When the body responds to a bacterial pathogen, fragments of bacterial peptidoglycan alert a human host to the presence of a microbial invader. A receptor named NOD2 recognizes the peptidoglycans and sounds the alarm, initiating immune responses by causing the production of pro-inflammatory cytokines via  NF-κB and MAP kinase pathways 1.

In this manner, NOD2 sets in motion a series of events that ripple throughout the immune system. But NOD2 also appears to play a role in facilitating the body’s ability to serve as a “good host” to beneficial bacterial comprising a healthy gut microbiome, because loss-of-function NOD2 mutations result in Crohn’s disease 2,3.

And as an immunomodulator, NOD2 gain-of-function mutations cause early-onset sarcoidosis—a granulomatous disease of the skin, joints, and eyes. NOD2 and its obligate kinase RIP2 (also known as RIPK2, RICK, or CARDIAK) are also involved in inflammatory arthritis, 4 multiple sclerosis 5, Blau syndrome 6, and allergic inflammation and asthma 7.

NOD2 Signals to RIPK2 Based on Bacterial Presence
NOD2 recognizes the bacterial peptidoglycans and activates RIPK2, initiating immune responses by causing the production of pro-inflammatory cytokines.

Clearly, inhibiting NOD2 holds clinical relevance. Unfortunately, screening for NOD2 inhibitors has been quite challenging; the multimer it forms is large, unwieldy, and structurally stubborn—in other words, NOD2 is not particularly “druggable.” 8

Since upon activation by NOD2, RIPK2 signals an inflammatory response, a potential alternative to inhibiting NOD2 is inhibiting its kinase partner, RIPK2. Several efforts have focused on identifying RIPK2 inhibitors, yielding Gefitinib, SB203580, WEHI-345, OD36 and OD38, Ponatinib, and GSK583199. Not all of these agents have a high degree of specificity to RIPK2, however. There remains a need for small molecule inhibitors of RIPK2 that are potent, selective, and that have good pharmacological properties.

Toward this end, researchers at Novartis took a virtual screening approach(read the paper here). They computationally evaluated 11 million compounds using a 2D profile quantitative structure-activity relationship (QSAR) method and a 3D pharmacophore search, narrowing down to about 100,000 hits 9.  In further rounds of computational evaluation, they used RIPK2 protein structure data for flexible docking analysis, then clustered and analyzed the 10,000 hits that had the highest docking scores 9.

At this point, researchers began in vitro analysis by testing a subset of these 10,000 compounds using a Trancreener ADP kinase assay with a TR-FRET readout. The Transcreener assay allowed researchers to discern promising hits at an early stage with speed and precision, identifying scaffolds with IC50 values ranging from nanomolar to micromolar 9.  (Please note: These researchers used CisBio’s Transcreener ADP assay, which relied on BellBrook’s first generation ADP antibody; this product is no longer available.  BellBrook’s Transcreener TR-FRET ADP2 Kinase Assay uses an improved ADP antibody that provides higher sensitivity and selectivity along with the advantages of a TR-FRET readout.

From this point, they selected a promising compound based on ligand efficiency, lipophilic efficiency, kinase selectivity, and binding mode 9. Compound 1 bound to the RIPK2 ATP binding site, and analysis of the co-crystal structure of this compound bound with RIPK2 inspired an informed structure-activity relationship approach to discover additional compounds and analogs with improved potency 9. Using in vivo pharmacokinetic assays in animal models and an IL8 secretion assay in human peripheral blood mononuclear cells, researchers evaluated metabolic stability, oral exposure, and cellular activity 9. Rodent and human liver microsomes were used to further refine the metabolic stability and pharmacokinetic profile 9. At every stage, researchers used the pharmacokinetic data to create analogs that were further optimized for desired qualities.

These efforts yielded compound 8, which the authors described as “a promising tool compound for mechanistically investigating the role of RIPK2 inhibition in vivo.” They conducted a battery of in vivo tests on this compound to:

  • evaluate the role of RIPK2 in IL-6 secretion,
  • identify downstream targets for RIPK2 (which led them to discover that ribosomal protein S6 is phosphorylated when the NOD2 pathway is activated, an event that is both biologically fascinating and practical as a potential biomarker for predicting activity of RIPK2 inhibitors in clinical studies),
  • assess the effect of the compound in a rat ex vivo pharmacokinetic/pharmacodynamic (PK/PD) model of cytokine secretion, and
  • assess pharmacodynamic activity in target tissues (blood and gut) using an in vivo rat challenge study to measure cytokine secretion.

Altogether, the work succeeded on several fronts. It identified previously unknown RIPK2 inhibitors and generated robust data on pharmacokinetics, pharmacodynamics, and biological activity. It identified S6 phosphorylation as a potential biomarker for future NOD2 pathway studies. And it served as an example of the power of high throughput Transcreener kinase assays in combination with virtual screening, which enabled the discovery of a potent, selective RIPK2 inhibitor with favorable oral availability from a starting point of 11 million compounds.

-Robyn M. Perrin, PhD

Learn More About the Transcreener ADP² Kinase Assay


[1] Nachbur U, et al. 2015. A RIPK2 inhibitor delays NOD signaling events yet prevents inflammatory cytokine production. Nat Commun. 6:6442.

[2] Balasubramanian I, Gao N. 2017. From sensing to shaping microbiota: insights into the role of NOD2 in intestinal homeostasis and progression of Crohn’s disease. Am J Physiol Gastrointest Liver Physiol. 313(1):G7-G13.

[3] Chen Y, Salem M, Boyd M, Bornholdt J, Li Y, Coskun M, Seidelin JB, Sandelin A, Nielsen OH. 2017. Relation between NOD2 genotype and changes in innate signaling in Crohn’s disease on mRNA and miRNA levels. NPJ Genom Med. eCollection 2017.

[4] Jun JC, Cominelli F, Abbott DW. 2013. RIP2 activity in inflammatory disease and implications for novel therapeutics. J Leukoc Biol. 94(5):927-32.

[5] Shaw PJ, Barr MJ, Lukens JR, McGargill MA, Chi H, Mak TW, Kanneganti TD. 2011. Signaling via the RIP2 Adaptor Protein in Central Nervous System-Infiltrating Dendritic Cells Promotes Inflammation and Autoimmunity. Immunity 34:75−84.

[6] Okafuji I, Nishikomori R, Kanazawa N, Kambe N, Fujisawa A, Yamazaki S, Saito M, Yoshioka T, Kawai T, Sakai H, Tanizaki H, Heike T, Miyachi Y, Nakahata T. 2009.  Role of the NOD2 Genotype in the Clinical Phenotype of Blau Syndrome and Early-onset Sarcoidosis. Arthritis Rheum. 60:242−250.

[7] Duan W, Mehta AK, Magalhaes JG, Ziegler SF, Dong C, Philpott DJ, Croft M. 2010. Innate Signals from NOD2 Block Respiratory Tolerance and Program Th2-Driven Allergic Inflammation. J. Allergy Clin. Immunol. 126:1284−1293.

[8] Maekawa S, Ohto U, Shibata T, Miyake K, Shimizu T. 2016. Crystal structure of NOD2 and its implications in human disease. Nat Commun. 7:11813.

[9]He, X., Da Ros, S., Nelson, J., Zhu, X., Jiang, T., Okram, B., … & Jia, Y. (2017). Identification of Potent and Selective RIPK2 Inhibitors for the Treatment of Inflammatory Diseases. ACS Medicinal Chemistry Letters, 8(10), 1048-1053.

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