ABSTRACT The NIH consortium focused on Illuminating the Druggable Genome (IDG) has identified 134 kinases (the `Dark Kinome') as members of a slightly larger group of biologically important proteins which are understudied. PKN3 (protein kinase N3) is an IDG-eligible member of the Dark Kinome. As of October 2018, Pharos lists only 23 publications for this kinase, and only a subset of these provide biochemical or functional data for PKN3 at the gene or protein level. Nonetheless, data from the Cancer Genome Atlas (TCGA) project and other func- tional studies highlight PKN3 as a potential drug target. There are currently no published, selective small mole- cule inhibitors for PKN3. In preliminary studies we developed and validated a first-in-class covalent inhibitor of PKN3. We went on to use this compound in elegant phosphoproteomic studies to identify potential PKN3 substrates. In parallel we used our well-established platform for tandem affinity purification followed by mass spectrometry (TAP-MS) to identify potential PKN3 protein binding partners. In this proposal we will extend these studies in order to pro- vide key biochemical characterization data for PKN3. Our study plan has two specific aims. Aim 1 is to interrogate signaling pathways relevant to PKN3, identify potential substrates and protein binding partners. We will use our well-established methods for phosphoproteomics along with our PKN3- focused reagents to identify PKN3-dependent signaling pathways as well as potential direct protein substrates. In parallel we will identify PKN3 protein binding partners by tandem affinity purification followed by mass spec- trometry (TAP-MS). Aim 2 is to utilize medicinal chemistry to optimize cellular potency and target selectivity of JZ128. We will diversify the structural elements of JZ128 to improve to create analogs with improved potency. Analogs will be screened in our in vitro PKN3 kinase assay, with the most promising candidates analyzed by CITe-Id to ver- ify selectivity for PKN3.