DESCRIPTION (provided by applicant): Breast cancer (BrCa) is the most frequently diagnosed cancer and the leading cause of cancer mortality in females. Estrogen Receptor (ER) is expressed in about 70% of BrCa and has therefore been the most targeted alternative to treat this disease. Effective therapy options include the anti-estrogen tamoxifen (TAM), aromatase inhibitors that block estrogen biosynthesis, or fulvestrant, which induces ER degradation. Unfortunately, many patients develop resistance to ER-targeting drugs, mainly TAM-resistance, which appears to involve ER phosphorylation by a variety of protein kinases. Many protein kinases are mutated or overexpressed in cancer. These hyperactivated kinases with oncogenic activity have been the target of signal transduction-based therapies that include small molecule inhibitors and biologics. Following the approval of Gleevec in 2001, several small molecule kinase inhibitors have been approved by the FDA for the treatment of various cancers, including Tykerb (lapatinib ditosylate), a dual inhibitor of Her2 and EGFR approved for the treatment of metastatic Her2-positive BrCa as stand-alone (2010) or in combination therapy (2007). Genome-wide screening efforts have been useful to identify novel kinases with oncogenic roles that could potentially be targeted for the treatment of BrCa. One such kinase is lemur tyrosine kinase 3 (LMTK3), which was found to phosphorylate and modulate ER¿ activity, and was essential for the growth of ER-positive BrCa cells. Most recently, LMTK3 has also been shown to be highly expressed in ER-negative BrCa cells and to promote invasion via induction of integrin ß1. LMTK3 abundance has been linked to shorter disease free and overall survival time and high nuclear expression was associated with more aggressive cancers. LMTK3 expression was also predictive of hormone resistance. Together, these studies reveal LMTK3 as a potential target for anti-BrCa drug development. With no LMTK3 inhibitors currently known, this proposal seeks to identify small molecules that show potent and selective inhibition of LMTK3 with the ultimate goal to pharmacologically validate this kinase as a novel target in BrCa. We have developed a homogeneous FRET assay to screen TPIMS mixture-based chemical libraries and have identified several mixture libraries with strong LMTK3 inhibitory activity. Individual compounds were found to inhibit LMTK3 at low micromolar dose, which encourage the proposed specific aims to i) complete the deconvolution of an active guanidine linked sulfonamide library to identify potent and selective LMTK3 inhibitors, which will be ii) tested against a panel of BrCa cells in vitro, followed by iii) SAR studies to optimize identified hits. Pharmacological validation of LMTK3 as a potential target in BrCa will support subsequent studies for lead optimization and in vivo testing, which could stimulate future development of novel strategies for the treatment of BrCa.