Mithramycin Analogues Generated by Combinatorial Biosynthesis Show Improved Bioactivity

Abstract

Plasmid pLNBIV was used to overexpress the biosynthetic pathway of nucleoside-diphosphate (NDP)-activatedL-digitoxose in the mithramycin producer Streptomyces argillaceus. This led to a "flooding" of the biosynthetic pathway of the antitumor drug mithramycin (MTM) with NDPactivated deoxysugars, which do not normally occur in the pathway, and consequently to the production of the four new mithramycin derivatives 1-4 with altered saccharide patterns. Their structures reflect that NDP sugars produced by pLNBIV, namely, L-digitoxose and its biosynthetic intermediates, influenced the glycosyl transfer to positions B, D, and E, while positions A and C remained unaffected. All four new structures have unique, previously not found sugar decoration patterns, which arise from either overcoming the substrate specificity or inhibition of certain glycosyltransferases (GTs) of the MTM pathway with the foreign NDP sugars expressed by pLNBIV. An apoptosis TUNEL (=terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay revealed that compounds 1 (demycarosyl-3D-β-D-digitoxosyl-MTM) and 3 (deoliosyl-3C-β-Dmycarosyl-MTM) show improved activity (64.8 ± 2% and 50.3 ± 2.5% induction of apoptosis, respectively) against the estrogen receptor (ER)-positive human breast cancer cell line MCF-7 compared with the parent drug MTM (37.8 ± 2.5% induction of apoptosis). In addition, compounds 1 and 4 (3A-deolivosyl-MTM) show significant effects on the ER-negative human breast cancer cell line MDA-231 (63.6 ± 2% and 12.6 ± 2.5% induction of apoptosis, respectively), which is not inhibited by the parent drug MTM itself (2.6 ± 1.5% induction of apoptosis), but for which chemotherapeutic agents are urgently needed.Mithramycin (MTM), a clinically used aureolic acid-type anti-cancer drug and calciumlowering agent produced by Streptomyces argillaceus as well as various other streptomycetes, consists of a polyketide-derived tricyclic core with a highly functionalized pentyl side chain at the 3-position and five deoxysugars linked as trisaccharide and disaccharide chains in the 2-and 6-positions, respectively. Its aglycon including the 3-side chain is biosynthesized from 10 acyl-CoA units by a type II polyketide synthase, and all five deoxyhexose moieties are successively attached to the tetracyclic intermediate premithramycinone, resulting in the fully glycosylated intermediate premithramycin B via premithramycin A 3 , 1-7 before an oxidative cleavage reaction, catalyzed by Baeyer-Villigerase MtmOIV, 3,5,7 followed by the reduction of a side chain keto group by MtmW, finishes the biosynthesis (Scheme 1). The glycosylation Since genes for only four GTs were found in the mtm gene cluster, which are responsible for five glycosylation steps, it was suggested from indirect evidence 8 that MtmGIV catalyzes the attachment of both sugars C (proven) and E (likely), while MtmGIII catalyzes the attachment of oliose D. MtmGI and MtmGII are responsible for the attachment of olivoses A and B, respectively. It is well-known that the glycos...

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