Pyrrolo[1,4]benzodiazepine antitumor antibiotics: evidence for two forms of tomaymycin bound to DNA

Tomaymycin is an antibiotic belonging to the pyrrolo[1,4]benzodiazepine group of antitumor compounds. Previous studies have shown that tomaymycin and other members of this group, which include anthramycin, sibiromycin, and the neothramycins, bind covalently through N-2 of guanine and lie within the minor groove of DNA. Two fluorescent ground-state species of tomaymycin were observed in protic solvents and on DNA. 1H NMR studies showed that the two fluorescent species in methanol are the 11R,11aS and 11S,11aS diastereomeric 11-methyl ethers of tomaymycin. On the basis of epimerization experiments and exchange of carbon-13 from 13CH3OH into the C-11 methoxy group of the tomaymycin methyl ether, a mechanism is proposed for their interconversion via 10,11-anhydrotomaymycin. Coupling information revealed that the solution conformations of the two diastereomers differ, with the C-5 carbonyl lying closer to the plane of the aromatic ring in the 11R,11aS diastereomer. The fluorescence excitation and emission spectra of the two emitting species in methanol were separated by time-resolved fluorescence spectroscopy and were associated with the diastereomeric forms identified by 1H NMR. Time-resolved fluorescence studies of tomaymycin in protic solvents and on DNA indicated that the absorption spectrum of the longer lifetime component (11R,11aS form) is red-shifted relative to the absorption spectrum of the shorter lifetime component (11S,11aS form), consistent with more extensive conjugation. The two conformational forms of tomaymycin on DNA were tentatively identified as the 11S,11aS and 11R,11aS diastereomeric adducts, which bind in opposite orientations in the minor groove. This proposal is supported by molecular modeling studies using a 6-mer duplex adduct of d(ATGCAT)2.     

Nuclear magnetic resonance solution structures of inter- and intrastrand adducts of DNA cross-linker SJG-136

SJG-136 (1) is a sequence-selective DNA-interactive agent that is about to enter phase II clinical trials for the treatment of malignant disease. Previous studies on the pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers, typified by SJG-136 and DSB-120 (2), have shown that these planar ligands react with the exocyclic NH(2) groups of two guanine bases in the base of the minor groove of DNA to form an irreversible interstrand cross-linked sequence-specific adduct. Using high-field NMR, we have characterized and modeled the previously predicted interstrand duplex adduct formed by SJG-136 with the self-complementary 5'-d(CICGATCICG)(2) duplex (4). This first SJG-136 NMR-refined adduct structure has been compared with previous high-field NMR studies of the adducts of the closely related PBD dimer DSB-120 with the same duplex and of the adduct of tomaymycin (3) formed with 5'-d(ATGCAT)(2). Surprisingly, the SJG-136 duplex adduct appears to be more closely related to the tomaymycin adduct than to the DSB-120 adduct with respect of the orientation and depth of insertion of the ligand within the minor groove. The intrastrand duplex adduct formed in the reaction of SJG-136 with the noncomplementary 5'-d(CTCATCAC)·(GTGATGAG) duplex (5) has also been synthesized and modeled. In this duplex adduct, the nature of the cross-link was confirmed, the central guanines were identified as the sites of alkylation, and the stereochemical configuration at C11 at both ends of the SJG-136 molecule was determined to be S. The NMR-refined solution structures produced for the intrastrand adduct confirm the previously proposed structure (which was based solely on mass spectroscopy). Both the inter- and intrastrand SJG-136 duplex adducts form with minimal distortion of the DNA duplex. These observations have an impact on the proposal for the mechanism of action of SJG-136 both in vitro and in vivo, on the repair of its adducts and mechanism of resistance in cells, and, potentially, on the type of pharmacodynamic assay to be used in clinical trials. SGJ-136 is currently in phase II clinical trials with several groups working on both dimeric cross-linking agents and monoalkylating ligands based on the PBD alkylating moiety. This study suggests subtle differences between the DNA binding of SJG-136 and the C2 unsubstituted analogue DSB-120 that are likely to be the origins of the differences in potency. Confirmation of the stereochemical configuration at the C11 position (particularly in the intrastrand adduct) provides confirmation of binding orientation that was previously only speculation in the HPLC MS study. Together, these observations are likely to be of value in the development of third-generation PBD-based cross-linkers and monoalkylating analogues.     

