Ewing sarcoma family of tumors (ESFT) is a family of resilient devastating cancers of bone and soft tissue affecting primarily children and young adults. Current highly cytotoxic combination therapy of five drugs provides only 30% overall survival. The aberrant transcription factor EWS-FLI1 present only in tumor cells is the oncogenic driver of EWS. However, transcription factors were believed to be “undruggable”, until a recent NCI screening found mithramycin (MTM) to act as a potent EWS-FLI1 antagonist. MTM has proven to be too toxic with a narrow therapeutic window and poor pharmacokinetic (PK) properties. Here we propose mechanistic and pharmacology studies of novel MTM analogues (MTM-SA) with significantly reduced toxicity, increased target specificity and greatly improved PK properties. In contrast to other analogues reported elsewhere, which still suffer from poor PK properties, the MTM-SA analogs display superior kinetics and reduced toxicity. The goal of this project is to gain molecular insights into the mode of action of MTM via structural, biochemical and pharmacological studies to generate a highly efficacious and selective anti-ESFT treatment. To aid synthetic efforts in Aim1 and identify analogues with clinical potential, we will perform molecular structure-function level studies in Aim 2 to determine how transcription factor EWS-FLI1 interacts with DNA microsatellite repeats and transcription factor Runx2 (each a necessary interaction for oncogenesis), and how these oncogenic functions are disrupted by MTM-SA. In Aim 3 will assess the in vitro cytotoxicity and target selectivity to identify analogues that will be evaluated in pharmacologic studies that will assess toxicity in humanized liver mice, PK and metabolism, as well as efficacy in xenograft and PDx models of Ewing Sarcoma. The project will be carried out by a team with an established collaboration who have extensive experience in fragment-based drug design and semi-synthetic routes of natural products, X-ray crystallographic, biophysical and molecular biology studies, and pharmacological evaluations. We expect that these structure-function studies will identify a lead-candidate that could enter a clinical trial for the treatment of ESFT.