ABSTRACT The human genome encodes 538 protein kinases, most of which are functionally important and are involved in the pathogenesis of many diseases. This new R01 is a discovery-based grant focused on the molecular validation of novel kinases that are upregulated in endometriosis. To address new therapeutic options for endometriosis, we will validate a subset of endometriosis-implicated kinases using siRNA knockdown strategies and available small molecule inhibitors and subsequently use DNA-Encoded Chemistry Technology (DEC-Tec) to identify novel therapeutic molecules for further chemical validation in vitro and in vivo. We identified dozens of kinases that are statistically upregulated in the lesions of patients with ovarian endometriosis, peritoneal endometriosis, and/or deep infiltrating endometriosis compared to control and patient endometrium. The hypothesis for this R01 is that inhibition of one or more of these novel kinase targets will be effective in the treatment of endometriosis. In this proposal, we outline a comprehensive approach to functionally characterize and target kinases in primary endometriotic stromal cells and organoid cultures derived from women. In parallel, we will utilize state-of-the-art DEC-Tec selection methods available in the Center for Drug Discovery (CDD) at Baylor College of Medicine (BCM) to identify novel kinase inhibitors that will then be re-synthesized without the DNA barcode and chemically optimized by our team. We currently have a screening collection of over 6 billion DNA-encoded molecules. The overarching objective is to identify potent and selective kinase inhibitors that can provide a foundation for new medicines to address the unmet clinical needs of endometriosis patients. The Specific Aims are: 1) Perform target validation of overexpressed kinases; 2) Use DEC-Tec to identify novel small-molecule inhibitors of validated kinases and optimize the selectivity, activity, and stability of high affinity kinase binders; and 3) Evaluate optimized high affinity kinase inhibitors in vitro and in vivo. Identifying new efficacious therapies for endometriosis requires a multifaceted approach that necessitates increased efforts to identify and screen novel targets for drug discovery. Our approach, which combines gene expression datasets for target identification with a novel DEC-Tec drug discovery platform, will rapidly advance key kinase targets through a well-established drug discovery pipeline. Our multidisciplinary team is seeking to discover new chemical entities that address the non-hormonal axes of endometriosis as stand-alone therapy or as sequential therapy during hiatus from endocrine-suppressing agents. Our DEC-Tec platform will help us to find the first specific inhibitors of unexplored kinases (a major druggable class of proteins) that are elevated in endometriosis to dissect these key physiological pathways and pathological mechanisms underlying endometriosis and provide new disease-specific treatm...