Accomodation of S-cis-tamoxifen-N(2)-guanine adduct within a bent and widened DNA minor groove

The non-steroidal anti-estrogen tamoxifen [TAM] has been in clinical use over the last two decades as a potent adjunct chemotherapeutic agent for treatment of breast cancer. It has also been given prophylactically to women with a strong family history of breast cancer. However, tamoxifen treatment has also been associated with increased endometrial cancer, possibly resulting from the reaction of metabolically activated tamoxifen derivatives with cellular DNA. Such DNA adducts can be mutagenic and the activities of isomeric adducts may be conformation-dependent. We therefore investigated the high resolution NMR solution conformation of one covalent adduct (cis-isomer, S-epimer of [TAM]G) formed from the reaction of tamoxifen [TAM] to N(2)-of guanine in the d(C-[TAM]G-C).d(G-C-G) sequence context at the 11-mer oligonucleotide duplex level. Our NMR results establish that the S-cis [TAM]G lesion is accomodated within a widened minor groove without disruption of the Watson-Crick [TAM]G. C and flanking Watson-Crick G.C base-pairs. The helix axis of the bound DNA oligomer is bent by about 30 degrees and is directed away from the minor groove adduct site. The presence of such a bulky [TAM]G adduct with components of the TAM residue on both the 5'- and the 3'-side of the modified base could compromise the fidelity of the minor groove polymerase scanning machinery.     

Solution structure of the (+)-cis-anti-benzo[a]pyrene-dA ([BP]dA) adduct opposite dT in a DNA duplex.

Minor adducts, derived from the covalent binding of anti-benzo[a]pyrene-7,8-dihydroxy-9,10-epoxide to cellular DNA, may play an important role in generating mutations and initiating cancer. We have applied a combined NMR-computational approach including intensity based refinement to determine the solution structure of the minor (+)-cis-anti-[BP]dA adduct positioned opposite dT in the d(C1-T2-C3-T4-C5-[BP]A6-C7-T8-T9-C10-C11). (d(G12-G13-A14-A15-G16-T17-G18-A19-G20+ ++-A21-G22) 11-mer duplex. The BP ring system is intercalated toward the 5'-side of the [BP]dA6 lesion site without disrupting the flanking Watson-Crick dC5.dG18 and [BP]dA6.dT17 base pairs. This structure of the (+)-cis-anti-[BP]dA.dT 11-mer duplex, containing a bay region benzo[a]pyrenyl [BP]dA adduct, is compared with the corresponding structure of the (+)-trans-anti-[BPh]dA.dT 11-mer duplex (Cosman et al., Biochemistry 32, 12488-12497, 1993), which contains a fjord region benzo[c]phenanthrenyl [BPh]dA adduct with the same R stereochemistry at the linkage site. The carcinogen intercalates toward the 5'-direction of the modified strand in both duplexes (the adduct is embedded within the same sequence context) with the buckling of the Watson-Crick [BP]dA6.dT17 base pair more pronounced in the (+)-cis-anti-[BP]dA.dT 11-mer duplex compared to its Watson-Crick [BPh]dA.dT17 base pair in the (+)-trans-anti-[BPh]dA.dT 11-mer duplex. The available structural studies of covalent polycyclic aromatic hydrocarbon (PAH) carcinogen-DNA adducts point toward the emergence of a general theme where distinct alignments are adopted by PAH adducts covalently linked to the N(6) of adenine when compared to the N(2) of guanine in DNA duplexes. The [BPh]dA and [BP]dA N(6)-adenine adducts intercalate their polycyclic aromatic rings into the helix without disruption of their modified base pairs. This may reflect the potential flexibility associated with the positioning of the covalent tether and the benzylic ring of the carcinogen in the sterically spacious major groove. By contrast, such an intercalation without modified base pair disruption option appears not to be available to [BP]dG N(2)-guanine adducts where the covalent tether and the benzylic ring are positioned in the more sterically crowded minor groove. In the case of [BP]dG adducts, the benzopyrenyl ring is either positioned in the minor groove without base pair disruption, or if intercalated into the helix, requires disruption of the modified base pair and displacement of the bases out of the helix.     

Solution structure of a 2:1 C2-(2-naphthyl) pyrrolo[2,1-c][1,4]benzodiazepine DNA adduct: molecular basis for unexpectedly high DNA helix stabilization

