The solution structure of the oligodeoxynucleotide 5′-d(CTCGGCXCCATC)-3′·5′-d (GATGGCGCCGAG)-3′ containing the heterocyclic amine 8- [(3-methyl-3H-imidazo[4,5-f] quinolin-2-yl)amino]-2′-deoxyguanosine adduct (IQ) at the third guanine in the NarI restriction sequence, a hot spot for −2 bp frameshifts, is reported. Molecular dynamics calculations restrained by distances derived from 24 1 H NOEs between IQ and DNA, and torsion angles derived from 3 J couplings, yielded ensembles of structures in which the adducted guanine was displaced into the major groove with its glycosyl torsion angle in the syn conformation. One proton of its exocyclic amine was approximately 2.8 Å from an oxygen of the 5′ phosphodiester linkage, suggesting formation of a hydrogen bond. The carcinogen-guanine linkage was defined by torsion angles α′ [N9-C8-N(IQ)-C2(IQ)] of 159 ± 7° and β′ [C8-N(IQ)-C2(IQ)-N3(IQ)] of −23 ± 8°. The complementary cytosine was also displaced into the major groove. This allowed IQ to intercalate between the flanking C·G base pairs. The disruption of Watson-Crick hydrogen bonding was corroborated by chemical-shift perturbations for base aromatic protons in the complementary strand opposite to the modified guanine. Chemical-shift perturbations were also observed for 31 P resonances corresponding to phosphodiester linkages flanking the adduct. The results confirmed that IQ adopted a base-displaced intercalated conformation in this sequence context but did not corroborate the formation of a hydrogen bond between the IQ quinoline nitrogen and the complementary dC.

The site-specific synthesis of oligonucleotides containing the C8-deoxyguanosine adduct of the highly mutagenic heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) has been achieved, and the oligonucleotides were characterized by UV melting temperature analysis, circular dichroism, and UV absorption spectroscopy. Examination of these data indicated that the IQ-adduct is accommodated in dramatically different environments. This sequence-dependent conformational preference is likely to play a key role in the mutagenicity and repair of IQ-modified oligonucleotides.

Abstract2-Amino-3-methylimidazo [4,5-f]quinoline (IQ) is found in cooked meats and forms DNA adducts at the C8-and N 2 -positions of dGuo after appropriate activation. IQ is a potent inducer of frameshift mutations in bacteria and is carcinogenic in laboratory animals. We have incorporated both IQ-adducts into the G 1 -and G 3 -positions of the NarI recognition sequence (5′-G 1 G 2 CG 3 CC-3′), which is a hotspot for arylamine modification. The in vitro replication of the oligonucleotides was examined with Escherichia coli pol I Klenow fragment exo − , E. coli pol II exo − , and Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), and the extension products were sequenced by tandem mass spectrometry. Replication of the C8-adduct at the G 3 -position resulted in two-base deletions with all three polymerases, whereas error-free bypass and extension was observed at the G 1 -position. The N 2 -adduct was bypassed and extended by all three polymerases when positioned at the G 1 -position, and the error-free product was observed. The N 2 -adduct at the G 3 -position was more blocking and was bypassed and extended only by Dpo4 to produce an error-free product. These results indicate that the replication of the IQ-adducts of dGuo is strongly influenced by the local sequence and the regioisomer of the adduct. These results also suggest a possible role for pol II and IV in the error-prone bypass of the C8-IQ-adduct leading to frameshift mutations in reiterated sequences, whereas noniterated sequences result in error-free bypass.

