Generation of new derivatives of the antitumor antibiotic mithramycin by altering the glycosylation pattern through combinatorial biosynthesis

Abstract

Mithramycin is an antitumor drug produced by Streptomyces argillaceus. It consists of a tricyclic aglycone and five deoxyhexoses that form a disaccharide and a trisaccharide chain, which are important for target interaction and therefore for the antitumor activity. Using a combinatorial biosynthesis approach, we have generated nine mithramycin derivatives, seven of which are new compounds, with alterations in the glycosylation pattern. The wild-type S. argillaceus strain and the mutant S. argillaceus M7U1, which has altered D-oliose biosynthesis, were used as hosts to express various "sugar plasmids", each one directing the biosynthesis of a different deoxyhexose. The newly formed compounds were purified and characterized by MS and NMR. Compared to mithramycin, they contained different sugar substitutions in the second (D-olivose, D-mycarose, or D-boivinose instead of D-oliose) and third (D-digitoxose instead of D-mycarose) sugar units of the trisaccharide as well as in the first (D-amicetose instead of D-olivose) sugar unit of the disaccharide. All compounds showed antitumor activity against different tumor cell lines. Structure-activity relationships are discussed on the basis of the number and type of deoxyhexoses present in these mithramycin derivatives.

Scheme 1

Chemical structures of A) mithramycin (1), premithramycinone (2), premithramycin A1 (3), and mithramycin derivatives with altered glycosylation profiles produced by recombinant strains of B) S. argillaceus wild-type and C) S. argillaceus M7U1. Demycarosyl-mithramycin (4), dideolivosyl-6-β-d-amicetosyl-mithramycin (5), demycarosyl-3D-β-d-digitoxosyl-mithramycin (6), deoliosyl-demycarosyl-3C-β-d-boivinosyl-mithramycin (7), deoliosyl-3C-β-d-mycarosyl-mithramycin (8), deoliosyl-demycarosyl-3C-β-d-olivosyl-3D-β-d-digitoxosyl-mithramycin (9), dideolivosyl-6-β-d-amicetosyl-deoliosyl-3C-β-d-olivosyl-mithramycin (10), dideolivosyl-6-β-d-amicetosyl-deoliosyl-demycarosyl-3C-β-d-olivosyl-3D-β-d-digitoxosyl-mithramycin (11), and dideolivosyl-6-β-d-amicetosyl-deoliosyl-demycarosyl-3C-β-d-boivinosyl-mithramycin (12). Structural differences from mithramycin are highlighted with gray circles.

Figure 1

HPLC analyses of cultures of S. argillaceus recombinant strains harboring different “sugar plasmids”. ✩ Premithramycin-like compounds. ★ Mithramycin-like compounds. Peaks corresponding to the different compounds are indicated as follows: M: mithramycin; A: premithramycin A1; P: premithramycinone; peaks a–i: mithramycin derivatives purified from the recombinant strains.

Scheme 2

Proposed pathways for the biosynthesis of the new deoxyhexoses produced in recombinant strains of S. argillaceus wild-type (A) and S. argillaceus M7U1 (B). Compound a: NDP-4-keto-2,3,6-trideoxyglucose; compound b: NDP-2,6-dideoxy-d-glycero-4-hexulose; compound c: NDP-4-keto-2,6-dideoxy-d-glucose. Gray boxes indicate enzymes codified by the “sugar plasmids”. White boxes indicate enzymes from the host. Plasmid names in bold or plain indicate that the biosynthesis of the corresponding DOH is codified by the plasmid itself or by the concerted action of plasmid and host-coded enzymes, respectively. Highlighted by a square is the substrate of MtmU 4-ketoreductase: DAMI, d-amicetose; DBOV, d-boivinose; DDIG, d-digitoxose; DOLV, d-olivose; DOLI, d-oliose.

Figure 2

HPLC analyses of cultures of S. argillaceus and S. argillaceus M7U1 containing pLNRHO and pFL845. ★ Mithramycin-like compounds. Peaks corresponding to the different compounds are indicated as follows: M: mithramycin; A: premithramycin A1; P: premithramycinone; peaks j–o: mithramycin derivatives purified from the recombinant strains.