Characterization of covalent DNA binding of morpholino and cyanomorpholino derivatives of doxorubicin.
BACKGROUND
The doxorubicin analogues cyanomorpholino doxorubicin (MRA-CN) and morpholino doxorubicin (MRA) were synthesized in an attempt to avoid the cardiotoxicity and drug resistance of doxorubicin therapy. MRA-CN forms interstrand DNA cross-links without requiring microsomal metabolic activation in the presence of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) to form a product that alkylates DNA, but MRA requires metabolic activation. Alkylation produces DNA cross-links, which are associated with potentiation of the cytotoxicity of some drugs.


OBJECTIVE
Our purpose was to study the DNA binding of MRA-CN and MRA with and without metabolic activation in order to better understand the mechanisms for cross-linking DNA.


METHODS
We used [3H]MRA and [3H]MRA-CN, with the 3H labeled at C-2 and C-6 of the morpholino ring. MRA (10 nM) was incubated with human liver microsomes with or without NADPH to measure DNA binding. In addition, a filter elution assay was used to determine the nature and extent of drug binding to DNA in the human ovarian carcinoma cell line ES-2. We studied the appearance of interstrand cross-links versus total DNA adducts in pBR322 plasmid DNA incubated with 100 nM MRA-CN in cell-free medium and then subjected to denaturation and agarose gel electrophoresis.


RESULTS
Regardless of the extracellular concentration of the drug (1-100 nM), 85% of intracellular MRA-CN was covalently bound to DNA, and the total amount of drug bound to DNA was proportional to extracellular drug concentration. No covalent binding of MRA to DNA was found in cells exposed to 10 nM MRA alone for 2 hours. In contrast, 10% of the intracellular drug was bound to DNA if the cells were exposed to MRA preincubated with human liver microsomes and NADPH. The percentage of plasmids containing at least one interstrand cross-link rose from 35% at 15 minutes to 92% at 2 hours. We estimate that eight molecules of MRA-CN were adducted per molecule of pBR322 DNA (or one drug adduct per 545 base pairs), with a minimum of 12% of the adducts forming interstrand cross-links.


CONCLUSIONS
These results suggest that the carbons at positions 2 and 6 of the morpholino ring of both MRA-CN and the activated metabolite of MRA are retained in the drug-DNA adduct. They also indicate that the formation of interstrand DNA cross-links by MRA-CN is preceded by formation of drug adducts to a single strand of DNA.
Lau
D H
DH
Department of Medicine, Stanford University School of Medicine, Calif.
Durán
G E
GE
Sikic
B I
BI
D000903:Antibiotics, Antineoplastic / Q000378:metabolism*; D001711:Biotransformation; D004273:DNA, Neoplasm / Q000378:metabolism*; D004317:Doxorubicin / Q000031:analogs & derivatives* / Q000378:metabolism; D005260:Female; D006801:Humans; D008862:Microsomes, Liver / Q000378:metabolism; D009249:NADP; D010051:Ovarian Neoplasms / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000903:Antibiotics, Antineoplastic; D004273:DNA, Neoplasm; D009249:NADP; D004317:Doxorubicin; C048396:NSC 354646; C038555:3'-deamino-3'-(3-cyano-4-morpholinyl)doxorubicin