Laurence Hurley

PMID: 10739869;Abstract:

G-quadruplexes are a family of secondary DNA structures formed in the presence of monovalent cations that consist of four-stranded structures in which Hoogsteen base-pairing stabilizes G-tetrad structures. These structures are proposed to exist in vivo, although direct confirmatory evidence is lacking. Guanine-rich regions of DNA capable of forming G-quadruplex structures are found in a variety of chromosomal regions, including telomeres and promoter regions of DNA. In this review, we describe the design of three separate groups of G-quadruplex-interactive compounds and their interaction with G-quadruplex DNA. Using the first group of compounds (anthraquinones), we describe experiments that provide the proof of concept that a G-quadruplex is required for inhibition of telomerase. Using the second group of compounds (perylenes), we describe the structure of a G-quadruplex-ligand complex and its effect on the dynamics of formation and enzymatic unwinding of the quadruplex. For the third group of compounds (porphyrins), we describe the experiments that relate the biological effects to their interactions with G-quadruplexes. Copyright (C) 2000 Elsevier Science Inc.

Activation of human telomerase reverse transcriptase (hTERT) is necessary for limitless replication in tumorigenesis. Whereas hTERT is transcriptionally silenced in normal cells, most tumor cells reactivate hTERT expression by alleviating transcriptional repression through diverse genetic and epigenetic mechanisms. Transcription-activating hTERT promoter mutations have been found to occur at high frequencies in multiple cancer types. These mutations have been shown to form new transcription factor binding sites that drive hTERT expression, but this model cannot fully account for differences in wild-type (WT) and mutant promoter activation and has not yet enabled a selective therapeutic strategy. Here, we demonstrate a novel mechanism by which promoter mutations activate hTERT transcription, which also sheds light on a unique therapeutic opportunity. Promoter mutations occur in a core promoter region that forms tertiary structures consisting of a pair of G-quadruplexes involved in transcriptional silencing. We show that promoter mutations exert a detrimental effect on the folding of one of these G-quadruplexes, resulting in a nonfunctional silencer element that alleviates transcriptional repression. We have also identified a small drug-like pharmacological chaperone (pharmacoperone) molecule, GTC365, that acts at an early step in the G-quadruplex folding pathway to redirect mutant promoter G-quadruplex misfolding, partially reinstate the correct folding pathway, and reduce hTERT activity through transcriptional repression. This transcription-mediated repression produces cancer cell death through multiple routes including both induction of apoptosis through inhibition of hTERT's role in regulating apoptosis-related proteins and induction of senescence by decreasing telomerase activity and telomere length. We demonstrate the selective therapeutic potential of this strategy in melanoma cells that overexpress hTERT.

PMID: 3030397;Abstract:

The design, construction, and characterization of a site-directed CC-1065-N3-adenine adduct in a 117 base pair segment of M13mpI DNA are described. CC-1065 is an extremely potent antitumor antibiotic produced by Streptomyces zelensis. Previous studies have demonstrated that the cyclopropyl ring of CC-1065 reacts quite specifically with N3 of adenine in double-stranded DNA to form a CC-1065-DNA adduct. Following alkylation, the drug molecule lies snugly within the minor groove of DNA, overlapping with five base pairs for which a marked sequence preference exists [Hurley, L. H., Reynolds, V. R., Swenson, D. H., Petzold, G. L., & Scahill, T. A. (1984) Science (Washington, D.C.) 226, 843-844]. On the basis of the unique characteristics of the reaction of CC-1065 with DNA and the structure of the resulting DNA adduct, we have designed a general strategy to construct a site-directed CC-1065-DNA adduct in a restriction fragment. The presence of unique AluI and HaeIII restriction enzymes sites on each side of a high-affinity CC-1065 binding sequence (5′-GATTA) permitted the preparation of a partial duplex DNA molecule containing the CC-1065 binding sequence in the duplex DNA region. Since CC-1065 only binds to duplex DNA, potential CC-1065 binding sequences in the long single-stranded regions were protected from drug binding during the construction process. After purification of the CC-1065 partial duplex DNA adduct by differential melting of the modified and unmodified partial duplex DNA, DNA polymerase I was used to generate the full duplex DNA molecule, which contained a single site-directed CC-1065-N3-adenine adduct at adenine 6229 of the 117 base pair MspI-BstNI DNA restriction fragment of the Escherichia coli lac insert of M13mpI DNA. A CC-1065 thermal strand scission assay was used to confirm the unique binding site on the covalently modified strand. Methidiumpropyl-EDTA-iron(II) [MPE-Fe(II)] digestions were used to locate the binding site and the orientation of CC-1065 in the minor groove of DNA. MPE-Fe(11) footprinting revealed a slight enhancement of digestion on both DNA strands, but just to one side of the CC-1065-DNA adduct. © 1986 American Chemical Society.

PMID: 6494915;Abstract:

Sequence-dependent variations in DNA revealed by x-ray crystallographic studies have suggested that certain DNA-reactive drugs may react preferentially with defined sequences in DNA. Drugs that wind around the helix and reside within one of the grooves of DNA have perhaps the greatest chance of recognizing sequence-dependent features of DNA. The antitumor antibiotic CC-1065 covalently binds through N-3 of adenine and resides within the minor groove of DNA. This drug overlaps with five base pairs for which a high sequence specificity exists.

PMID: 6406412;Abstract:

The biosynthesis is the antitumor antibiotic, CC-1065, has been investigated by radioactive isotope techniques, in combination with chemical degradation of CC-1065. Tyrosine, dopa, serine and methionine (S-CH3 group) have been shown to be precursors of CC-1065. Tyrosine is proposed to be a precursor of all three benzodipyrrole subunits, while dopa is only apparently incorporated into subunits B and C. Serine is postulated to contribute three 2C units, with loss of C-1, to all three subunits of CC-1065. The S-CH3 group of methionine probably contributes four C-1 units to CC-1065 of which one is incorporated with considerable loss of tritium, most probably into the cyclopropane ring of subunit A.