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Proceedings of the National Academy of... Apr 1995Key studies defining the DNA alkylation properties and selectivity of a new class of exceptionally potent, naturally occurring antitumor antibiotics including CC-1065,... (Comparative Study)
Comparative Study Review
Key studies defining the DNA alkylation properties and selectivity of a new class of exceptionally potent, naturally occurring antitumor antibiotics including CC-1065, duocarmycin A, and duocarmycin SA are reviewed. Recent studies conducted with synthetic agents containing deep-seated structural changes and the unnatural enantiomers of the natural products and related analogs have defined the structural basis for the sequence-selective alkylation of duplex DNA and fundamental relationships between chemical structure, functional reactivity, and biological properties. The agents undergo a reversible, stereoelectronically controlled adenine-N3 addition to the least substituted carbon of the activated cyclopropane within selected AT-rich sites. The preferential AT-rich non-covalent binding selectivity of the agents within the narrower, deeper AT-rich minor groove and the steric accessibility to the alkylation site that accompanies deep AT-rich minor groove penetration control the sequence-selective DNA alkylation reaction and stabilize the resulting adduct. For the agents that possess sufficient reactivity to alkylate DNA, a direct relationship between chemical or functional stability and biological potency has been defined.
Topics: Alkylating Agents; Antibiotics, Antineoplastic; Base Sequence; Binding Sites; Consensus Sequence; DNA; Duocarmycins; Indoles; Leucomycins; Models, Molecular; Molecular Sequence Data; Molecular Structure; Nucleic Acid Conformation; Pyrroles; Structure-Activity Relationship
PubMed: 7731958
DOI: 10.1073/pnas.92.9.3642 -
Anti-cancer Drug Design Apr 1998Three different groups of analogs of the sequence-specific minor groove alkylator tallimustine (2) have been synthesized and investigated. Within group I, the dibromo...
Three different groups of analogs of the sequence-specific minor groove alkylator tallimustine (2) have been synthesized and investigated. Within group I, the dibromo nitrogen mustard (3) and the half-mustard (4) are more cytotoxic (IC50 = 0.6 and 40 ng/ml respectively) than tallimustine (IC50 = 50.3 ng/ml) against L1210 cells with high reactivity against the region 5'-TTTTGA. The diol derivative (6) and the difluoro nitrogen mustard (5) were not cytotoxic against L1210 cells and did not show any detectable DNA alkylation. The two compounds modified in the propionamidine terminus (7 and 8, group II), showed lower cytotoxic potency (IC50 = 130 and 94 ng/ml respectively) against L1210 cells than tallimustine (IC50 = 50.3 ng/ml) and a loss of in vitro sequence specificity for DNA alkylation. Considering the compounds in which the pyrrole rings were replaced by one (9) or two (10) pyrazole rings, compound 9 was not significantly cytotoxic against L1210 cell line and was apparently unable to produce alkylation on the DNA fragments tested, while compound 10 showed decreased cytotoxicity (IC50 = 114 ng/ml) and no modification in the pattern and intensity of DNA alkylation. The data obtained in this work suggest that it is possible to increase tallimustine potency by modifying the nitrogen mustard moiety. Moreover, the sequence specificity of DNA alkylation appears to be affected by the modification of the propionamidino moiety but not by the isosteric modification of the pyrrole rings. The correlation between cytotoxicity and alkylation pattern suggests that tallimustine exerts its cytotoxicity through DNA sequence-specific alkylation of the adenine located in the sequence 5'-TTTTGA.
Topics: Animals; Antineoplastic Agents, Alkylating; Autoradiography; DNA; DNA Footprinting; Distamycins; Electrophoresis, Agar Gel; Humans; Leukemia L1210; Nitrogen Mustard Compounds; Tumor Cells, Cultured
PubMed: 9595033
DOI: No ID Found -
Biochemistry May 1994In this study, we have mapped the intracellular alkylation sites of adozelesin and bizelesin, two potent analogs of CC-1065, in individual genes at the single-nucleotide...
