Sequence-Specific DNA Cleavage by Dipeptides Disubstituted with Chlorambucil and 2,6-Dimethoxyhydroquinone-3-mercaptoacetic Acid

• Published in: Bioconjugate chemistry 2001-11, Vol.12 (6), p.870-882
• Main Authors: Minnock, Andrew, Lin, Li-Seng, Morgan, Julie, Crow, Stephen D. G, Waring, Michael J, Sheh, Leung
• Format: Article
• Language: English
• Subjects: Antineoplastic Agents; Antineoplastic Agents, Alkylating - chemistry; Antineoplastic Agents, Alkylating - metabolism; Autoradiography; Base Sequence; Binding Sites; Chlorambucil - chemistry; Chlorambucil - metabolism; Cross-Linking Reagents - chemistry; Dipeptides - chemistry; DNA - metabolism; Dose-Response Relationship, Drug; Drug Design; Escherichia coli - genetics; Hydrolysis; Hydroquinones - chemistry; Hydroquinones - metabolism; Iron - pharmacology; Lysine - chemistry; Molecular Sequence Data
• ISSN: 1043-1802; 1520-4812
• DOI: 10.1021/bc000151r

Two dipeptides, each containing a lysyl residue, were disubstituted with chlorambucil (CLB) and 2,6-dimethoxyhydroquinone-3-mercaptoacetic acid (DMQ-MA):  DMQ-MA-Lys(CLB)-Gly-NH2 (DM-KCG) and DMQ-MA-β-Ala-Lys(CLB)-NH2 (DM-BKC). These peptide−drug conjugates were designed to investigate sequence-specificity of DNA cleavage directed by the proximity effect of the DNA cleavage chromophore (DMQ-MA) situated close to the alkylating agent (CLB) inside a dipeptide moiety. Agarose electrophoresis studies showed that DM-KCG and DM-BKC possess significant DNA nicking activity toward supercoiled DNA whereas CLB and its dipeptide conjugate Boc-Lys(CLB)-Gly-NH2 display little DNA nicking activity. ESR studies of DMQ-MA and DM-KCG both showed five hyperfine signals centered at g = 2.0052 and are assigned to four radical forms at equilibrium, which may give rise to a semiquinone radical responsible for DNA cleavage. Thermal cleavage studies at 90 °C on a 265-mer test DNA fragment showed that besides alkylation and cleavage at G residues, reactions with DM-KCG and DM-BKC show a preference for A residues with the sequence pattern:  5‘-G-(A) n -Pur-3‘ > 5‘-Pyr-(A) n -Pyr-3‘ (where n = 2−4). By contrast, DNA alkylation and cleavage by CLB occurs at most G and A residues with less sequence selectivity than seen with DM-KCG and DM-BKC. Thermal cleavage studies using N7-deazaG and N7-deazaA-substituted DNA showed that strong alkylation and cleavage at A residues by DM-KCG and DM-BKC is usually flanked on the 3‘ side by a G residue whereas strong cleavage at G residues is flanked by at least one purine residue on either the 5‘ or 3‘ side. At 65 °C, it is notable that the preferred DNA cleavage by DM-KCG and DM-BKC at A residues is significantly more marked than for G residues in the 265-mer DNA; the strongest sites of A-specific reaction occur within the sequences 5‘-Pyr-(A) n -Pyr-3‘; 5‘-Pur-(A) n -G-3‘ and 5‘-Pyr-(A) n -G-3‘. In pG4 DNA, cleavage by DM-KCG and DM-BKC is much greater than that by CLB at room temperature and at 65 °C. It was also observed that DM-KCG and DM-BKC cleaved at certain pyrimidine residues:  C40, T66, C32, T34, and C36. These cleavages were also sequence selective since the susceptible pyrimidine residues were flanked by two purine residues on both the 5‘ and 3‘ sides or by a guanine residue on the 5‘ side. These findings strongly support the proposal that once the drug molecule is positioned so as to permit alkylation by the CLB moiety, the DMQ-MA moi