Aclacinomycin B

The genes rdmB and rdmC of Streptomyces purpurascens encoding aclacinomycin modifying enzymes RdmB and RdmC were expressed in Streptomyces lividans TK24. In contrast to the earlier suggestion that RdmC may be an esterase that causes the removal of the carbomethoxy group from the 10 position of aclacinomycins, RdmC functions as an aclacinomycin methyl esterase and catalyzes the removal of the methoxy group from the C-15 position of aclacinomycin T producing 15-demethoxyaclacinomycin T. RdmB acts upon C-10 of 15-demethoxyaclacinomycin T and is able to remove the carboxylic group from the C-10 position. It functions also as an aclacinomycin-10-hydroxylase being able to add a hydroxyl group at the same, C-10 position in vitro. Aclacinomycin methyl esterase was purified to apparent homogeneity from S. lividans carrying the rdmC and aclacinomycin-10-hydroxylase as a glutathione S-transferase fusion construct from Escherichia coli carrying the rdmB gene, respectively. Aclacinomycin methyl esterase functions as a monomer and aclacinomycin-10-hydroxylase as a tetramer. Aclacinomycin methyl esterase has an exceptionally high temperature stability and has an apparent K(m) for aclacinomycin T of 15.5 microM. The introduction of rdmC and rdmB in a Streptomyces galilaeus mutant HO38 produced the same modifications of aclacinomycin T in vivo as aclacinomycin methyl esterase and aclacinomycin-10-hydroxylase in vitro.

Anthracyclines including doxorubicin, epirubicin, daunorubicin, aclarubicin are extensively used as chemotherapeutic agents for treatment of hematological and other malignancies. In cancer therapy anthracyclines are often used in combinations with other chemotherapeutic drugs and agents for molecularly targeted therapy. Anthracyclines are effective and powerful antineoplastic drugs with wide spectrum of application but active use of preparations of this group is limited because of such side effects as cardiotoxicity, myelotoxicity, thromboembolism, alopecia, etc. Cardiotoxicity is the most severe side effect of anthracycline administration. Clinical studies have shown that it is progressive and irreversible. Therefore, early detection and prevention of anthracycline cardiotoxicity has become an important trend in cardiology.

Aclacinomycins A and B are anthracycline antibiotics with potent antitumor activity. Each consists of an alkavinone aglycon chromophore and a trisaccharide (rhodosamine-deoxyfucose-cinerulose A or B) tail attached at the C7 of ring A of the alkavinone. The structures of the 2:1 aclacinomycin-d(CGTACG) complexes have been studied in solution by 2D NMR spectroscopy using nuclear Overhauser effect data. SPEDREF refinement procedure (incorporating simulated annealing within the program X-PLOR) was used to obtain an ensemble of refined structures which reveal that the elongated alkavinone is intercalated between the CpG steps and the trisaccharide lies in the minor groove. In the complex, the two GC Watson-Crick base pairs (C1:G12 and G2:C11) that wrap around the aglycon have large buckles, consistent with those seen in the crystal structures of other anthracycline-DNA complexes. The intercalation geometry of aclcainomycin is a hybrid between those of daunorubicin and nogalamycin. Ring D of alkavinone is sandwiched by the C1 and C11 bases. The deoxyfucose ring of the trisaccharide is close to the DNA backbone at the A4 nucleotide, forcing the DNA helix to kink toward the major groove (with the opening in the minor groove). The kink between two adjacent A-T base pairs (T3-A10 and A4-T9) causes the adenine A4N6 to form two hydrogen bonds to T9O4 (interstrand) and T3O4 (intrastrand) simultaneously. There is a small unwinding of the helix resulting from the intercalated aclacinomycin. Several potential hydrogen bonds exist between the drug and the guanine bases in the minor groove of the helix.