In this study, we have mapped the intracellular alkylation sites of adozelesin and bizelesin, two potent analogs of CC-1065, in individual genes at the single-nucleotide level. Human colon carcinoma cells were treated with adozelesin and bizelesin, and the position of adducts were mapped within the PGK-1 and p53 genes by means of ligation-mediated polymerase chain reaction. The monofunctional alkylating agent adozelesin was found to alkylate genomic DNA predominantly within 5'-(A/T)(A/T)A* sequences. Additional sites of alkylation were observed within 5'-(A/T)(G/C)(A/T)A* sequences; however, these were considered to represent sites of medium to low preference. Bizelesin, a bifunctional analog capable of both DNA monofunctional alkylation and DNA interstrand cross-link formation, was also found to alkylate 5'-(A/T)(A/T)A* sequences. Putative bizelesin DNA interstrand cross-link sites indicated that AT-rich sequences are preferred in the intervening sequence between the two cross-linked adenines. Both six- and seven-nucleotide regions were identified as putative sites of DNA interstrand cross-link formation with 5'-TTTTTTA*, 5'-TTTATCA* and 5'-GTACTAA* sequences being preferred. Non-adenine bases are not observed as potential intracellular sites of either DNA interstrand cross-linking formation or monofunctional alkylation. Thus, the patterns of alkylation induced by adozelesin and bizelesin in genomic DNA are similar but not identical to that observed in purified cell-free DNA.
Topics: Alkylating Agents; Base Sequence; Benzofurans; Cyclohexanecarboxylic Acids; Cyclohexenes; DNA; Duocarmycins; Genes, p53; Humans; Indoles; Molecular Sequence Data; Phosphoglycerate Kinase; Polymerase Chain Reaction; Tumor Cells, Cultured; Urea
PubMed: 8180230
DOI: 10.1021/bi00185a043 -
European Journal of Biochemistry Nov 1972
Topics: Alkanesulfonates; Alkylating Agents; Alkylation; Animals; Chemical Phenomena; Chemistry; Chromatography, Ion Exchange; Coliphages; DNA; DNA, Viral; Drug Stability; Esters; Hot Temperature; Phosphoric Acids; Salmon; Tritium
PubMed: 4344908
DOI: 10.1111/j.1432-1033.1972.tb02506.x -
Anti-cancer Drug Design Aug 2000Interstrand DNA cross-links have been considered essential to the activity of current clinical DNA-alkylating antitumour drugs, which generally alkylate in the major... (Comparative Study)
Comparative Study
Interstrand DNA cross-links have been considered essential to the activity of current clinical DNA-alkylating antitumour drugs, which generally alkylate in the major groove. However, the relationship between cross-linking adducts located in the minor groove of DNA with cytotoxicity and antitumour activity has not been extensively investigated. Previous studies have shown that cross-linking ability is not correlated with cytotoxicity in a novel series of polybenzamide-linked nitrogen mustard compounds which alkylate DNA at adenines in the minor groove. In the present study the nature of these cross-linking adducts was explored for a related pair of compounds which are both highly effective cross-linkers but which differ in antitumour potential. Both of these drugs effectively interact with adenines in the minor groove, although their sequence specificity differs. However, the cross-linking event was not inhibited by pre-treatment with Hoechst 33258, although this pre-treatment effectively prevented adenine alkylation. The primary cross-links detected may thus represent guanine N7 alkylations in the major groove. Whether minor groove cross-linking adducts can be formed is uncertain, since the effect of background guanine N7 alkylation may complicate analysis. The cytotoxicity of the polybenzamides may therefore be related to other factors such as their interaction with cellular repair systems.
Topics: Adenine; Alkylation; Antineoplastic Agents, Alkylating; Benzamides; Bisbenzimidazole; Chlorambucil; Cross-Linking Reagents; DNA; Dose-Response Relationship, Drug; Drug Interactions; Fluorescent Dyes; Nitrogen Mustard Compounds; Nucleic Acid Conformation; Taq Polymerase
PubMed: 11200500
DOI: No ID Found -
Neurotoxicology 2000Treatment of pregnant guinea pigs with trichlorfon causes cerebellar hypoplasia in offspring. The most sensitive period for treatment is days 42-47 of gestation, which...
Brain hypoplasia caused by exposure to trichlorfon and dichlorvos during development can be ascribed to DNA alkylation damage and inhibition of DNA alkyltransferase repair.
