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Bioorganic & Medicinal Chemistry Sep 2014Introducing novel building blocks to solid-phase peptide synthesis, we readily synthesized long-chain hairpin pyrrole-imidazole (PI) polyamide-chlorambucil conjugates 3...
Sequence-specific DNA alkylation and transcriptional inhibition by long-chain hairpin pyrrole-imidazole polyamide-chlorambucil conjugates targeting CAG/CTG trinucleotide repeats.
Introducing novel building blocks to solid-phase peptide synthesis, we readily synthesized long-chain hairpin pyrrole-imidazole (PI) polyamide-chlorambucil conjugates 3 and 4 via the introduction of an amino group into a GABA (γ-turn) contained in 3, to target CAG/CTG repeat sequences, which are associated with various hereditary disorders. A high-resolution denaturing polyacrylamide sequencing gel revealed sequence-specific alkylation both strands at the N3 of adenines or guanines in CAG/CTG repeats by conjugates 3 and 4, with 11bp recognition. In vitro transcription assays using conjugate 4 revealed that specific alkylation inhibited the progression of RNA polymerase at the alkylating sites. Chiral substitution of the γ-turn with an amino group resulted in higher binding affinity observed in SPR assays. These assays suggest that conjugates 4 with 11bp recognition has the potential to cause specific DNA damage and transcriptional inhibition at the alkylating sites.
Topics: Alkylation; Chlorambucil; DNA; Imidazoles; Nylons; Pyrroles; Structure-Activity Relationship; Transcription, Genetic; Trinucleotide Repeats
PubMed: 25127467
DOI: 10.1016/j.bmc.2014.07.019 -
Scientific Reports Feb 2021Sperm DNA contains a range of DNA base damage that can arise, in part, from exposure to methylating agents. However, the effects are not fully characterized and so the...
Sperm DNA contains a range of DNA base damage that can arise, in part, from exposure to methylating agents. However, the effects are not fully characterized and so the aim of this study was to investigate associations between semen quality and the levels of N7-methyldeoxyguanosine (N7-MedG), a marker of exposure to methylating agents, and other markers of DNA damage and DNA methylation. Sperm samples were collected from 105 men attending an assisted reproduction clinic as part of a couple undergoing treatment for infertility and semen quality assessed manually according to WHO guidelines. Semen levels of N7-MedG, quantified by immunoslotblot, were significantly higher in men with sperm concentration < 15 × 10/ml (p ≤ 0.01), semen volume < 1.5 ml (p ≤ 0.05) and also in men with any aspect of semen quality below WHO reference levels (p ≤ 0.001). Measures of neutral Comet DNA damage were correlated with semen quality in a univariate analysis but not after adjustment for N7-MedG levels. Sperm concentration was negatively associated with % methylation at the gene for DAZL but no other marker of global or gene-specific DNA methylation. Results support the hypothesis that the known toxic and DNA damaging properties of alkylating agent exposure may have direct deleterious consequences on semen quality.
Topics: Adult; Alkylating Agents; Biomarkers; Comet Assay; DNA; DNA Adducts; DNA Damage; DNA Methylation; Deoxyguanosine; Gene Expression; Humans; Infertility, Male; Male; Middle Aged; RNA-Binding Proteins; Semen; Semen Analysis; Sperm Count; Spermatozoa
PubMed: 33542261
DOI: 10.1038/s41598-021-81674-x -
Pediatric Blood & Cancer May 2012Fanconi anemia (FA) patients are hypersensitive to DNA alkylating agents and require lower doses than non-FA patients to minimize serious toxicity. The mechanism by...
BACKGROUND
Fanconi anemia (FA) patients are hypersensitive to DNA alkylating agents and require lower doses than non-FA patients to minimize serious toxicity. The mechanism by which hypersensitivity occurs is thought to be due to the inability of these individuals to effectively repair drug-induced interstrand DNA-DNA crosslinks. We recently developed a highly sensitive assay for cyclophosphamide specific interstrand DNA-DNA crosslinks (G-NOR-G) and are able to quantify and compare formation of these adducts in the blood of patients. Therefore we sought to determine whether FA patients have higher in vivo exposure to the cyclophosphamide specific interstrand DNA crosslink, G-NOR-G, relative to patients without FA.
PROCEDURE
Cyclophosphamide interstrand DNA crosslinks were measured with the first dose of cyclophosphamide in FA and non-FA patients receiving a cyclophosphamide based preparative regimen prior to hematopoietic cell transplantation (HCT). FA patients received a lower cyclophosphamide dose than the non-FA patients (5-10 mg/kg/day vs. 50-60 mg/kg/day).
