Sequence-specific DNA cleavage by dipeptides disubstituted with chlorambucil and 2,6-dimethoxyhydroquinone-3-mercaptoacetic acid.
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-beta-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 degrees 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 degrees 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 degrees 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 moiety is held close to the alkylation site, resulting in markedly enhanced sequence-specific cleavage.
Minnock
A
A
Department of Chemistry, Tunghai Christian University, Taichung, Taiwan 407, Republic of China.
Lin
L S
LS
Morgan
J
J
Crow
S D
SD
Waring
M J
MJ
Sheh
L
L
D000970:Antineoplastic Agents; D018906:Antineoplastic Agents, Alkylating / Q000737:chemistry* / Q000378:metabolism; D001345:Autoradiography; D001483:Base Sequence; D001665:Binding Sites; D002699:Chlorambucil / Q000737:chemistry* / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry; D004247:DNA / Q000378:metabolism*; D004151:Dipeptides / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D006873:Hydroquinones / Q000737:chemistry* / Q000378:metabolism; D007501:Iron / Q000494:pharmacology; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data
D000970:Antineoplastic Agents; D018906:Antineoplastic Agents, Alkylating; D003432:Cross-Linking Reagents; D004151:Dipeptides; D006873:Hydroquinones; C081862:2,6-dimethoxyhydroquinone-3-mercaptoacetic acid; D002699:Chlorambucil; D004247:DNA; D007501:Iron; D008239:Lysine