The naturally occurring pyrrolo[2,1- c][1,4]benzodiazepine (PBD) monomers such as sibiromycin, anthramycin, and tomaymycin form stable covalent adducts with duplex DNA at purine-guanine-purine sites. A correlative relationship between DNA-binding affinity, as measured by enhanced thermal denaturation temperature of calf thymus DNA ( T m), and cytotoxicity is well documented for these naturally occurring compounds and a range of synthetic analogues with sibiromycin having the highest Delta T m value (16.3 degrees C), reflecting favorable hydrogen-bonding interactions between the molecule and DNA bases. We report here that, surprisingly, the structurally simple synthetic C2-(2-naphthyl)-substituted pyrrolo[2,1- c][1,4]benzodiazepine monomer ( 5) has a Delta T m value (15.8 degrees C) similar to that of sibiromycin and significantly higher than the values for either anthramycin (13.0 degrees C) or tomaymycin (2.6 degrees C). 5 also has similar cytotoxic potency to sibiromycin which is widely regarded as the most potent naturally occurring PBD monomer. To investigate this, we have used NMR in conjunction with molecular dynamics to study the 2:1 adduct formed between 5 and the DNA duplex d(AATCTTTAAAGATT) 2. In contrast to the hydrogen-bonding interactions which predominate in the case of sibiromycin and anthramycin adducts, we have shown that the high binding affinity of 5 is due predominantly to hydrophobic (van der Waals) interactions. The high-resolution 2D NOESY, TOCSY, and COSY data obtained have also allowed unequivocal determination of the orientation of the PBD molecule (A-ring toward 3'-end of covalently bound strand), the stereochemistry at the C11 position of the PBD (C11 S), and the conformation of the C2-naphthyl ring which extends along the floor of the minor groove thus optimizing hydrophobic interactions with DNA. These results provide opportunities for future drug design in terms of extending planar hydrophobic groups at the C2 position of PBDs to maximize binding affinity.     

Structure of the anthramycin-d(ATGCAT)2 adduct from one- and two-dimensional proton NMR experiments in solution

One- and two-dimensional 400-MHz proton NMR experiments are used to examine the solution structure of the covalent adduct formed by the interaction of anthramycin methyl ether with the self-complementary deoxyoligonucleotide d(ATGCAT)2. The concentration dependence of chemical shifts and nuclear Overhauser enhancement (NOE) experiments are utilized to assign the adenine H2 protons within the minor groove for both free d(ATGCAT)2 and the adduct. These studies demonstrate that one of the four adenine H2 protons is in close proximity to the bound anthramycin and this results in its upfield shift of 0.3 ppm compared to the adenine H2 protons of the free duplex. Effects of the covalent attachment of anthramycin to the d(ATGCAT)2 duplex result in an increased shielding of selected deoxyribose protons located within the minor groove of the adduct, as demonstrated by two-dimensional autocorrelated (COSY) NMR techniques. Interactions between the protons of the covalently attached anthramycin and the d(ATGCAT)2 duplex are determined by utilizing two-dimensional NOE (NOESY) techniques. Analysis of these data reveals NOE cross-peaks between the anthramycin methyl, H6, and H7 protons with specific deoxyoligonucleotide protons within the minor groove, thus allowing the orientation of the drug within the minor groove to be determined. Nonselective inversion recovery (T1) relaxation experiments are used to probe the structural and dynamic properties of the anthramycin-d(ATGCAT)2 adduct. These data suggest that the binding of anthramycin alters the correlation time of the d(ATGCAT)2 duplex and stabilizes both of the internal A X T base pairs with respect to solvent exchange.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solution structure of the covalent sterigmatocystin-DNA adduct.

Sterigmatocystin and aflatoxin are potent mutagens that contaminate foodstuffs stored under conditions that permit fungal growth. These food mycotoxins can be metabolically activated to their epoxides, which subsequently form covalent adducts with DNA and can eventually induce tumor development. We have generated the sterigmatocystin-d(A1-A2-T3-G4-C5-A6-T7-T8) covalent adduct (two sterigmatocystins per duplex) by reacting sterigmatocystin-1,2-epoxide with the self-complementary d(A-A-T-G-C-A-T-T) duplex and determined its solution structure by the combined application of two-dimensional NMR experiments and molecular dynamics calculations. The self-complementary duplex retains its 2-fold symmetry following covalent adduct formation of sterigmatocystin at the N7 position of G4 residues on each strand of the duplex. The H8 proton of [ST]G4 exchanges rapidly with water and resonates at 9.58 ppm due to the presence of the positive charge on the guanine ring following adduct formation. We have assigned the exchangeable and nonexchangeable proton resonances of sterigmatocystin and the duplex in the covalent adduct and identified the intermolecular proton-proton NOEs that define the orientation and mode of binding of the mutagen to duplex DNA. The analysis was aided by intermolecular NOEs between the sterigmatocystin protons with both the major groove and minor groove protons of the DNA. The molecular dynamics calculations were aided by 180 intramolecular nucleic acid constraints, 16 intramolecular sterigmatocystin constraints, and 56 intermolecular distance constraints between sterigmatocystin and the nucleic acid protons in the adduct. The sterigmatocystin chromophore intercalates between the [ST]G4.C5 and T3.A6 base pairs and stacks predominantly over the modified guanine ring in the adduct duplex. The overall conformation of the DNA remains right-handed on adduct formation with unwinding of the helix, as well as widening of the minor groove. Parallel NMR studies on the sterigmatocystin-d(A1-A2-A3-G4-C5-T6-T7-T8) covalent adduct (two sterigmatocystins per duplex) provide supportive evidence that the mutagen covalently adducts the N7 position of G4 and its chromophore intercalates to the 5' side of the guanine and stacks over it. The present NMR-molecular dynamics studies that define a detailed structure for the sterigmatocystin-DNA adduct support key structural conclusions proposed previously on the basis of a qualitative analysis of NMR parameters for the adduct formed by the related food mutagen aflatoxin B1 and DNA [Gopalakrishnan, S., Harris, T. M., & Stone, M. P. (1990) Biochemistry 29, 10438-10448].     

