Sequence specificity of alkylation for a series of nitrogen mustard-containing analogs of distamycin of increasing binding site size: evidence for increased cytotoxicity with enhanced sequence specificity

• Published in: Biochemistry (Easton) 1995-10, Vol.34 (40), p.13034-13041
• Main Authors: Wyatt, Michael D, Lee, Moses, Garbiras, Bonnie J, Souhami, Robert L, Hartley, John A
• Format: Article
• Language: English
• Subjects: Antineoplastic Agents, Alkylating - chemistry; Base Sequence; Benzoates - chemistry; Benzoic Acid; Distamycins - chemistry; DNA - biosynthesis; DNA - chemistry; DNA Adducts - chemistry; DNA Damage; DNA Primers - chemistry; Mechlorethamine - chemistry; Molecular Sequence Data; Nitrogen Mustard Compounds - chemistry; Structure-Activity Relationship
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00040a014

The covalent sequence specificity of a series of nitrogen mustard-containing analogues of distamycin was determined using modified sequencing techniques. The analogues tether benzoic acid mustard (BAM) and possess either one, two, or three pyrrole-amide units. Previous characterization of the biological profile of the series revealed an increase in cytotoxicity for each corresponding increase in the number of pyrrole units, while showing poor cross-link formation in isolated and cellular DNA. Examination of the sequence specificity revealed that BAM produced guanine-N7 lesions in similar manner to other conventional nitrogen mustards. The monopyrrole BAM conjugate also produced guanine-N7 alkylation in a similar pattern to BAM. However, alkylation of adenines was also seen that was found to be minor groove adenine-N3 lesions. The dipyrrole and tripyrrole conjugates did not produce detectable guanine-N7 alkylation but only alkylated in AT tracts. In addition, the tripyrrole conjugate preferentially alkylated only a subset of those sites alkylated by the monopyrrole and dipyrrole conjugates. Two sites, 5'-TTTTGG and 5'-TTTTGA, confirmed as guanine-N3 and adenine-N3 lesions, respectively, were strongly alkylated by the tripyrrole conjugate in preference to other similar sites including three occurrences of 5'-TTTTAA. Footprinting studies comparing distamycin and the tripyrrole conjugate showed identical non-covalent recognition of AT-rich sites. Hence, the drug that possessed the most enhanced sequence specificity for alkylation was also the most cytotoxic of this series.