ABSTRACT The NOD2:RIPK2 complex helps a cell recognize and respond to an intracellular bacterial infection. Activation of this pathway initiates a cytokine response that helps tailor the adaptive immune system to eradicate the offending pathogen. Signaling from this complex must be precisely tailored. Too little inflammation can cause immunodeficiency that can manifest in dysbiosis while too much inflammation can manifest in inflammatory disease. Over the past decade, my lab has worked to understand this signaling system with the goal of identifying drug targets such that when the NOD2:RIPK2 complex is hyperactive and therefore hyperinflammatory, we have pharmacologic tools to inhibit it. To this end, we have now developed agents for RIPK2 that inhibit its kinase activity. These inhibitors are now at the Pre-IND stage and we anticipate filing for an IND in late 2020 or early 2021. Despite this chemical success, key questions surrounding RIPK2’s kinase activity remain. While some studies suggest that kinase activity is necessary for function, others suggest it is not. We don’t know the kinase-dependent versus kinase-independent signaling pathways that are activated or inhibited. We don’t know any bone fide RIPK2 substrates, and we don’t know which patients will particularly benefit from RIPK2 inhibition. To this end, we have developed phosphoproteomic and phospho-substrate databases utilizing mass spectrometry and chemical genetics. This grant application aims to leverage these unpublished databases to i) identify novel RIPK2 substrates, ii) identify novel RIPK2 kinase-regulated signaling pathways and iii) identify patient populations in which RIPK2 targeting might be most efficacious. Better understanding RIPK2’s kinase activity is highly significant as RIPK2 inhibitors enter the clinic over the next few years.