Abstract2-Amino-3-methylimidazo [4,5-f]quinoline (IQ) is a highly mutagenic heterocyclic amine found in cooked meats. The major DNA adduct of IQ is at the C8-position of dGuo. We have previously reported the incorporation of the C8-IQ adduct into oligonucleotides, namely, the G 1 -position of codon 12 of the N-ras oncogene sequence (G 1 G 2 T) and the G 3 -position of the NarI recognition sequence (G 1 G 2 CG 3 CC) (Elmquist et al. (2004) J. Am. Chem. Soc. 126, 11189-11201). Ultraviolet spectroscopy and circular dichroism studies indicated that the conformation of the adduct in the two oligonucleotides was different, and they were assigned as groove-bound and base-displaced intercalated, respectively. The conformation of the latter was subsequently confirmed through NMR and restrained molecular dynamics studies (Wang et al. (2006) J. Am. Chem. Soc. 128, 10085-10095). We report here the incorporation of the C8-IQ adduct into the G 1 -and G 2 -positions of the NarI sequence. A complete analysis of the UV, CD, and NMR chemical shift data for the IQ protons are consistent with the IQ adduct adopting a minor groove-bound conformation at the G 1 -and G 2 -positions of the NarI sequence. To further correlate the spectroscopic data with the adduct conformation, the C8-aminofluorene (AF) adduct of dGuo was also incorporated into the NarI sequence; previous NMR studies demonstrated that the AF-modified oligonucleotides were in a sequence-dependent conformational exchange between major groove-bound and base-displaced intercalated conformations. The spectroscopic data for the IQ-and AF-modified oligonucleotides are compared. The sequence-dependent conformational preferences are likely to play a key role in the repair and mutagenicity of C8-arylamine adducts.

The conformations of C8-dG adducts of 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) positioned in the C-X 1 -G, G-X 2 -C, and C-X 3 -C contexts in the C-G 1 -G 2 -C-G 3 -C-C recognition sequence of the NarI restriction enzyme were compared, using the oligodeoxynucleotides 5′- d (CTCXGCGCCATC)-3′·5′-d(GATGGCGCCGAG)-3′, 5′-d(CTCGXCGCCATC)-3′·5′-d (GATGGCGCCGAG)-3′, and 5′-d(CTCGGCXCCATC)-3′·5′-d(GATGGCGCCGAG)-3′ (X is the C8-dG adduct of IQ). These were the NarIIQ1, NarIIQ2, and NarIIQ3 duplexes, respectively. In each instance, the glycosyl torsion angle χ for the IQ-modified dG was in the syn conformation. The orientations of the IQ moieties were dependent upon the conformations of torsion angles α′ [N9-C8- N(IQ)-C2(IQ)] and β′ [C8-N(IQ)-C2(IQ)-N3(IQ)], which were monitored by the patterns of 1 H NOEs between the IQ moieties and the DNA in the three sequence contexts. The conformational states of IQ torsion angles α′ and β′ were predicted from the refined structures of the three adducts obtained from restrained molecular dynamics calculations, utilizing simulated annealing protocols. For the NarIIQ1 and NarIIQ2 duplexes, the α′ torsion angles were predicted to be −176 ± 8° and −160 ± 8°, respectively, whereas for the NarIIQ3 duplex, torsion angle α′ was predicted to be 159 ± 7°. Likewise, for the NarIIQ1 and NarIIQ2 duplexes, the β′ torsion angles were predicted to be −152 ± 8° and −164 ± 7°, respectively, whereas for the NarIIQ3 duplex, torsion angle β′ was predicted to be −23 ± 8°. Consequently, the conformations of the IQ adduct in the NarIIQ1 and NarIIQ2 duplexes were similar, with the IQ methyl protons and IQ H4 and H5 protons facing outward in the minor groove, whereas in the NarIIQ3 duplex, the IQ methyl protons and the IQ H4 and H5 protons faced into the DNA duplex, facilitating the base-displaced intercalated orientation of the IQ moiety [Wang, F., Elmquist, C. E., Stover, J. S., Rizzo, C. J., and Stone, M. P. (2006) J. Am. Chem. Soc. 128, 10085 † This work was supported by NIH Grant CA-55678 (M.P.S.). The Vanderbilt Center for Molecular Toxicology is funded by a center grant from the National Institute of Environmental Health Sciences (NIEHS) (ES-00267). J.S.S. was supported by NIEHS Predoctoral Traineeship ES-07028. Funding for the NMR spectrometers was supplied in part by NIH Grant RR-05805, the Vanderbilt Center in Molecular Toxicology, and Vanderbilt University. The Vanderbilt-Ingram Cancer Center is funded by a center grant from the National Center Institute (NCI) (CA-68485). ‡ The PDB ID code for the NarIIQ1 duplex structure is 2Z2G and the PDB ID code for the NarIIQ2 duplex structure is 2Z2H.© 2007 American Chemical Society * To whom correspondence should be addressed. C.J.R.: telephone, (615) 322−6100; fax, (615) 343−1234; e-mail, c.rizzo@vanderbilt.edu. M.P.S.: telephone, (615) 322−2589; fax, (615) 322−7591; e-mail, michael.p.stone@vanderbilt.edu.. SUPPORTING INFORMATION AVAILABLE Nonexchangeable proton chemical shifts of the modified NarIIQ1 and NarIIQ2 duplexes (Table S1), exchangeabl...

