Laurence Hurley

PMID: 1322736;Abstract:

The solution structure of the GC-rich non-self-complementary DNA 12-mer duplex (I), which contains a (+)-CC-1065 highly reactive bonding sequence 5′AGTTA* (where * denotes the (Matrix Presented) covalent modification site), has been examined thoroughly by one- and two-dimensional proton and phosphorus NMR spectroscopy, hydroxyl-radical footprinting, and NOESY restrained molecular mechanics and dynamics calculations. The assignments of the nonexchangeable proton resonances (except some of the H5′ and H5″ protons due to severe resonance overlap), phosphorus resonances, and the exchangeable resonances (except amino protons of adenosine and guanosine) of this 12-mer duplex have been made. The results show that this 12-mer duplex maintains an overall B-form DNA with all anti base orientation throughout in aqueous solution at room temperature. Hydroxyl-radical footprinting experiments on a 21-mer sequence that contains this 12-mer duplex used for NMR studies showed that the minor groove is somewhat narrowed at the 7G-8T and 17A-18C steps, as indicated by the inhibition of cleavage at these locations. Although both high-field NMR and hydroxyl-radical footprinting experiments supported a bent-like structure for this 12-mer duplex, nondenaturing gel electrophoresis on the ligated 21-mer sequence that contains this 12-mer duplex did not show the abnormally slow migration characteristic of a bent DNA duplex. Analysis of the NMR data sets reveals several local structural perturbations similar to those found on an (A)n tract DNA duplex. For example, the existence of a propeller twist was detected within the A·T-rich region for both the 12-mer and the (A)n tract DNA duplexes. The 18CH5 aromatic resonance that is directly adjacent to the 3′ side of the 5′TAA segment was significantly shifted upfield with a chemical shift of 5.10 ppm, which is almost within the region normally associated with sugar H3′ protons. The sugar geometries for 18C and 7G, which are located to the 3′ side of the 5′TAA segment, are proposed to be in the neighborhood of C3′-endo and O1′-endo ⇔ C3′-endo, respectively. We propose that this unusually upfield-shifted resonance signal for 18CH5 and the average C3′-endo sugar geometry for 18C nucleotide on the 12-mer duplex is connected with the peculiar conformation, possibly a transient kink, within the 5′AC/GT step. The results of the NOESY restrained molecular mechanics and dynamics calculations on the 12-mer sequence reveal two kinks, which are located on either side of the 18C nucleotide that has an average C3′-endo sugar geometry. The two phosphorus resonance signals that are located at the 7G-8T and the 18C-19T steps, where the minor groove is narrowed as indicated by the hydroxyl-radical footprinting experiments, displayed unusual upfield chemical shifts. Also identified were two unusually broadened base protons of the 16A nucleotide and one imino proton belonging to the 9T·16A base pair within the A·T-rich segment. We proposed that this broadening phenomenon is most likely due to a unique internal motion characterized by a rapid local conformational equilibrium between microstates of the 12-mer duplex in aqueous solution at room temperature. This local conformational flexibility, a transient kink, and the bent-like structure are proposed to play a critical role in the sequence-specific recognition of the DNA duplex by (+)-CC-1065. © 1992 American Chemical Society.

PMID: 3718937;Abstract:

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 115,11aS 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. © 1986 American Chemical Society.

Abstract:

The addition of L-methionine to cultures of Streptomyces refuineus producing anthramycin, results in an apparent decrease in the antibiotic titer. It has been shown that this effect is not due to the inhibition of tyrosinase, an enzyme implicated in anthramycin biosynthesis. The inhibitory effect also appears unlikely to be due to a regulatory role played by methionine in a non-precursor fashion. Experiments with radiolabeled anthramycin have revealed that the reduced yields of the antibiotic are due to the interaction of anthramycin with generated reactive metabolites produced in the methionine-supplemented cultures. © 1976 Springer-Verlag.

Secondary DNA structures are uniquely poised as therapeutic targets due to their molecular switch function in turning gene expression on or off and scaffold-like properties for protein and small molecule interaction. Strategies to alter gene transcription through these structures thus far involve targeting single DNA conformations. Here we investigate the feasibility of simultaneously targeting different secondary DNA structures to modulate two key oncogenes, cellular-myelocytomatosis (MYC) and B-cell lymphoma gene-2 (BCL2), in diffuse large B-cell lymphoma (DLBCL). Cotreatment with previously identified ellipticine and pregnanol derivatives that recognize the MYC G-quadruplex and BCL2 i-motif promoter DNA structures lowered mRNA levels and subsequently enhanced sensitivity to a standard chemotherapy drug, cyclophosphamide, in DLBCL cell lines. In vivo repression of MYC and BCL2 in combination with cyclophosphamide also significantly slowed tumor growth in DLBCL xenograft mice. Our findings demonstrate concurrent targeting of different DNA secondary structures offers an effective, precise, medicine-based approach to directly impede transcription and overcome aberrant pathways in aggressive malignancies.

PMID: 15079086;PMCID: PMC395936;Abstract:

We have demonstrated that a parallel G-quadruplex structure in the c-MYC promoter functions as a transcriptional repressor element. Furthermore, a specific G-to-A mutation in this element results in destabilization of the G-quadruplex repressor element and an increase in basal transcriptional activity. To validate this model in an in vivo context, we have examined the sequence of this region in human colorectal tumors and the surrounding normal tissue. We have found that ≈30% of tumors contain one of two specific G-to-A mutations, not present in the surrounding normal tissue, that destabilize the parallel G-quadruplex, which would be expected to give rise to abnormally high expression of c-MYC in these cells. In contrast, G-quadruplex-disruptive mutations were absent in 20 colon adenomas, suggesting that these mutations occur late in tumorigenesis. We have also demonstrated that these same mutations are found in established colorectal cell lines. NM23-H2 levels are lower in cancer tissues and cell lines that harbor these mutations. In cells with repressed levels of NM23-H2, the mutated and destabilized G-quadruplex silencer element can be reinstated by the addition of G-quadruplex-stabilizing compounds, providing an opportunity for therapeutic intervention for patients carrying these mutations.