Solution structures of aminofluorene [AF]-stacked conformers of the syn [AF]-C8-dG adduct positioned opposite dC or dA at a template-primer junction.

A solution structural study has been undertaken on the aminofluorene-C8-dG ([AF]dG) adduct located at a single-strand-double-strand d(A1-A2-C3-[AF]G4-C5-T6-A7-C8-C9-A10-T11-C12-C13). d(G14-G15-A16-T17-G18-G19-T20- A21-G22-N23) 13/10-mer junction (N = C or A) using proton-proton distance restraints derived from NMR data in combination with intensity-based relaxation matrix refinement computations. This single-strand-double-strand junction models one arm of a replication fork composed of a 13-mer template strand which contains the [AF]dG modification site and a 10-mer primer strand which has been elongated up to the modified guanine with either its complementary dC partner or a dA mismatch. The solution structures establish that the duplex segment retains a minimally perturbed B-DNA conformation with Watson-Crick hydrogen-bonding retained up to the dC5.dG22 base pair. The guanine ring of the [AF]dG4 adduct adopts a syn glycosidic torsion angle and is displaced into the major groove when positioned opposite dC or dA residues. This base displacement of the modified guanine is accompanied by stacking of one face of the aminofluorene ring of [AF]dG4 with the dC5.dG22 base pair, while the other face of the aminofluorene ring is stacked with the purine ring of the nonadjacent dA2 residue. By contrast, the dC and dA residues opposite the junctional [AF]dG4 adduct site adopt distinctly different alignments. The dC23 residue positioned opposite the adduct site is looped out into the minor groove by the aminofluorene ring. The syn displaced orientation of the modified dG with stacking of the aminofluorene and the looped out position of the partner dC could be envisioned to cause polymerase stalling associated with subsequent misalignment leading to frameshift mutations in appropriate sequences. The dA23 residue positioned opposite the adduct site is positioned in the major groove with its purine ring aligned face down over the van der Waals surface of the major groove and its amino group directed toward the T6.A21 base pair. The Hoogsteen edge of the modified guanine of [AF]dG4 and the Watson-Crick edge of dA23 positioned opposite it are approximately coplanar and directed toward each other but are separated by twice the hydrogen-bonding distance required for pairing. This structure of [AF]dG opposite dA at a model template-primer junctional site can be compared with a previous structure of [AF]dG opposite dA within a fully paired duplex [Norman, D., Abuaf, P., Hingerty, B. E., Live, D. , Grunberger, D., Broyde, S., and Patel, D. J. (1989) Biochemistry 28, 7462-7476]. The alignment of the Hoogsteen edge of [AF]dG (syn) positioned opposite the Watson-Crick edge of dA (anti) has been observed for both systems with the separation greater in the case of the junctional alignment in the model template-primer system. However, the aminofluorene ring is positioned in the minor groove in the fully paired duplex while it stacks over the junctional base pair in the template-primer system. This suggests that the syn [AF]dG opposite dA junctional alignment can be readily incorporated within a duplex by a translation of this entity toward the minor groove.     

Steady-state fluorescence and molecular-modeling studies of tomaymycin-DNA adducts

The interaction of tomaymycin and 8-O-methyltomaymycin with calf thymus DNA was studied by steady-state fluorescence techniques. The 8-phenolic proton of tomaymycin has a pK = 8.0, and the phenolate anion is essentially nonfluorescent. However, the fluorescence of the DNA adduct does not decrease until pH greater than 10.5, when the DNA double helix denatures. Acrylamide quenches the fluorescence of the free antibiotic with a quenching rate constant kq = 7 x 10(9) M-1 s-1. In DNA adducts, the quenching rate constant is reduced about 50-fold, indicating that the aromatic ring of the drug is shielded from the solvent. The four possible binding modes of the antibiotics were modeled on a 6-mer duplex by molecular mechanics calculations in the absence and presence of water and counterions. The modeling studies show that the antibiotic is buried in the minor groove in all binding modes, with the 8-substituent pointing away from the DNA core. Three or five waters are displaced from the minor groove, depending on the orientation of the drug on the DNA.     

