Laurence Hurley

PMID: 8672503;Abstract:

The T-antigen-induced structural changes of the SV40 replication origin were probed with three DNA-reactive antitumor agents: (+)-CC-1065, bizelesin, and pluramycin, (+)-CC-1065 is an N3 adenine minor groove alkylating agent that selectively reacts with AT-rich DNA sequences with a bent conformation; bizelesin also reacts with the minor groove of AT-rich sequences but is selective for a straight DNA conformation. Pluramycin is an intercalative guanine alkylator whose reactivity is increased by unwinding and decreased by compression of the minor and/or major grooves of DNA. We show that while binding of T-antigen reduced the ability of (+)-CC-1065 to alkylate the AT tract in the SV40 replication origin, it did not interfere with bizelesin modification of the same sequence. These unexpected results suggest that when T-antigen binds to the SV40 origin the AT tract is in a straight DNA conformation. High-resolution DNase I footprinting experiments indicate that at least three helically in-phase T-antigen binding sites exist in the GC box region located immediately downstream of the AT tract. The binding of T- antigen enhances the reactivity of (+)-CC-1065 to the two 5'-AGTTA* (the asterisk indicates the covalent bonding site) drug modification sites in the GC box region, demonstrating that these sites are in a bent conformation. In contrast, T-antigen inhibited the reactivity of pluramycin at sequences within the GC box region that are known not to bind T-antigen. These data, in combination with the DNase I footprinting results, suggest that T-antigen binding induces a conformational change in the DNA that no longer favors pluramycin intercalation. Based on our results, we propose that T-antigen binds tightly to the upstream region of the AT tract of SV40 replication origin forming double hexamers. In the downstream region, binding of T- antigen to the helically in-phase sites in the GC box region induces DNA bending in the opposite direction of the natural AT tract bending, while simultaneously transforming the naturally bent AT tract DNA into a straight conformation.

PMID: 10956013;Abstract:

The quinobenzoxazines, a group of structural analogues of the antibacterial fluoroquinolones, are topoisomerase II inhibitors that have demonstrated promising anticancer activity in mice. It has been proposed that the quinobenzoxazines form a 2:2 drug-Mg²⁺ self-assembly complex on DNA. The quinobenzoxazine (S)-A-62176 is photochemically unstable and undergoes a DNA-accelerated photochemical reaction to afford a highly fluorescent photoproduct. Here we report that the irradiation of both supercoiled DNA and DNA oligonucleotides in the presence of (S)-A-62176 results in photochemical cleavage of the DNA. The (S)-A-62176-mediated DNA photocleavage reaction requires Mg²⁺. Photochemical cleavage of supercoiled DNA by (S)-A-62176 is much more efficient that the DNA photocleavage reactions of the fluoroquinolones norfloxacin, ciprofloxacin, and enoxacin. The photocleavage of supercoiled DNA by (S)-A-62176 is unaffected by the presence of SOD, catalase, or other reactive oxygen scavengers, but is inhibited by deoxygenation. The photochemical cleavage of supercoiled DNA is also inhibited by 1 mM KI. Photochemical cleavage of DNA oligonucleotides by (S)-A-62176 occurs most extensively at DNA sites bound by drug, as determined by DNase I footprinting, and especially at certain G and T residues. The nature of the DNA photoproducts, and inhibition studies, indicate that the photocleavage reaction occurs by a free radical mechanism initiated by abstraction of the 4'- and 1'-hydrogens from the DNA minor groove. These results lend further support for the proposed DNA binding model for the quinobenzoxazine 2:2 drug-Mg²⁺ complex and serve to define the position of this complex on the minor groove of DNA.

PMID: 7151226;Abstract:

The reaction of anthramycin with DNA has been examined to determine the chemical identity of the adduct which forms in a living cell and to observe the effects of the nucleosome structure of chromatin on drug binding. The chemical identity of the cellular adduct was probed by comparing various properties of the cellular adduct to properties of the known, in vitro adduct. The effect of the histones on anthramycin binding was investigated by time-course binding reactions. Results indicate that the properties of the cellular anthramycin-DNA adduct are similar to the in vitro adduct. The histone proteins associated with DNA in chromatin were found to decrease both the reaction kinetics and the final levels of anthramycin binding. Anthramycin reacts appreciably with nucleosome core DNA, but appears to exhibit a preference for linker DNA. © 1982.

B-Raf protein kinase, which is a key signaling molecule in the RAS-RAF-MEK-ERK signaling pathway, plays an important role in many cancers. The B-Raf V600E mutation represents the most frequent oncogenic kinase mutation known and is responsible for increased kinase activity in approximately 7% of all human cancers, establishing B-Raf as an important therapeutic target for inhibition. Through the use of an iterative program that utilized a chemocentric approach and a rational structure based design, we have developed novel, potent, and specific DFG-out allosteric inhibitors of B-Raf kinase. Here, we present efficient and versatile chemistry that utilizes a key one pot, [3+2] cycloaddition reaction to obtain highly substituted imidazoles and their application in the design of allosteric B-Raf inhibitors. Inhibitors based on this scaffold display subnanomolar potency and a favorable kinase profile.

PMID: 19601575;PMCID: PMC2761083;Abstract:

The hTERT core promoter contains a G-rich region of 12 consecutive G-tracts, embracing 3 Sp1 binding sites, and has the potential to form multiple G-quadruplexes. From the 12 runs of guanines, 9 putative hTERT G-quadruplex-forming sequences were selected to assay for G-quadruplex formation and stability using circular dichroism and a Taq polymerase stop assay. Results from biophysical and chemical assays demonstrate an approximate inverse correlation between total loop size and structure stability. Investigation of the full-length hTERT G-rich sequence using a Taq polymerase stop assay and dimethyl sulfate footprinting revealed the formation of a unique end-to-end stacked G-quadruplex structure from this sequence. This structure consists of an all parallel G-quadruplex, formed by four consecutive G-tracts, linked to another, atypical G-quadruplex, formed by two pairs of consecutive G-tracts separated by a 26-base loop. This 26-base loop likely forms a stable hairpin structure, which would explain the unexpected stability of this G-quadruplex. Significantly, the formation of this tandem G-quadruplex structure in the full-length sequence masks all three Sp1 binding sites, which is predicted to produce significant inhibition of hTERT promoter activity. Furthermore, our study implies that inhibition of telomerase activity by some G-quadruplex ligands is not only produced by targeting telomeric G-quadruplexes but also by stabilization of the hTERT promoter G-quadruplexes. © 2009 American Chemical Society.