Transcreener ADP Assay Aids in Discovery of RIPK2 Inhibitors for Autoinflammation

GÖTTINGEN, Germany - Jan. 25, 2018 - PRLog -- 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.

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.

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, multiple sclerosis, Blau syndrome, and allergic inflammation and asthma.

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."

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 GSK58319. 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. 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.  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.

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.  (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.

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