Two-dimensional NMR studies on the anthramycin-d(ATGCAT)2 adduct

Two-dimensional NMR experiments were performed on the adduct of anthramycin with d(ATGCAT)2 to obtain the assignments of the nucleotide base and sugar protons as well as the anthramycin protons. Anthramycin is covalently attached to a guanine 2-amino group, forming the d(ATamGCAT).d(ATGCAT) modified duplex. The anthramycin protons in the minor groove exhibit NOEs to several nucleotide protons. The network of anthramycin-nucleotide NOEs and the measurement of the 10-Hz coupling constant between the anthramycin H11 and H11a protons shows that anthramycin is covalently attached as the S stereoisomer at the anthramycin C11 position with the side chain of anthramycin oriented toward the 5' end of the modified strand. The NOE data show that the anthramycin-modified duplex is in a right-handed conformation with all bases in an anti conformation. Analysis of the J1'-2' coupling constants for the resolved H1' resonances shows that the S-type conformation of the sugars is highly preferred.     

Solution structure of an O6-[4-oxo-4-(3-pyridyl)butyl]guanine adduct in an 11 mer DNA duplex: evidence for formation of a base triplex

The pyridyloxobutylating agents derived from metabolically activated tobacco-specific nitrosamines can covalently modify guanine bases in DNA at the O(6) position. The adduct formed, O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine ([POB]dG), results in mutations that can lead to tumor formation, posing a significant cancer risk to humans exposed to tobacco smoke. A combined NMR-molecular mechanics computational approach was used to determine the solution structure of the [POB]dG adduct within an 11mer duplex sequence d(CCATAT-[POB]G-GCCC).d(GGGCCATATGG). In agreement with the NMR results, the POB ligand is located in the major groove, centered between the flanking 5'-side dT.dA and the 3'-side dG.dC base pairs and thus in the plane of the modified [POB]dG.dC base pair, which is displaced slightly into the minor groove. The modified base pair in the structure adopts wobble base pairing (hydrogen bonds between [POB]dG(N1) and dC(NH4) amino proton and between [POB]dG(NH2) amino proton and dC(N3)). A hydrogen bond appears to occur between the POB carbonyl oxygen and the partner dC's second amino proton. The modified guanine purine base, partner cytosine pyrimidine base, and POB pyridyl ring form a triplex via this unusual hydrogen-bonding pattern. The phosphodiester backbone twists at the lesion site, accounting for the unusual phosphorus chemical shift differences relative to those for the control DNA duplex. The helical distortions and wobble base pairing induced by the covalent binding of POB to the O(6)-position of dG help explain the significant decrease of 17.6 degrees C in melting temperature of the modified duplex relative to the unmodified control.     

Solution structure of the N-(deoxyguanosin-8-yl)-1-aminopyrene ([AP]dG) adduct opposite dA in a DNA duplex.

Solution structural studies have been undertaken on the aminopyrene-C(8)-dG ([AP]dG) adduct in the d(C5-[AP]G6-C7). d(G16-A17-G18) sequence context in an 11-mer duplex with dA opposite [AP]dG, using proton-proton distance and intensity restraints derived from NMR data in combination with distance-restrained molecular mechanics and intensity-restrained relaxation matrix refinement calculations. The exchangeable and nonexchangeable protons of the aminopyrene and the nucleic acid were assigned following analysis of two-dimensional NMR data sets on the [AP]dG.dA 11-mer duplex in H2O and D2O solution. The broadening of several resonances within the d(G16-A17-G18) segment positioned opposite the [AP]dG6 lesion site resulted in weaker NOEs, involving these protons in the adduct duplex. Both proton and carbon NMR data are consistent with a syn glycosidic torsion angle for the [AP]dG6 residue in the adduct duplex. The aminopyrene ring of [AP]dG6 is intercalated into the DNA helix between intact Watson-Crick dC5.dG18 and dC7.dG16 base pairs and is in contact with dC5, dC7, dG16, dA17, and dG18 residues that form a hydrophobic pocket around it. The intercalated AP ring of [AP]dG6 stacks over the purine ring of dG16 and, to a lesser extent dG18, while the looped out deoxyguanosine ring of [AP]dG6 stacks over dC5 in the solution structure of the adduct duplex. The dA17 base opposite the adduct site is not looped out of the helix but rather participates in an in-plane platform with adjacent dG18 in some of the refined structures of the adduct duplex. The solution structures are quite different for the [AP]dG.dA 11-mer duplex containing the larger aminopyrene ring (reported in this study) relative to the previously published [AF]dG.dA 11-mer duplex containing the smaller aminofluorene ring (Norman et al., Biochemistry 28, 7462-7476, 1989) in the same sequence context. Both the modified syn guanine and the dA positioned opposite it are stacked into the helix with the aminofluorene chromophore displaced into the minor groove in the latter adduct duplex. By contrast, the aminopyrenyl ring participates in an intercalated base-displaced structure in the present study of the [AP]dG.dA 11-mer duplex and in a previously published study of the [AP]dG.dC 11-mer duplex (Mao et al., Biochemistry 35, 12659-12670, 1996). Such intercalated base-displaced structures without hydrogen bonding between the [AP]dG adduct and dC or mismatched dA residues positioned opposite it, if present at a replication fork, may cause polymerase stalling and formation of a slipped intermediate that could produce frameshift mutations, the most dominant mutagenic consequence of the [AP]dG lesion.     