RESULTS
Despite the lower cyclophosphamide dose and lower plasma concentrations in FA patients, they had G-NOR-G amounts similar to the non-FA patients (area under the curve (AUC)(0-∞) , 99.8 vs. 144.9 G-NOR-G adducts/10(6) nucleotides hour, respectively, P = 0.47). When G-NOR-G AUC was normalized for cyclophosphamide plasma concentrations, FA study subjects produced 15-fold higher adducts than non-FA patients (P = 0.05).
CONCLUSIONS
FA patients are hypersensitive to DNA alkylating agents possibly as a result of greater formation of cyclophosphamide specific interstrand DNA crosslinks and/or diminished capacity for DNA repair. Identification and quantification of these adducts may be important determinant of cyclophosphamide related toxicity.
Topics: Adult; Antineoplastic Agents, Alkylating; Child; Child, Preschool; Cyclophosphamide; DNA Adducts; Fanconi Anemia; Female; Humans; Infant; Male; Middle Aged
PubMed: 21793181
DOI: 10.1002/pbc.23254 -
PLoS Genetics Apr 2013Alkylating agents comprise a major class of front-line cancer chemotherapeutic compounds, and while these agents effectively kill tumor cells, they also damage healthy...
Alkylating agents comprise a major class of front-line cancer chemotherapeutic compounds, and while these agents effectively kill tumor cells, they also damage healthy tissues. Although base excision repair (BER) is essential in repairing DNA alkylation damage, under certain conditions, initiation of BER can be detrimental. Here we illustrate that the alkyladenine DNA glycosylase (AAG) mediates alkylation-induced tissue damage and whole-animal lethality following exposure to alkylating agents. Aag-dependent tissue damage, as observed in cerebellar granule cells, splenocytes, thymocytes, bone marrow cells, pancreatic β-cells, and retinal photoreceptor cells, was detected in wild-type mice, exacerbated in Aag transgenic mice, and completely suppressed in Aag⁻/⁻ mice. Additional genetic experiments dissected the effects of modulating both BER and Parp1 on alkylation sensitivity in mice and determined that Aag acts upstream of Parp1 in alkylation-induced tissue damage; in fact, cytotoxicity in WT and Aag transgenic mice was abrogated in the absence of Parp1. These results provide in vivo evidence that Aag-initiated BER may play a critical role in determining the side-effects of alkylating agent chemotherapies and that Parp1 plays a crucial role in Aag-mediated tissue damage.
Topics: Alkylation; Animals; Antineoplastic Agents, Alkylating; Bone Marrow Cells; DNA Glycosylases; DNA Repair; Humans; Insulin-Secreting Cells; Mice; Mice, Transgenic; Neoplasms; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Thymocytes
PubMed: 23593019
DOI: 10.1371/journal.pgen.1003413 -
Nature Nov 2017DNA repair is essential to prevent the cytotoxic or mutagenic effects of various types of DNA lesions, which are sensed by distinct pathways to recruit repair factors...
DNA repair is essential to prevent the cytotoxic or mutagenic effects of various types of DNA lesions, which are sensed by distinct pathways to recruit repair factors specific to the damage type. Although biochemical mechanisms for repairing several forms of genomic insults are well understood, the upstream signalling pathways that trigger repair are established for only certain types of damage, such as double-stranded breaks and interstrand crosslinks. Understanding the upstream signalling events that mediate recognition and repair of DNA alkylation damage is particularly important, since alkylation chemotherapy is one of the most widely used systemic modalities for cancer treatment and because environmental chemicals may trigger DNA alkylation. Here we demonstrate that human cells have a previously unrecognized signalling mechanism for sensing damage induced by alkylation. We find that the alkylation repair complex ASCC (activating signal cointegrator complex) relocalizes to distinct nuclear foci specifically upon exposure of cells to alkylating agents. These foci associate with alkylated nucleotides, and coincide spatially with elongating RNA polymerase II and splicing components. Proper recruitment of the repair complex requires recognition of K63-linked polyubiquitin by the CUE (coupling of ubiquitin conjugation to ER degradation) domain of the subunit ASCC2. Loss of this subunit impedes alkylation adduct repair kinetics and increases sensitivity to alkylating agents, but not other forms of DNA damage. We identify RING finger protein 113A (RNF113A) as the E3 ligase responsible for upstream ubiquitin signalling in the ASCC pathway. Cells from patients with X-linked trichothiodystrophy, which harbour a mutation in RNF113A, are defective in ASCC foci formation and are hypersensitive to alkylating agents. Together, our work reveals a previously unrecognized ubiquitin-dependent pathway induced specifically to repair alkylation damage, shedding light on the molecular mechanism of X-linked trichothiodystrophy.
