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 (MTMox) conjugation strategy for rapid
MTM diversification. Comparative cytotoxicity assays of 41 MTMox 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 MTMox32E (a Phe–Trp dipeptide-based 2′-conjugate)
for in vivo testing. Relative to MTM, MTMox32E 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.