Intercalation of aflatoxin B1 in two oligodeoxynucleotide adducts: comparative 1H NMR analysis of d(ATCAFBGAT).d(ATCGAT) and d(ATAFBGCAT)2

8,9-Dihydro-8-(N7-guanyl-[d(ATCGAT)])-9-hydroxyaflatoxin B1.d(ATCGAT) and 8,9-dihydro-8-(N7-guanyl-[d(ATGCAT)])-9-hydroxyaflatoxin B1.8,9-dihydro-8-(N7-guanyl-[d(ATGCAT)])-9-hydroxyaflatoxin B1 were prepared by direct addition of afltoxin B1 8,9-epoxide to d(ATCGAT)2 and d(ATGCAT)2, respectively. In contrast to reaction of aflatoxin B1 8,9-epoxide with d(ATCGAT)2 which exhibits a limiting stoichiometry of 1:1 aflatoxin B1:d(ATCGAT)2 [Gopalakrishnan, S., Stone, M. P., & Harris, T. M. (1989) J. Am. Chem. Soc. 111, 7232-7239], reaction of aflatoxin B1 8,9-epoxide with d(ATGCAT)2 exhibits a limiting stoichiometry of 2:1 aflatoxin B1:d(ATGCAT)2. 1H NOE experiments, nonselective 1H T1 relaxation measurements, and 1H chemical shift perturbations demonstrate that in both modified oligodeoxynucleotides the aflatoxin moiety is intercalated above the 5'-face of the modified guanine. The oligodeoxynucleotides remain right-handed, and perturbation of the B-DNA structure is localized adjacent to the adducted guanine. Aflatoxin-oligodeoxynucleotide 1H NOEs are observed between aflatoxin and the 5'-neighbor base pair and include both the major groove and the minor groove. The aflatoxin methoxy and cyclopentenone ring protons face into the minor groove; the furofuran ring protons face into the major groove. No NOE is observed between the imino proton of the modified base pair and the imino proton of the 5'-neighbor base pair; sequential NOEs between nucleotide base and deoxyribose protons are interrupted in both oligodeoxynucleotide strands on the 5'-side of the modified guanine. The protons at C8 and C9 of the aflatoxin terminal furan ring exhibit slower spin-lattice relaxation as compared to other oligodeoxynucleotide protons, which supports the conclusion that they face into the major groove. Increased shielding is observed for aflatoxin protons; chemical shift perturbations of the oligodeoxynucleotide protons are confined to the immediate vicinity of the adducted base pair. The imidazole proton of the modified guanine exchanges with water and is observed at 9.75 ppm. The difference in reaction stoichiometry is consistent with an intercalated transition-state complex between aflatoxin B1 8,9-epoxide and B-DNA. Insertion of aflatoxin B1-8,9 epoxide above the 5'-face of guanine in d(ATCGAT)2 would prevent the binding of a second molecule of aflatoxin B1 8,9-epoxide. In contrast, two intercalation sites would be available with d(ATGCAT)2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of adduct formation derived from ethylene dibromide on DNA conformation.

Spectral properties of DNA oligomers containing the single modified guanine, S-[2-(N7-guanyl)ethyl]-glutathione, the major adduct derived from 1,2-dibromoethane, were investigated using UV, CD, and NMR. Two palindromic hexamers, d(ATGCAT) and d(ATCGAT), did not form a duplex with guanine bases modified. When the non self-complementary heptamer, d(CATGCCT), was modified at the single guanine, it formed a duplex with its normal complement d(AGGCATG), although the melting temperature was lowered. However, no duplex formation was observed when a non complementary base other than cytosine was placed in d(AGGXATG), suggesting that non Watson-Crick type base pairs are not stabilized by formation of this adduct.     

