Inflammatory Signaling by NOD-RIPK2 Is Inhibited by Clinically Relevant Type II Kinase Inhibitors

Abstract

SummaryRIPK2 mediates pro-inflammatory signaling from the bacterial sensors NOD1 and NOD2, and is an emerging therapeutic target in autoimmune and inflammatory diseases. We observed that cellular RIPK2 can be potently inhibited by type II inhibitors that displace the kinase activation segment, whereas ATP-competitive type I inhibition was only poorly effective. The most potent RIPK2 inhibitors were the US Food and Drug Administration-approved drugs ponatinib and regorafenib. Their mechanism of action was independent of NOD2 interaction and involved loss of downstream kinase activation as evidenced by lack of RIPK2 autophosphorylation. Notably, these molecules also blocked RIPK2 ubiquitination and, consequently, inflammatory nuclear factor κB signaling. In monocytes, the inhibitors selectively blocked NOD-dependent tumor necrosis factor production without affecting lipopolysaccharide-dependent pathways. We also determined the first crystal structure of RIPK2 bound to ponatinib, and identified an allosteric site for inhibitor development. These results highlight the potential for type II inhibitors to treat indications of RIPK2 activation as well as inflammation-associated cancers.

Structures of the inactive and active states of RIP2 kinase inform on the mechanism of activation

“…However in chain A of the RIP2K K47R model the loop is structured and contains a short helix (Fig 3A). A similarly structured loop is observed in one monomer of the RIP2K D146N -STAU bound dimer and in recently described structures with bound inhibitors ponatinib, SB2 or biphenyl-sulfonamide (PDB codes: 4C8B, 5AR4, 5AR8 respectively) [38, 39]. These observations suggest that the Lys209 loop is in a dynamic equilibrium between a helical or disordered conformation.…”

Synthetic Biology Reveals the Uniqueness of the RIP Kinase Domain

“…RIPK2 is critically required for NOD1/2 signaling in response to intracellular bacterial exposure (35), and RIPK4 is required for proper development (9). Despite this, recent structural work has shown that a number of broad-spectrum kinase inhibitors target RIPK1, RIPK2 and RIPK3 with similar potency (16–18). This same structural work has shown that while there are subtle structural differences that may help direct medicinal chemistry toward specific inhibitors; these three kinases overlap significantly in a 3D structural context, potentially make such efforts futile (18).…”

“…Despite this, recent structural work has shown that a number of broad-spectrum kinase inhibitors target RIPK1, RIPK2 and RIPK3 with similar potency (16–18). This same structural work has shown that while there are subtle structural differences that may help direct medicinal chemistry toward specific inhibitors; these three kinases overlap significantly in a 3D structural context, potentially make such efforts futile (18). Additionally, work presented in this manuscript shows that they have overlapping molecular and biochemical activities.…”

“…RIPK2 contains an extended, deep ATP binding pocket, which allows a pharmacologic manipulation likely not afforded by the other RIPKs(11, 16, 18). While molecular modeling and crystal structures have shown largely superimposable kinase domains between RIPK1, 2 and 3, there are subtle structural differences between these three kinases, which can help explain pharmacologic specificity(18). Lastly, pharmacologic inhibitors for RIPK1, RIPK2 and RIPK3 have been developed that independently target these three kinases(10–19).…”

Allergic asthma: RIPK2 takes the lead

“…If RIPK2 kinase activity is confirmed to be important, an FDA approved drug Ponatinib, which is shown to inhibit RIPK2 phosphorylation could provide an alternative to treat allergic asthma. 11 …”