Treatment of pregnant guinea pigs with trichlorfon causes cerebellar hypoplasia in offspring. The most sensitive period for treatment is days 42-47 of gestation, which coincides with the rapid brain growth spurt and with the development of cerebellar granule cells. When rat granule cells were exposed in vitro to trichlorfon and dichlorvos for 24 hours they died, whereas trichloroethanol had no effect. When the cells were exposed to trichlorfon and dichlorvos for 3 hours, only dichlorvos was lethal indicating that the metabolite dichlorvos was more potent than trichlorfon itself. Cultured cerebellar granule cells were also found to be quite sensitive to other DNA-alkylating agents such as methylazoxymethanol and methylmethane sulphonate and to O6-benzylguanine; a potent and specific inhibitor of the DNA alkyltransferase involved in the repair of DNA alkylation damage. The organophosphorous compounds were also found to cause inhibition of the alkyltransferase and the lethal effects of the tested compounds on granule cell culture correlated well with the potency of inhibition. In a bacterial test system for monitoring alkylation effects on the DNA, dichlorvos was demonstrated to have a strong DNA alkylation effect. These results suggest that alkylation of DNA and inhibition of its repair can contribute to the brain hypoplasia observed after exposure to trichlorfon and dichlorvos during brain development.
Topics: Alkyl and Aryl Transferases; Alkylating Agents; Alkylation; Animals; Bacteria; Brain; Brain Chemistry; Cell Survival; Cells, Cultured; DNA; DNA Damage; DNA Repair; Dichlorvos; Female; Guinea Pigs; Insecticides; Methyl Methanesulfonate; Methylazoxymethanol Acetate; Neurons; Pregnancy; Rats; Rats, Wistar; Trichlorfon
PubMed: 10794396
DOI: No ID Found -
Anti-cancer Drug Design Dec 1998A series of bisbenzimidazoles bearing a variety of alkylating agents [ortho- and meta-mustards, imidazolebis(hydroxymethyl), imidazolebis(methylcarbamate) and...
A series of bisbenzimidazoles bearing a variety of alkylating agents [ortho- and meta-mustards, imidazolebis(hydroxymethyl), imidazolebis(methylcarbamate) and pyrrolebis(hydroxymethyl)], appended by a propyl linker chain, were prepared and investigated for sequence-specificity of DNA alkylation and their cytotoxicity. Previous work has shown that, for para-aniline mustards, a propyl linker is optimal for cytotoxicity. Alkaline cleavage assays using a variety of different labelled oligonucleotides showed that the preferred sequences for adenine alkylation were 5'-TTTANANAANN and 5'-ATTANANAANN (underlined bases show the drug alkylation sites), with AT-rich sequences required on both the 5' and 3' sides of the alkylated adenine. The different aniline mustards showed little variation in alkylation pattern and similar efficiencies of DNA cross-link formation despite the changes in orientation and positioning of the mustard, suggesting that the propyl linker has some flexibility. The imidazole- and pyrrolebis(hydroxymethyl) alkylators showed no DNA strand cleavage following base treatment, indicating that no guanine or adenine N3 or N7 adducts were formed. Using the PCR-based polymerase stop assay, these alkylators showed PCR blocks at 5'-C*G sites (the * nucleotide indicates the blocked site), particularly at 5'-TAC*GA 5'-AGC*GGA, and 5'-AGCC*GGT sequences, caused by guanine 2-NH2 lesions on the opposite strand. Only the (more reactive) imidazolebis(methylcarbamoyl) and pyrrolebis(hydroxymethyl) alkylators demonstrated interstrand cross-linking ability. All of the bifunctional mustards showed large (approximately 100-fold) increases in cytotoxicity over chlorambucil, with the corresponding monofunctional mustards being 20- to 60-fold less cytotoxic. These results suggest that in the mustards the propyl linker provides sufficient flexibility to achieve delivery of the alkylator to favoured (adenine N3) sites in the minor groove, regardless of its exact geometry with respect to the bisbenzimidazole carrier. The 'targeted' bisbenzimidazole bis(hydroxymethyl)pyrrole- and imidazole analogues showed very similar patterns of alkylation to the corresponding 'untargeted' compounds, with little evidence of additional selectivity imposed by this AT-preferring carrier.
Topics: Animals; Antineoplastic Agents, Alkylating; Base Sequence; Benzimidazoles; Cattle; DNA Adducts; Drug Screening Assays, Antitumor; Leukemia P388; Mustard Compounds
PubMed: 10335264
DOI: No ID Found -
Carcinogenesis Jan 1980Cultures of Escherichia coli were treated with alkylnitrosoureas. The rates of removal of methylation and ethylation products from the DNA of strains defective in...
The removal of alkylation products from the DNA of Escherichia coli cells treated with the carcinogens N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea: influence of growth conditions and DNA repair defects.
