Mithramycin 2′-Oximes with Improved Selectivity, Pharmacokinetics, and Ewing Sarcoma Antitumor Efficacy

Mithramycin A (MTM) inhibits the oncogenic transcription factor EWS-FLI1 in Ewing sarcoma, but poor pharmacokinetics (PK) and toxicity limit its clinical use. To address this limitation, we report an efficient MTM 2′-oxime (MTM(ox)) conjugation strategy for rapid MTM diversification. Comparative cytotoxicity assays of 41 MTM(ox) analogues using E-twenty-six (ETS) fusion-dependent and ETS fusion-independent cancer cell lines revealed improved ETS fusion-independent/dependent selectivity indices for select 2′-conjugated analogues as compared to MTM. Luciferase-based reporter assays demonstrated target engagement at low nM concentrations, and molecular assays revealed that analogues inhibit the transcriptional activity of EWS-FLI1. These in vitro screens identified MTM(ox)32E (a Phe–Trp dipeptide-based 2′-conjugate) for in vivo testing. Relative to MTM, MTM(ox)32E displayed an 11-fold increase in plasma exposure and improved efficacy in an Ewing sarcoma xenograft. Importantly, these studies are the first to point to simple C3 aliphatic side-chain modification of MTM as an effective strategy to improve PK.