Towards understanding the mechanism of action of mithramycin and its analogues: dimerization and DNA binding studies

There has been a recent resurgence of interest in the natural product mithramycin (MTM) following the discovery of MTM as a potent inhibitor of the oncogenic transcription factor EWS-FLI1 [1] present only in Ewing sarcoma cells. MTM is currently in clinical trials for this rare bone and soft tissue cancer primarily affecting children and young adults. It is understood that MTM binds the minor groove of G/C rich DNA as an Mg²⁺-coordinated dimer [2] disrupting transcription of proto-oncogenes; however, this is in disagreement with the binding properties of EWS-FLI1, which prefers GGAA microsatellites [3]. Thus, it was unclear how MTM inhibits EWS-FLI1 and therefore necessary to further investigate MTM's DNA binding preferences. In an effort to elucidate MTM's DNA recognition rules, we investigated dimerization of MTM, its biosynthetic precursor premithramycin B (PreMTM B), and several MTM analogues with modified 3-side chains: mithramycin SDK (MTM SDK) [4], mithramycin SA tryptophan (MTM SA-Trp) [5], and mithramycin SA alanine (MTM SA-Ala) [6]. We also studied DNA specificity of these compounds with various sequences and conformations of double-stranded DNA oligomers. We show that MTM and its analogues dimerize even in the absence of DNA. All compounds except PreMTM B bind DNA with the following rank order binding affinities: MTM = MTM SDK >MTM SA-Trp > MTM SA-Ala [5], demonstrating that modification of the 3-side chain modulates DNA binding affinity of MTM analogues. We determined that the minimum MTM binding site on DNA is an X(G/C)(G/C)X motif, where X can be any base, and established that MTM DNA recognition is driven by direct (sequence) and not indirect (conformation) readout [5]. This study has laid the foundation for subsequent research based on the interaction between MTM, DNA, and the oncogenic transcription factor EWS-FLI1 in the rational design of new MTM analogues for the treatment of Ewing sarcoma.

Acknowledgements: Funding by NIH CA 091901, NIH GM 105977, and Markey Cancer Pilot Award.

Keywords: DNA binding, Chemotherapeutic, Divalent metal ion coordination.

References:

[1] Grohar PJ, Woldemichael GM, Griffin LB, Mendoza A, Chen QR, Yeung C, Currier DG, Davis S, Khanna C, Khan J, McMahon JB, Helman LJ. Identification of an inhibitor of the EWS-FLI1 oncogenic transcription factor by high-throughput screening. J Natl Cancer Inst 2011; 103: 962 – 978

[2] Sastry M, Patel DJ. Solution structure of the mithramycin dimer-DNA complex. Biochemistry 1993; 32: 6588 – 6604

[3] Gangwal K, Sankar S, Hollenhorst PC, Kinsey M, Haroldsen SC, Shah AA, Boucher KM, Watkins WS, Jorde LB, Graves BJ, Lessnick SL. Microsatellites as EWS/FLI response elements in Ewing's sarcoma. Proc Natl Acad Sci U S A 2008; 105: 10149 – 10154

[4] Albertini V, Jain A, Vignati S, Napoli S, Rinaldi A, Kwee I, Nur-e-Alam M, Bergant J, Bertoni F, Carbone GM, Rohr J, Catapano CV. Novel GC-rich DNA-binding compound produced by a genetically engineered mutant of the mithramycin producer Streptomyces argillaceus exhibits improved transcriptional repressor activity: implications for cancer therapy. Nuc Acids Res 2006; 34: 1721 – 1734

[5] Weidenbach S, Hou C, Chen JM, Tsodikov OV, Rohr J. Dimerization and DNA recognition rules of mithramycin and its analogues. J Inorg Biochem 2015; 156: 40 – 47

[6] Scott D, Chen JM, Bae Y, Rohr J. Semi-synthetic mithramycin SA derivatives with improved anticancer activity. Chem Biol Drug Des 2013; 81: 615 – 624