Topics: AlkB Enzymes; AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase; Alkylating Agents; Alkylation; Amino Acid Sequence; DNA Adducts; DNA Helicases; DNA Repair; DNA-Binding Proteins; Endoplasmic Reticulum; Genes, X-Linked; Humans; Kinetics; Models, Molecular; Multiprotein Complexes; Nuclear Proteins; Polyubiquitin; RNA Polymerase II; RNA Splicing; Signal Transduction; Trichothiodystrophy Syndromes; Ubiquitin; Ubiquitination
PubMed: 29144457
DOI: 10.1038/nature24484 -
Cell Chemical Biology Apr 2022Mutations in mitochondrial DNA (mtDNA) cause mitochondrial diseases, characterized by abnormal mitochondrial function. Although eliminating mutated mtDNA has potential...
Mutations in mitochondrial DNA (mtDNA) cause mitochondrial diseases, characterized by abnormal mitochondrial function. Although eliminating mutated mtDNA has potential to cure mitochondrial diseases, no chemical-based drugs in clinical trials are capable of selective modulation of mtDNA mutations. Here, we construct a class of compounds encompassing pyrrole-imidazole polyamides (PIPs), mitochondria-penetrating peptide, and chlorambucil, an adenine-specific DNA-alkylating reagent. The sequence-selective DNA binding of PIPs allows chlorambucil to alkylate mutant adenine more efficiently than other sites in mtDNA. In vitro DNA alkylation assay shows that our compound 8950A-Chb(Cl/OH) targeting a nonpathogenic point mutation in HeLa S3 cells (m.8950G>A) can specifically alkylate the mutant adenine. Furthermore, the compound reduces the mtDNA possessing the target mutation in cultured HeLa S3 cells. The programmability of PIPs to target different sequences could allow this class of compounds to be developed as designer drugs targeting pathogenic mutations associated with mitochondrial diseases in future studies.
Topics: Adenine; Alkylation; Chlorambucil; DNA, Mitochondrial; Humans; Mitochondria; Mutation; Nylons
PubMed: 34450110
DOI: 10.1016/j.chembiol.2021.08.003 -
Journal of the American Chemical Society Sep 2007A systematic examination of the impact of the yatakemycin left and right subunits and their substituents is detailed along with a study of its unique three subunit...
A systematic examination of the impact of the yatakemycin left and right subunits and their substituents is detailed along with a study of its unique three subunit arrangement (sandwiched vs extended and reversed analogues). The examination of the ca. 50 analogues prepared illustrate that within the yatakemycin three subunit structure, the subunit substituents are relatively unimportant and that it is the unique sandwiched arrangement that substantially increases the rate and optimizes the efficiency of its DNA alkylation reaction. This potentiates the cytotoxic activity of yatakemycin and its analogues overcoming limitations typically observed with more traditional compounds in the series (CC-1065, duocarmycins). Moreover, a study of the placement of the alkylation subunit within the three subunit arrangement (sandwiched vs extended and reversed analogues) indicates that it not only has a profound impact on the rate and efficiency of DNA alkylation but also controls and establishes the DNA alkylation selectivity as well, where both enantiomers of such sandwiched agents alkylate the same adenine sites exhibiting the same DNA alkylation selectivity independent of their absolute configuration.
Topics: Alkylation; Antibiotics, Antineoplastic; DNA; Duocarmycins; Indoles; Models, Molecular; Pyrroles; Structure-Activity Relationship
PubMed: 17691783
DOI: 10.1021/ja072777z -
Nucleic Acids Research Dec 1998The levels of N-alkyl purine and DNA interstrand crosslink formation, produced by the clinically used nitrogen mustard antitumour drug mechlorethamine (HN2), were...
The levels of N-alkyl purine and DNA interstrand crosslink formation, produced by the clinically used nitrogen mustard antitumour drug mechlorethamine (HN2), were quantitated at the level of specific genes in a panel of human tumour cell lines using modified Southern blotting methods. When purified genomic DNA was treated with HN2 in vitro, no significant difference in the extent of N-alkyl purine or interstrand crosslink formation in the N-ras, c-myc or CD3delta genes was observed. When the cell lines LS174T, Colo320HSR, J6 and U937 were treated with HN2, however, there was significant heterogeneity in the levels of N-alkyl purine formation in the three genes. The rank order of the extent of damage in the three genes was also different in the cell lines. The level of alkylation did not correlate with either the transcriptional activity of a gene or drug sensitivity. Crosslinks were not detectable in the N-ras or c-myc genes of LS174T, J6 or U937 cells treated with HN2, and only detectable in the amplified c-myc gene of the Colo320HSR cell line. In the related cell line Colo320DM, which has both native and translocated c-myc alleles which are both amplified and episomal, crosslinks were detected in the amplified native and rearranged c-myc alleles, and also in the N-ras gene which is also amplified in this cell line. For bifunctional alkylating agents such as HN2, therefore, heterogeneity of DNA damage can occur between different genes in human cells and can also vary for different lesions produced by the same agent. In addition, this heterogeneity can differ between human tumour cell lines.
