RIPK2 mediates inflammatory signaling by the bacteria‐sensing receptors NOD1 and NOD2. Kinase inhibitors targeting RIPK2 are a proposed strategy to ameliorate NOD‐mediated pathologies. Here, we reveal that RIPK2 kinase activity is dispensable for NOD2 inflammatory signaling and show that RIPK2 inhibitors function instead by antagonizing XIAP‐binding and XIAP‐mediated ubiquitination of RIPK2. We map the XIAP binding site on RIPK2 to the loop between β2 and β3 of the N‐lobe of the kinase, which is in close proximity to the ATP‐binding pocket. Through characterization of a new series of ATP pocket‐binding RIPK2 inhibitors, we identify the molecular features that determine their inhibition of both the RIPK2‐XIAP interaction, and of cellular and in vivoNOD2 signaling. Our study exemplifies how targeting of the ATP‐binding pocket in RIPK2 can be exploited to interfere with the RIPK2‐XIAP interaction for modulation of NOD signaling.

RIPK 2 通过细菌感应受体NOD 1 和NOD 2 介导炎症信号传导。靶向RIPK 2 的激酶抑制剂是改善NOD介导的病理的建议策略。在这里，我们揭示了RIPK 2 激酶活性对于NOD 2 炎症信号传导是可有可无的，并表明RIPK 2 抑制剂通过拮抗XIAP结合和XIAP介导的RIPK 2泛素化发挥作用。我们将XIAP结合位点映射到RIPK2到激酶N叶的β2和β3之间的环，该环非常靠近ATP结合袋。通过对一系列新的ATP口袋结合RIPK 2 抑制剂的表征，我们确定了决定它们抑制RIPK 2 - XIAP相互作用以及细胞和体内NOD 2 信号传导的分子特征。我们的研究举例说明了如何利用RIPK 2 中ATP结合口袋的靶向来干扰RIPK 2 - XIAP相互作用以调节NOD信号传导。