The preparation and evaluation of a series of inhibitors of Myc—Max dimerization and Myc-induced cell transformation are described providing mycmycin-1 (3) and mycmycin-2 (4).

The design, synthesis, and evaluation of a predictably more potent analogue of CC-1065 entailing the substitution replacement of a single skeleton atom in the alkylation subunit are disclosed and was conducted on the basis of design principles that emerged from a fundamental parabolic relationship between chemical reactivity and cytotoxic potency. Consistent with projections, the MeCTI (7-methyl-1,2,8,8a-tetrahydrocyclopropa[c]thieno[3,2-e]indol-4-one) alkylation subunit as well as its isomer iso-MeCTI (6-methyl-1,2,8,8a-tetrahydrocyclopropa[c]thieno[2,3-e]indol-4-one) were found to be 5-6 times more stable than the MeCPI alkylation subunit found in CC-1065 and slightly more stable than even the DSA alkylation subunit found in duocarmycin SA placing it at the point of optimally balanced stability and reactivity for this class of antitumor agents. Their incorporation into the key analogues of the natural products provided derivatives that surpassed the potency of MeCPI derivatives (3-10 fold) matching or slightly exceeding the potency of the corresponding DSA derivatives consistent with projections made based on the parabolic relationship. Notable of these, MeCTI-TMI proved to be as potent or slightly more potent than the natural product duocarmycin SA (DSA-TMI, IC 50 = 5 vs 8 pM) and MeCTI-PDE 2 proved to be 3-fold more potent than the natural product CC-1065 (MeCPI-PDE 2 , IC 50 = 7 vs 20 pM). Both exhibited efficiencies of DNA alkylation that correlate with this enhanced potency without impacting the intrinsic selectivity characteristic of this class of antitumor agents.

Heterocyclic arylamines are highly mutagenic and cause tumors in animal models. The mutagenicity is attributed to the C 8 -and N 2 -G adducts, the latter of which accumulates due to slower repair. The C 8 -and N 2 -G adducts derived from 2-amino-3-methylimidazo[4,5-f ]quinoline (IQ) were placed at the G 1 and G 3 sites of the NarI sequence, in which the G 3 site is an established hot spot for frameshift mutation with the model arylamine derivative 2-acetylaminofluorene but G 1 is not. Human DNA polymerase (pol) extended primers beyond template G-IQ adducts better than did pol and much better than pol or ␦. In 1-base incorporation studies, pol inserted C and A, pol inserted T, and pol inserted G. Steady-state kinetic parameters were measured for these dNTPs opposite the C 8 -and N 2 -IQ adducts at both sites, being most favorable for pol . Mass spectrometry of pol extension products revealed a single major product in each of four cases; with the G 1 and G 3 C 8 -IQ adducts, incorporation was largely error-free. With the G 3 N 2 -IQ adduct, a ؊2 deletion occurred at the site of the adduct. With the G 1 N 2 -IQ adduct, the product was error-free at the site opposite the base and then stalled. Thus, the pol products yielded frameshifts with the N 2 but not the C 8 IQ adducts. We show a role for pol and the complexity of different chemical adducts of IQ, DNA position, and DNA polymerases.