Time-resolved fluorescence studies of tomaymycin bonding to synthetic DNAs.

Tomaymycin reacts covalently with guanine in the DNA minor groove, exhibiting considerable specificity for the flanking bases. The sequence dependence of tomaymycin bonding to DNA was investigated in synthetic DNA oligomers and polymers. The maximum extent of bonding to DNA is greater for homopurine and natural DNA sequences than for alternating purine-pyrimidine sequences. Saturation of DNA with tomaymycin has little effect on the melting temperature in the absence of unbound drug. Fluorescence lifetimes were measured for DNA adducts at seven of the ten unique trinucleotide bonding sites. Most of the adducts had two fluorescence lifetimes, representing two of the four possible binding modes. The lifetimes cluster around 2-3 ns and 5-7 ns; the longer lifetime is the major component for most bonding sites. The two lifetime classes were assigned to R and S diastereomeric adducts by comparison with previous NMR results for oligomer adducts. The lifetime difference between binding modes is interpreted in terms of an anomeric effect on the excited-state proton transfer reaction that quenches tomaymycin fluorescence. Bonding kinetics of polymer adducts were monitored by fluorescence lifetime measurements. Rates of adduct formation vary by two orders of magnitude with poly(dA-dG).poly(dC-dT), reacting the fastest at 4 x 10(-2) M-1 s-1. The sequence specificity of tomaymycin is discussed in light of these findings and other reports in the literature.     

DNA sequence specificity of the pyrrolo[1,4]benzodiazepine antitumor antibiotics. Methidiumpropyl-EDTA-iron(II) footprinting analysis of DNA binding sites for anthramycin and related drugs

Anthramycin, tomaymycin, and sibiromycin are members of the pyrrolo[1,4]benzodiazepine [P(1,4)B] antitumor antibiotic group. These drugs bind covalently through N2 of guanine and lie within the minor groove of DNA [Petrusek, R. L., Anderson, G. L., Garner, T. F., Fannin, Q. L., Kaplan, D. J., Zimmer, S. G., & Hurley, L. H. (1981) Biochemistry 20, 1111-1119]. The DNA sequence specificity of the P(1,4)B antibiotics has been determined by a footprinting method using methidiumpropyl-EDTA-iron(II) [MPE.Fe(II)], and the results show that each of the drugs has a two to three base pair sequence specificity that includes the covalently modified guanine residue. While 5'PuGPu is the most preferred binding sequence for the P(1,4)Bs, 5'PyGPy is the least preferred sequence. Footprinting analysis by MPE.Fe(II) reveals a minimum of a three to four base pair footprint size for each of the drugs on DNA with a larger than expected offset (two to three base pairs) on opposite strands to that observed in previous analyses of noncovalently bound small molecules. There is an extremely large enhancement of MPE.Fe(II) cleavage between drug binding sites in AT rich regions, probably indicating a drug-induced change in the conformational features of DNA which encourages interaction with MPE.Fe(II). In the presence of sibiromycin or tomaymycin the normally guanine-specific methylene blue reaction used in Maxam and Gilbert sequencing cleaves at other bases in defined positions relative to the drug binding sites. Finally, modeling studies are used to rationalize the differences and similarities in sequence specificities between the various drugs in the P(1,4)B group and their reactions with DNA.     

Determination of the structural role of the linking moieties in the DNA binding of adozelesin

