Development of Mithramycin Analogues with Increased Selectivity toward ETS Transcription Factor Expressing Cancers

Abstract

Mithramycin A (1) was identified as the top potential inhibitor of the aberrant ETS transcription factor EWS-FLI1, which causes Ewing sarcoma. Unfortunately, 1 has a narrow therapeutic window, compelling us to seek less toxic and more selective analogues. Here, we used MTMSA (2) to generate analogues via peptide coupling and fragment-based drug development strategies. Cytotoxicity assays in ETS and non-ETS dependent cell lines identified two dipeptide analogues, 60 and 61, with 19.1- and 15.6-fold selectivity, respectively, compared to 1.5-fold for 1. Importantly, the cytotoxicity of 60 and 61 is <100 nM in ETS cells. Molecular assays demonstrated the inhibitory capacity of these analogues against EWS-FLI1 mediated transcription in Ewing sarcoma. Structural analysis shows that positioning the tryptophan residue in a distal position improves selectivity, presumably via interaction with the ETS transcription factor. Thus, these analogues may present new ways to target transcription factors for clinical use.

Discovery of a Cryptic Intermediate in Late Steps of Mithramycin Biosynthesis

“…Neither iso‐MTM nor MTM were active against the Gram‐negative Salmonella enterica . Bioactivity against the EWS‐FLI1 containing Ewing sarcoma cancer cell line TC‐32 was also assayed: iso‐MTM and MTM had an IC 50 of 209 n m and 45 n m , respectively. From these observations it appeared that iso‐MTM was not as cytotoxic as MTM.…”

“…In accordance with this mechanism of action, MTM was recently found to potently inhibit growth of cancer cells that depend on oncogenic transcription. Specifically, MTM is a highly potent antagonist of the bone and soft tissue cancer Ewing sarcoma, which is driven by transcription factor EWS‐FLI1 and prostate cancers driven by TMPRSS2‐ERG . MTM binds DNA at X(G/C)(G/C)X sequences, explaining its preference for G/C‐rich promoters .…”

Bioanalytical method for quantitative determination of mithramycin analogs in mouse plasma by HPLC–QTOF

“…High‐purity MTM, MTMSA‐Trp, MTMSK and MTMSA‐Phe (Figure ) were prepared as described previously (Hou et al, ; Mitra et al, ; Weidenbach, Hou, Chen, Tsodikov, & Rohr, ). Compounds were purified by HPLC and fully characterized by high‐resolution MS and NMR prior to use.…”

Discovery of a Cryptic Intermediate in Late Steps of Mithramycin Biosynthesis

“…Neither iso-MTM nor MTM were active against the Gramnegative Salmonella enterica. Bioactivity against the EWS-FLI1 containing Ewing sarcoma cancer cell line TC-32 [4] was also assayed:iso-MTM and MTM had an IC 50 of 209 nm and 45 nm,respectively.F rom these observations it appeared that iso-MTM was not as cytotoxic as MTM. However, when butanol extractions of the culture supernatant samples containing iso-MTM were analyzed by LC/MS,t hey were found to contain % 50 %MTM, which was likely generated by spontaneous isomerization from iso-MTM.…”

“…Specifically,M TM is ah ighly potent antagonist of the bone and soft tissue cancer Ewing sarcoma, [3] which is driven by transcription factor EWS-FLI1 and prostate cancers driven by TMPRSS2-ERG. [4] MTM binds DNAatX(G/C)(G/C)X sequences,explaining its preference for G/C-rich promoters. [5] Structurally,M TM is ap olyketide drug, composed of at ricyclic aglycone,C 2-and C6-linked tri-and disaccharide chains,r espectively,a nd am ultifunctional C3-pentyl side chain (Scheme 1).…”