DNA interstrand cross-linking, DNA sequence specificity, and induced conformational changes produced by a dimeric analog of (+)-CC-1065.
U-77779 is a symmetrical dimer of the spirocyclopropyl alkylating subunit of (+)-CC-1065 in which the linker consists of two indole subunits separated by a ureido group. This compound was synthesized by scientists of the Upjohn Company and was found to be more active in both anti-tumor efficacy and cytotoxicity than its mono-alkylating analogs. Using three different 21-base pair DNA duplexes containing U-77779 reactive sequences, we have shown that U-77779 produces a stable interstrand cross-linked species that loses its internal self complementarity. A comparison of U-77779 with the mono-alkylating analogs of (+)-CC-1065 shows that it appears to have an increased sequence selectivity such that, while mono-alkylating compounds like (+)-CC-1065 react at more than one site, U-77779 reacts only at sites where there are two suitably positioned alkylation sites. Chemical footprinting with 1,10-phenanthroline-copper complex revealed a six base pair cross-linked region between the two covalently modified adenines with modulated cleavage outside this region. In the case of hydroxyl radical footprinting, considerable variability of the extent of cleavage within the cross-linked sequence was found. These results are discussed in terms of likely induced conformational changes in DNA. In contrast to (+)-CC-1065, non-denaturing gel electrophoresis did not reveal any net bending of DNA due to U-77779, which we believe is due to the 180 degrees out-of-phase bending produced on opposite strands of DNA by the cross-linker.
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Drug Dynamics Institute, College of Pharmacy, University of Texas, Austin 78712.
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D000225:Adenine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D001483:Base Sequence; D003300:Copper; D003432:Cross-Linking Reagents / Q000378:metabolism / Q000494:pharmacology*; D004247:DNA / Q000032:analysis / Q000187:drug effects* / Q000378:metabolism; D004249:DNA Damage; D016172:DNA Fingerprinting; D000080890:Duocarmycins; D004586:Electrophoresis / Q000379:methods; D006878:Hydroxides; D017665:Hydroxyl Radical; D007211:Indoles / Q000378:metabolism / Q000494:pharmacology*; D007933:Leucomycins / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation / Q000187:drug effects; D009841:Oligonucleotides / Q000494:pharmacology*; D010618:Phenanthrolines; D013329:Structure-Activity Relationship; D014508:Urea / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology
D000903:Antibiotics, Antineoplastic; D003432:Cross-Linking Reagents; D000080890:Duocarmycins; D006878:Hydroxides; D007211:Indoles; D007933:Leucomycins; D009841:Oligonucleotides; D010618:Phenanthrolines; C014741:bis(1,10-phenanthroline)copper(2+) ion; D017665:Hydroxyl Radical; C018782:CC 1065; D003300:Copper; D014508:Urea; D004247:DNA; D000225:Adenine; C072257:bizelesin