The Position of Indole Methylation Controls the Structure, DNA Binding, and Cellular Functions of Mithramycin SA‐Trp Analogues

Mithramycin (MTM) is a polyketide anticancer natural product, which functions by noncovalent binding to DNA in the minor groove without intercalation, resulting in inhibiting transcription at G/C‐rich promoters. MTM is a potent inhibitor of cancer cells, such as Ewing sarcoma, driven by abnormal fusions involving E26 transformation‐specific (ETS) family transcription factors friend leukemia integration 1 (FLI1) and ETS‐related gene (ERG). However, MTM is rather toxic and nonselective; therefore, safer, selective analogues of MTM are required for use in the clinic as anticancer drugs. Herein, by using a combination of X‐ray crystallographic, biophysical, and cell and molecular biological techniques, the structural and functional consequences of 3‐side chain methylation at positions 5, 6, and 7 of the indole ring of the potent analogue MTM SA‐Trp are explored. The conformation of the analogues in complexes with DNA, their DNA binding function, cytotoxicity, selectivity, and potency as transcription antagonists depended on the position of the methylation. MTM SA‐5‐methyl‐Trp emerged as the most selective analogue, presumably due to the right balance of the DNA binding and the solvent exposure of the 3‐side chain. This study demonstrates that minor chemical changes can have strong effects in analogue development and paves the way to further development of next‐generation MTM analogues.