Adozelesin (formerly U73975, The Upjohn Co.) is a monofunctional DNA alkylating analogue of the antitumor antibiotic (+)-CC-1065. Adozelesin consists of a cyclopropa[c]pyrrolo[3,2-e]indol-4(5H)-one (CPI) alkylating subunit of (+)-CC-1065 and a indole and benzofurans subunit replacing the more complex pyrroloindole B and C subunits, respectively, of (+)-CC-1065. Previous studies have shown that adozelesin forms a reversible covalent DNA duplex adduct via a reaction between the N3 of adenine and the cyclopropyl of the cyclopropapyrroloindole (CPI) subunit. Gel electrophoresis studies have shown that adozelesin, like all the monofunctional (CPI)-based antitumor antibiotics, has a sequence preference for 5'-TTA-3' [the asterisk () indicates covalently modified base]. Molecular-modeling studies have shown that the bound adozelesin ligand spans a total of five base pairs including the modified adenine. These studies have also indicated that, owing to the orientation of the ligand within the base minor groove, there should be an overall preference for sequences rich in A.T base pairs, thus avoiding steric crowding around the exocyclic NH(2) of any guanines present. In this study, we have prepared and studied, by high-field NMR and restrained molecular mechanics (rMM) and dynamics (rMD), the duplex adduct formed between adozelesin and 5'-CGTAAGCGCTTACG-3'. Previous molecular-modeling studies suggested that this sequence should be less preferred, since the two GC base pairs should lead to extensive steric crowding within the adduct, and this hypothesis has, however, never been supported by DNA-footprinting data. (1)H NMR of the adozelesin duplex adduct has reveals that, although Watson-Crick base pairing is maintained throughout the DNA duplex, there is significant distortion around the central base pairs. This distortion is the result of strong hydrogen-bonding between the amide linker of the indole and benzofuran subunits, and the carbonyl of a central thymine base and second, weaker, hydrogen bond to the exocyclic NH(2) of the central guanine was also observed. (1)H NMR and rMD also indicate that, to accommodate this hydrogen-bond system, the bound adozelesin is not positioned centrally within the minor groove but pushed toward the modified DNA strand. Previous studies on the dimeric CPI analogue bizelesin have indicated the important role the ureylene linker plays in the DNA binding. This study indicates that a similar situation exists in the reaction of adozelesin with double-stranded DNA and provides a possible explanation into the unpredicted sequence selectivity of these ligands.     

Determination of the major tautomeric form of the covalently modified adenine in the (+)-CC-1065-DNA adduct by 1H and 15N NMR studies

(+)-CC-1065 is an extremely potent antitumor antibiotic produced by Streptomyces zelensis. The potent cytotoxic effects of the drug are thought to be due to the formation of a covalent adduct with DNA through N3 of adenine. Although the covalent linkage sites between (+)-CC-1065 and DNA have been determined, the tautomeric form of the covalently modified adenine in the (+)-CC-1065-DNA duplex adduct was not defined. A [6-15N]deoxyadenosine-labeled 12 base pair non-self-complementary oligomer, d(GGCGGAGTT*AGG).d(CCTAACTCCGCC) (asterisk indicates 15N-labeled base), containing the (+)-CC-1065 most preferred binding sequence 5'AGTTA, was synthesized and modified with (+)-CC-1065. This [6-15N]deoxyadenosine-labeled 12-mer duplex adduct was then studied by 1H and 15N NMR. One-dimensional NOE difference and two-dimensional NOESY 1H NMR experiments on the nonisotopically labeled 12-mer duplex adduct demonstrate that the 6-amino protons of the covalently modified adenine exhibit two signals at 9.19 and 9.08 ppm. Proton NMR experiments on the [6-15N]deoxyadenosine-labeled 12-mer duplex adduct show that the two resonance signals for adenine H6 observed on the nonisotopically labeled duplex adduct were split into doublets by the 15N nucleus with coupling constants of 91.3 Hz for non-hydrogen-bonded and 86.8 Hz for hydrogen-bonded amino protons. Parallel 15N NMR experiments on the [6-15N]deoxyadenosine-labeled (+)-CC-1065-12-mer duplex adduct show a triplet-like signal around -276.9 ppm and coupling constants of 91.5 and 85.6 Hz.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of Molecules with Nucleic Acids. X. Covalent Intercalat e Binding of the Carcinogenic BPDE I(+) to Kinked DNA

Abstract

A theoretical model is proposed for the covalent binding of (+) 7 β,8α-dihydroxy-9α, 10α- epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene denoted by BPDE I(+), to N2 on guanine. The DNA must kink a minimum of 39° to allow proper hybrid configurations about the C10 and N2 atoms involved in bond formation and to allow stacking of the pyrene moiety with the non-bonded adjacent base pair. Conservative (same sugar puckers and glycosidic angles as in B-DNA) and non-conservative (alternating sugar puckers as in intercalation sites) conformations are found and they are proposed structures in pathways connecting B-DNA, an intercalation site, and a kink site in the formation of a covalently intercalative bound adduct of BPDE I(+) to N2 on guanine. Stereographic projections are presented for (3′) and (5′) binding in the DNA. Experimental data for bending of DNA by BPDE, orientation of BPDE in DNA and unwinding of superhelical DNA is explained. The structure of a covalent intercalative complex is predicted to result from the reaction. Also, an anti ? syn transition of guanine results in a structure which allows the DNA to resume its overall B-form. The only change is that guanine has been rotated by 200° about its glycosidic bond so that the BPDE I(+) is bound in the major groove. The latter step may allow the DNA to be stored with an adduct which may produce an error in the genetic code.