Hurley, L., Kaiser, C. E., Gokhale, V., Yang, D., & Hurley, L. -. (2013). Gaining Insights into the Small Molecule Targeting of the G-Quadruplex in the c-MYC Promoter Using NMR and an Allele-Specific Transcriptional Assay. Topics in current chemistry, 330.
G-quadruplexes (four-stranded DNA secondary structures) are showing promise as new targets for anticancer therapies. Specifically, G-quadruplexes in the proximal promoter region of regulatory genes have the potential to act as silencer elements and thereby turn off transcription. Thus, compounds that are capable of binding to and stabilizing G-quadruplexes would be of great benefit. In this chapter we describe two recent studies from our labs. In the first case, we use NMR to elucidate the structure of a 2:1 complex between a small molecule and the G-quadruplex in the c-MYC promoter. In the second case, we use an allele-specific transcription assay to demonstrate that the effect of a G-quadruplex-interactive compound is mediated directly through the G-quadruplex. Finally, we use this information to propose models for the interaction of various small molecules with the c-MYC G-quadruplex.
Fedoroff, O. Y., Salazar, M., Han, H., Chemeris, V. V., Kerwin, S. M., & Hurley, L. H. (1998). NMR-based model of a telomerase-inhibiting compound bound to G- quadruplex DNA. Biochemistry, 37(36), 12367-12374.
PMID: 9730808;Abstract:
The single-stranded (TTAGGG)(n) tail of human telomeric DNA is known to form stable G-quadruplex structures. Optimal telomerase activity requires the nonfolded single-stranded form of the primer, and stabilization of the G- quadruplex form is known to interfere with telomerase binding. We have identified 3,4,9,10-perylenetetracarboxylic diimide-based ligands as potent inhibitors of human telomerase by using a primer extension assay that does not use PCR-based amplification of the telomerase primer extension products. A set of NMR titrations of the ligand into solutions of G-quadruplexes using various oligonucleotides related to human telomeric DNA showed strong and specific binding of the ligand to the G-quadruplex. The exchange rate between bound and free DNA forms is slow on the NMR time scale and allows the unequivocal determination of the binding site and mode of binding. In the case of the 5'-TTAGGG sequence, the ligand-DNA complex consists of two quadruplexes oriented in a tail-to-tail manner with the ligand sandwiched between terminal G4 planes. Longer telomeric sequences, such as TTAGGGTT, TTAGGGTTA, and TAGGGTTA, form 1:1 ligand-quadruplex complexes with the ligand bound at the GT step by a threading intercalation mode. On the basis of 2D NOESY data, a model of the latter complex has been derived that is consistent with the available experimental data. The determination of the solution structure of this telomerase inhibitor bound to telomeric quadruplex DNA should help in the design of new anticancer agents with a unique and novel mechanism of action.
Lee, S., & Hurley, L. H. (1999). A thymine: Thymine mismatch enhances the pluramycin alkylation site downstream of the TBP - TATA box complex. Journal of the American Chemical Society, 121(39), X.
Abstract:
The DNA groove interactions of the pluramycins determine the base-pair specificity to the 5′-side of the covalently modified guanine. The DNA reactivity of these drugs at defined sites can be further increased by structural and dynamic DNA distortion induced by TATA binding protein (TBP) binding to the TATA box. This enhanced drug reactivity has led to the proposal that protein-induced DNA conformational dynamics might be responsible for the more selective biological consequences of the pluramycins. To identify the structural and/or dynamic determinants that account for the enhanced drug reactivity, DNA heteroduplexes that contain base mismatches were examined for enhanced alkylation by altromycin B. The results demonstrate that base mismatches located at the 5′-side of the target guanine enhance drug reactivity. An analysis of the structural and dynamic properties of the base mismatches demonstrates that the pluramycin reactivities are not only determined by dynamic conformation of the base mismatch, which improves the accessibility of the drug to the DNA helix, but also by the specific groove interactions between the DNA and the drug, which results in stabilization of the precovalent drug - DNA complex. Having established that the highest pluramycin reactivity with heteroduplex DNA is produced by a thymine:thymine (T:T) mismatch, we next addressed how the same mismatch affects pluramycin reactivity in the flanking region to the TBP - TATA box complex. When this mismatch is introduced into the downstream flanking sequence of the TATA box, TBP binding cooperatively enhances the drug alkylation on a downstream guanine adjacent to the inserted mismatch. While an obvious structural similarity between the T:T mismatch and the TBP-induced effects at a downstream site of the TATA box does not exist, we propose that the base pair destabilizing effects of the T:T mismatch may resemble the dynamically accessible intercalation site on the downstream side of the TATA box induced by binding of TBP to the TATA box.
Lubawy, W. C., Whaley, J., & Hurley, L. H. (1979). Coenzyme Q10 or α-tocopherol reduce the acute toxicity of anthramycin in mice. Research Communications in Chemical Pathology and Pharmacology, 24(2), 401-404.
Herzig, M. C., Rodriguez, K. A., Trevino, A. V., Dziegielewski, J., Arnett, B., Hurley, L., & Woynarowski, J. M. (2002). The genome factor in region-specific DNA damage: The DNA-reactive drug U-78779 prefers mixed A/T-G/C sequences at the nucleotide level but is region-specific for long pure AT islands at the genomic level. Biochemistry, 41(5), 1545-1555.
PMID: 11814348;Abstract:
Bizelesin is the first anticancer drug capable of damaging specific regions of the genome with clusters of its binding sites T(A/T)4A. This study characterized the sequence- and region-specificity of a bizelesin analogue, U-78779, designed to interact with mixed A/T-G/C motifs. At the nucleotide level, U-78779 was found to prefer runs of A/Ts interspersed with 1 or 2 G/C pairs, although 25% of the identified sites corresponded to pure AT motifs similar to bizelesin sites. The in silico computational analysis showed that the preferred mixed A/T-G/C motifs distribute uniformly at the genomic level. In contrast, the secondary, pure AT motifs (A/T)6A were found densely clustered in the same long islands of AT-rich DNA that bizelesin targets. Mapping the sites and quantitating the frequencies of U-78779 adducts in model AT island and non-AT island naked DNAs demonstrated that clusters of pure AT motifs outcompete isolated mixed A/T-G/C sites in attracting drug binding. Regional preference of U-78779 for AT island domains was verified also in DNA from drug-treated cells. Thus, while the primary sequence preference gives rise to non-region-specific scattered lesions, the clustering of the minor pure AT binding motifs seems to determine region-specificity of U-78779 in the human genome. The closely correlated cytotoxic activities of U-78779 and bizelesin in several cell lines further imply that both drugs may share common cellular targets. This study underscores the significance of the genome factor in a drug's potential for region-specific DNA damage, by showing that it can take precedence over drug binding preferences at the nucleotide level.
