Dimerization and DNA recognition rules of mithramycin and its analogues

Abstract

The antineoplastic and antibiotic natural product mithramycin (MTM) is used against cancer-related hypercalcemia and, experimentally, against Ewing sarcoma and lung cancers. MTM exerts its cytotoxic effect by binding DNA as a divalent metal ion (Me2+)-coordinated dimer and disrupting the function of transcription factors. A precise molecular mechanism of action of MTM, needed to develop MTM analogues selective against desired transcription factors, is lacking. Although it is known that MTM binds G/C-rich DNA, the exact DNA recognition rules that would allow one to map MTM binding sites remain incompletely understood. Towards this goal, we quantitatively investigated dimerization of MTM and several of its analogues, MTM SDK (for Short side chain, DiKeto), MTM SA-Trp (for Short side chain and Acid), MTM SA-Ala, and a biosynthetic precursor premithramycin B (PreMTM B), and measured the binding affinities of these molecules to DNA oligomers of different sequences and structural forms at physiological salt concentrations. We show that MTM and its analogues form stable dimers even in the absence of DNA. All molecules, except for PreMTM B, can bind DNA with the following rank order of affinities (strong to weak): MTM = MTM SDK > MTM SA-Trp > MTM SA-Ala. An X(G/C)(G/C)X motif, where X is any base, is necessary and sufficient for MTM binding to DNA, without a strong dependence on DNA conformation. These recognition rules will aid in mapping MTM sites across different promoters towards development of MTM analogues as useful anticancer agents.

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

“…[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). [6] The specific structure of the aglycone is critical for DNAbinding, because MTM binds DNAa sadimer,i nw hich the two molecules of MTM are bound to each other by ad ivalent metal ion coordination to aglycone oxygens.…”

“…[9] In aseries of post-PKS tailoring steps, 2 is glycosylated and methylated resulting in premithramycin B (PMB, 3), presumably the last non-bioactive intermediate of this pathway. [5] Thefourth ring of 3 is oxidized by the Baeyer-Villiger monooxygenase MtmOIV to form premithramycin B-lactone (5), which upon opening provides the C3-pentyl side chain in mithramycin-DKA (6). [10] Ad ecarboxylation reaction, which produces MTM DK (4), is followed by the reduction of the 4'-keto group of the pentyl side chain catalyzed by MtmW,w hich finalizes the biosynthesis of MTM.…”

Discovery of a Cryptic Intermediate in Late Steps of Mithramycin Biosynthesis

“…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 . Structurally, MTM is a polyketide drug, composed of a tricyclic aglycone, C2‐ and C6‐linked tri‐ and disaccharide chains, respectively, and a multifunctional C3‐pentyl side chain (Scheme ) .…”

“…The biosynthesis of MTM begins with the synthesis of the decaketide backbone assembly by a type‐II polyketide synthase (PKS), which includes cyclizing reactions, oxidation/hydroxylation, ketoreduction and a methyl transfer step, to produce the first isolable pathway products, the tetracyclic intermediates 4‐ O ‐demethyl‐premithramycinone and premithramycinone ( 2 ) . In a series of post‐PKS tailoring steps, 2 is glycosylated and methylated resulting in premithramycin B (PMB, 3 ), presumably the last non‐bioactive intermediate of this pathway . The fourth ring of 3 is oxidized by the Baeyer–Villiger monooxygenase MtmOIV to form premithramycin B‐lactone ( 5 ), which upon opening provides the C3‐pentyl side chain in mithramycin‐DKA ( 6 ) .…”