Laurence Hurley

PMID: 2271598;Abstract:

(+)-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. The large downfield shift (24.05 ppm) of the ¹⁵N signal of the (+)-CC-1065-12-mer duplex adduct is in accord with the formation of an extra positive charge on the exocyclic nitrogen at the N6 position and a partial double bond character between N6 and C6 atoms of the covalently modified adenine upon drug modification. We conclude that the covalently modified adenine N6 of the (+)-CC-1065-12-mer duplex adduct is predominantly in the doubly protonated form, in which calculations predict that the C6-N6 bond is shortened and the positive charge is delocalized over the entire adenine molecule. © 1990 American Chemical Society.

PMID: 19400591;PMCID: PMC2749488;Abstract:

I-motifs are four-stranded DNA secondary structures formed in C-rich DNA sequences and consist of parallel-stranded DNA duplexes zipped together in an antiparallel orientation by intercalated, hemiprotonated cytosine ⁺-cytosine base pairs. I-motif structures have been indicated to form in various regions of the human genome as well as in nanotechnological applications. While NMR is a major tool for structural studies of I-motifs, the determination of the folding topologies of unimolecular I-motifs has been a challenging and arduous task using conventional NMR spectral assignment strategies, due to the inherent sequence redundancy of the C-rich strands in the formation of unimolecular I-motif structures. We report here a direct and nondestructive method that can be utilized to unambiguously determine the hemiprotonated C ⁺-C base pairs and thus the folding topology of unimolecular I-motif structures formed from native C-rich DNA sequences. The reported approach uses affordable low-enrichment site-specific labeling. More significantly, the reported method can directly and unambiguously determine the equilibrating multiple conformations coexisting in a single DNA sequence, which would be a very difficult task using conventional assignment strategies. Additionally, this method can be applied to the direct detection of the base-paired thymines that are involved in the capping structures. © 2009 American Chemical Society.

PMID: 3002434;Abstract:

CC-1065 is a unique antitumor antibiotic produced by Streptomyces zelensis. The potent cytotoxic effects of this drug are thought to be due to its ability to form a covalent adduct with DNA through N3 of adenine. Thermal treatment of CC-1065-DNA adducts leads to DNA strand breakage. We have shown that the CC-1065 structural modification of DNA that leads to DNA strand breakage is related to the primary alkylation site on DNA. The thermally induced DNA strand breakage occurs between the deoxyribose at the adenine covalent binding site and the phosphate on the 3′ side. No residual modification of DNA is detected on the opposite strand around the CC-1065 lesion. Using the early promoter element of SV40 DNA as a target, we have examined the DNA sequence specificity of CC-1065. A consensus sequence analysis of CC-1065 binding sites on DNA reveals two distinct classes of sequences for which CC-1065 is highly specific, i.e., 5′PuNTTA and 5′AAAAA. The orientation of the DNA sequence specificity relative to the covalent binding site provides a basis for predicting the polarity of drug binding in the minor groove. Stereo drawings of the CC-1065-DNA adduct are proposed that are predictive of features of the CC-1065-DNA adduct elucidated in this investigation. © 1985 American Chemical Society.

PMID: 12449379;Abstract:

Apyrases are enzymes that efficiently hydrolyze ATP and ADP and may operate both inside and outside the cell. Although apyrases are important to a variety of cellular mechanisms and uses in industry, there are no available apyrase-specific inhibitors. Colorimetric assays based on the Fiske-Subbarow method for measuring inorganic phosphate are able to detect the release of inorganic phosphate, from ATP and other nucleotides. We found that this type of assay could be automated and used to screen, for apyrase-inhibiting compounds by assaying for a reduction in released phosphate in the presence of potential inhibitors. The automation of this assay allowed for the successful screening of a commercially available compound library. Several low molecular weight compounds were identified that, when used at micromolar concentrations, effectively inhibited apyrase activity.