Topics: Alkylation; Antineoplastic Agents, Alkylating; Cross-Linking Reagents; DNA; DNA, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Mechlorethamine; Neoplasms; Tumor Cells, Cultured
PubMed: 9837991
DOI: 10.1093/nar/26.24.5617 -
Science (New York, N.Y.) Feb 2019Certain strains residing in the human gut produce colibactin, a small-molecule genotoxin implicated in colorectal cancer pathogenesis. However, colibactin's chemical...
Certain strains residing in the human gut produce colibactin, a small-molecule genotoxin implicated in colorectal cancer pathogenesis. However, colibactin's chemical structure and the molecular mechanism underlying its genotoxic effects have remained unknown for more than a decade. Here we combine an untargeted DNA adductomics approach with chemical synthesis to identify and characterize a covalent DNA modification from human cell lines treated with colibactin-producing Our data establish that colibactin alkylates DNA with an unusual electrophilic cyclopropane. We show that this metabolite is formed in mice colonized by colibactin-producing and is likely derived from an initially formed, unstable colibactin-DNA adduct. Our findings reveal a potential biomarker for colibactin exposure and provide mechanistic insights into how a gut microbe may contribute to colorectal carcinogenesis.
Topics: Alkylating Agents; Alkylation; Animals; Carcinogenesis; Colorectal Neoplasms; Cyclopropanes; DNA Adducts; DNA Damage; Escherichia coli; Gastrointestinal Microbiome; Germ-Free Life; HT29 Cells; HeLa Cells; Humans; Mice; Mice, Inbred C57BL; Mutagens; Peptides; Polyketides
PubMed: 30765538
DOI: 10.1126/science.aar7785 -
Chemical Research in Toxicology Feb 2020DNA methylating agents are abundant in the environment and are sometimes used in cancer chemotherapy. They react with DNA to form methyl-DNA adducts and byproduct...
DNA methylating agents are abundant in the environment and are sometimes used in cancer chemotherapy. They react with DNA to form methyl-DNA adducts and byproduct lesions that can be both toxic and mutagenic. Foremost among the mutagenic lesions is -methylguanine (m6G), which base pairs with thymine during replication to cause GC → AT mutations. The delta C57BL/6J mouse strain of Nohmi et al. ( , , 465-70) reliably produces mutational spectra of many DNA damaging agents. In this work, mouse embryo fibroblasts (MEFs) were made from delta C57BL/6J mice and evaluated as a screening tool to determine the qualitative and quantitative features of mutagenesis by -methyl--nitrosourea (MNU), a direct-acting DNA alkylator that serves as a model for environmental -nitrosamines, such as -nitrosodimethylamine and therapeutic agents such as Temozolomide. The DNA repair protein MGMT (-methylguanine DNA methyltransferase) protects against environmental mutagenesis by DNA methylating agents and, by removing m6G, limits the therapeutic potential of Temozolomide in cancer therapy. The delta MEFs were treated with MNU to establish dose-dependent toxicity. In parallel, MNU mutagenicity was determined in the presence and absence of the MGMT inhibitor AA-CW236 (4-(2-(5-(chloromethyl)-4-(4-(trifluoromethoxy)phenyl)-1H-1,2,3-triazol-1-yl)ethyl)-3,5-dimethylisoxazole). With and without the inhibitor, the principal mutagenic event of MNU was GC → AT, but more mutations were observed when the inhibitor was present. Evidence that the mutagenic lesion was m6G was based on mass spectral data collected using -methyl--guanine as an internal standard; m6G levels were higher in AA-CW236 treated MEFs by an amount proportional to the higher mutation frequency seen in the same cells. This work establishes delta MEFs as a versatile tool for probing mutagenesis by environmental and therapeutic agents and as a cell culture model in which chemical genetics can be used to determine the impact of DNA repair on biological responses to DNA damaging agents.
Topics: Alkylating Agents; Animals; DNA Modification Methylases; DNA Repair Enzymes; Enzyme Inhibitors; Fibroblasts; Methylnitrosourea; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutagenesis; Tumor Suppressor Proteins
PubMed: 31841318
DOI: 10.1021/acs.chemrestox.9b00444