Laurence Hurley

Abstract:

Anthramycin is a member of the pyrrolo[1,4]benzodiazepine group of antitumor antibiotics. Previous studies have demonstrated that anthramycin binds covalently through N-2 of guanine within the minor groove of DNA, resulting in a relatively nondistortive DNA adduct. From the nuclear Overhauser effect spectroscopy (NOESY) proton NMR spectra of the anthramycin-d(ATGCAT)2 adduct, we have obtained results that unambiguously assign the orientation of the drug molecule in the minor groove of DNA. Four sets of NOE cross-peaks between anthramycin protons and nucleotide protons on either the covalently or the noncovalently modified strands reveal that the drug is specifically oriented with the aromatic ring to the 3′-side of the covalently modified guanine. Unequivocal assignment of the geometry at the site of attachment of anthramycin to d(ATGCAT)2 cannot be made by J-correlated spectroscopy (COSY). However, when combined with the results of modeling with the molecular mechanics program AMBER, an 11S stereochemistry at this site can be confidently predicted. ³¹P NMR studies show that two of the resonance signals in the anthramycin-d(ATGCAT)2 adduct have moved significantly downfield. Both downfield ³¹P NMR signals have been assigned by ¹⁷O isotopic labeling and ¹H-³¹P two-dimensional J-correlation experiments and shown to correspond to the phosphates on the 5′-sides of the covalently modified deoxyguanine and the deoxycytosine on the opposite strand. Assignment of resonance signals of nonexchangeable base and sugar protons of the anthramycin-d(ATGCAT)2 has been made with two-dimensional Fourier transform NMR methods (COSY and NOESY). Conformational details about the sugar puckers, the glycosidic dihedral angles, and the effect of anthramycin bonding on secondary structure of the duplex have been obtained from the relative intensities of cross-peaks in the two-dimensional NMR spectra in aqueous solution. All of the sugars that are amenable to this analysis possess a conformation consistent with B-type DNA. Molecular mechanics calculations with AMBER are predictive of the orientation and stereochemistry of anthramycin bound to d(ATGCAT)2. The species having an 11S stereochemistry at the covalent bonding site and oriented with the aromatic ring of anthramycin to the 3′-side of the covalently modified guanine of anthramycin-d(ATGCAT)2 appears to be favored over the three other possible species. This is because of the greater intermolecular binding for this species rather than lower helix distortion energies. The molecular modeling is also in accord with the experimentally determined nondistortive nature of the anthramycin-d-(ATGCAT)2 adduct.

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.

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.

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.