 2 hours of incubation at 25 C, well fluorescence was measured on the EnVision plate reader (PerkinElmer Life Sciences, Turku, Finland) at an excitation wavelength of 405 nm and emission wavelengths of 535 nm and 450 nm. Fluorescence values measured for each channel were corrected by subtracting "background" fluorescence, i.e. fluorescence measured in wells containing media only. Background corrected fluorescence emission values were then used to calculate a ratio for each well, according to the following mathematical expression:
Ratio = I450 nm/I535 nm
I represents the measured fluorescence emission intensity at the enumerated wavelength in nm.
Percent inhibition was calculated from the median ratio as follows:
%_Inhibition = 100 * ( ( Ratio_Test_Compound - Median_Ratio_Low_Control ) / ( Median_Ratio_High_Control - Median_Ratio_Low_Control ) ) )
Test_Compound is defined as wells containing test compound
Low_Control is defined as wells containing 0.5% DMSO (0% inhibition)
High_Control is defined as wells containing 200 uM clavulanate in 0.5% DMSO (100% inhibition).
PubChem Activity Outcome and Score:
A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-6, and for inactive compounds 6-0.
List of Reagents:
Stably-transfected CHO cell line coexpressing NOD2-Bla(b) and RIPK2-Bla(a) (supplied by Assay Provider)
LiveBLAzer FRET-B/G Loading Kit (Invitrogen, part K1030)
Potassium Clavulanate (Sigma, part 33454)
DMEM, High Glucose (Invitrogen, part 11965-092)
DMEM, High Glucose, Phenol Red Free (Invitrogen, part 21063-029)
Fetal Bovine Serum (Invitrogen, part 26140-079)
NEAA (Invitrogen, part 11140-050)
Hepes (Invitrogen, part 15630-080)
Pen/Strep (Cellgro, part 30-002-CI)
L-Glutamine (Invitrogen, part 25030-081)
Sodium Pyruvate (Invitrogen, part 11360-070)
Geneticin (Invitrogen, part 10131027)
500CM2 Triple Flasks (Nunc, part 132913)
1536-well plates (Corning, part 7338)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.
** Test Concentration.
Data Table (Concise)