Cultures of Escherichia coli were treated with alkylnitrosoureas. The rates of removal of methylation and ethylation products from the DNA of strains defective in various repair pathways were compared with those of their respective wild-type strains. It was found that the removal of O6-methylguanine did not depend upon xth gene function or the uvr endonuclease. However, the rate of elimination of this product was markedly decreased in polA strains. O6-Ethylguanine (in contrast to its methyl analogue) was removed more slowly from the DNA of uvrA(-) than from that of uvrA(+) strains, indicating that the removal of O6-ethylguanine can be initiated by the uvr endonuclease. The composition of the medium in which methylated cells were resuspended following treatment with N-methyl-N-nitrosourea was also found to influence the rate at which O6-methylguanine was removed from the DNA of treated bacteria. No significant removal of this product from bacterial DNA occurred during treatment of cells in buffer, or when treated bacteria were resuspended in salts medium or in growth medium containing chloramphenicol. The results indicate that the elimination of O6-methylguanine, but not of 3-methyladenine, requires protein synthesis. Only very limited constitutive activity capable of removing O6-MeGua was detected.
Topics: Adenine; Alkylating Agents; Alkylation; Carcinogens; DNA Repair; DNA, Bacterial; Escherichia coli; Ethylnitrosourea; Guanine; Methylation; Methylnitrosourea
PubMed: 22282983
DOI: 10.1093/carcin/1.1.67 -
Acta Neuropathologica Aug 1978The role of DNA alkylation by the neurooncogenic agent 3,3-dimethyl-1-phenyltriazene (DMPT) was investigated perinatally and in adult rats. Following a single...
The role of DNA alkylation by the neurooncogenic agent 3,3-dimethyl-1-phenyltriazene (DMPT) was investigated perinatally and in adult rats. Following a single subcutaneous injection of 14C-DMPT (100 mg/kg) on the 21 day of gestation, the concentration of methylated purines was similar in both fetal liver and brain whereas during postnatal growth this treatment resulted in an increasingly preferential methylation of liver DNA. In 30-day-old and adult rats the concentration of 7-methylguanine in liver was about 8 times higher in brain DNA, suggesting that during prenatal development both liver and brain DNA are transplacentally methylated by a proximate carcinogen produced by maternal organs. Multiple doses of 14C-DMPT (50 mg/kg) to adult rats led to a preferential accumulation of O6-methylguanine in cerebral DNA. This supports the hypothesis that the deficient repair excision capacity of the hypothesis that the deficient repair excision capacity of the central nervous system is a significant factor in the organ-specific carcinogenicity of DMPT and related carcinogens.
Topics: Alkylating Agents; Animals; Brain Chemistry; Carbon Radioisotopes; DNA; Female; Guanine; Liver; Male; Maternal-Fetal Exchange; Methylation; Pregnancy; Purines; Rats
PubMed: 676674
DOI: 10.1007/BF00685004 -
Chemical Research in Toxicology 1993Bizelesin, an intrahelical DNA-DNA interstrand cross-linker related to (+)-CC-1065, has been shown to alkylate DNA through guanine in restriction enzyme sequences in...
Alkylation of guanine and cytosine in DNA by bizelesin. Evidence for a covalent immobilization leading to a proximity-driven alkylation of normally unreactive bases by a (+)-CC-1065 cross-linking compound.
Bizelesin, an intrahelical DNA-DNA interstrand cross-linker related to (+)-CC-1065, has been shown to alkylate DNA through guanine in restriction enzyme sequences in which there is a suitably positioned adenine contained in a highly reactive monoalkylation sequence on the opposite strand. Oligomers containing the sequence 5'-TTTTTN*, in which "N" was either G, C, or T, were synthesized to evaluate the cross-linking potential of bizelesin at nonadenine bases. Kinetic analysis of monoalkylation and cross-linking events demonstrates that it is the reaction at "N" (guanine or cytosine) that results in the cross-link which is the slow step. On the basis of this analysis and the normal unreactivity of guanine and cytosine to alkylation by the cyclopropapyrroloindole alkylating moiety of (+)-CC-1065, we propose that the molecular mechanism for this type of cross-linking reaction most likely involves a covalent immobilization of the second alkylating arm, resulting in a "proximity-driven" reaction.
Topics: Alkylating Agents; Alkylation; Antibiotics, Antineoplastic; Base Composition; Base Sequence; Cross-Linking Reagents; Cytosine; DNA; Duocarmycins; Guanine; Indoles; Kinetics; Leucomycins; Molecular Sequence Data; Oligonucleotides; Urea
PubMed: 8117929
DOI: 10.1021/tx00036a020