Laurence Hurley

PMID: 19922264;Abstract:

c-MYC is an important regulator of a wide array of cellular processes necessary for normal cell growth and differentiation, and its dysregulation is one of the hallmarks of many cancers. Consequently, understanding c-MYC transcriptional activation is critical for understanding developmental and cancer biology, as well as for the development of new anticancer drugs. The nuclease hypersensitive element (NHE) III1 region of the c-MYC promoter has been shown to be particularly important in regulating c-MYC expression. Specifically, the formation of a G-quadruplex structure appears to promote repression of c-MYC transcription. This review focuses on what is known about the formation of a G-quadruplex in the NHE III1 region of the c-MYC promoter, as well as on those factors that are known to modulate its formation. Last, we discuss the development of small molecules that stabilize or induce the formation of G-quadruplex structures and could potentially be used as anticancer agents. Copyright © 2010 by Annual Reviews. All rights reserved.

PMID: 16998187;PMCID: PMC1636422;Abstract:

BCL2 protein functions as an inhibitor of cell apoptosis and has been found to be aberrantly expressed in a wide range of human diseases. A highly GC-rich region upstream of the P1 promoter plays an important role in the transcriptional regulation of BCL2. Here we report the NMR solution structure of the major intramolecular G-quadruplex formed on the G-rich strand of this region in K⁺ solution. This well-defined mixed parallel/antiparallel-stranded G-quadruplex structure contains three G-tetrads of mixed G-arrangements, which are connected with two lateral loops and one side loop, and four grooves of different widths. The three loops interact with the core G-tetrads in a specific way that defines and stabilizes the overall G-quadruplex structure. The loop conformations are in accord with the experimental mutation and footprinting data. The first 3-nt loop adopts a lateral loop conformation and appears to determine the overall folding of the BCL2 G-quadruplex. The third 1-nt double-chain-reversal loop defines another example of a stable parallel-stranded structural motif using the G3 NG3 sequence. Significantly, the distinct major BCL2 promoter G-quadruplex structure suggests that it can be specifically involved in gene modulation and can be an attractive target for pathway-specific drug design. © 2006 Oxford University Press.

PMID: 11439034;Abstract:

Ecteinascidin 743 (Et 743), one of a series of structurally related antitumor antibiotics isolated from a marine tunicate, is currently in phase II clinical trials. Et 743 alkylates guanine N2 through the minor groove of DNA. Hydrogen-bonding networks that associate the drug with a three base pair DNA recognition site have been proposed to contribute to both the reactivity and the stability of the Et 743-DNA adduct. Here, we report that the reaction of Et 743 with DNA is reversible under nondenaturing conditions and that the rate of this reverse reaction depends critically upon the DNA-modified sequence. Quite unexpectedly, it was found that although the rates of alkylation are similar for the 5′-AGT and 5′-AGC sequences, reversal from the 5′-AGT sequence occurs faster than from the 5′-AGC sequence. Consequently, it is the differences in the rate of the reverse reaction that dictate the sequence selectivity of Et 743 toward its favored target sequence. As a direct consequence of the reversible nature of Et 743 with DNA, Et 743 can migrate from the nonfavored bonding sequence (e.g., 5′-AGT) to the favored DNA target site (e.g., 5′-AGC). The data suggest that the observed differences in the rate of reversibility arise from differences in the stability of the Et 743-DNA adduct at the 5′-AGT and 5′-AGC target sequences. On the basis of gel electrophoresis and ^IH NMR experiments, the Et 743-AGT adduct is less stable, has more dynamic motion, and produces different conformational changes in the DNA than the more stable Et 743-AGC adduct. The shuffling of Et 743-DNA adducts to the more stable alkylation sites has important implications for understanding the underlying relationship between the structural modification of DNA by Et 743 and its biological potency and efficacy in tumor cells.

PMID: 12825936;Abstract:

Topoisomerase II, an enzyme that catalyzes changes in the topology of DNA, plays several key roles in DNA metabolism and chromosome structure, and it is the primary cytotoxic target for a number of clinically important DNA intercalating agents such as doxorubicin. It seems likely that if these intercalating topoisomerase II poisons are structurally modified to also be DNA alkylating agents, they will have increased dwell time on the topoisomerase II-DNA complex and increased potency and selectivity for cancer cells, On the basis of insights into the mechanisms of action of psorospermin and the quinobenzoxazine A-62176 and molecular modeling studies of these compounds with duplex DNA, we have designed and synthesized a series of novel hybrid DNA-interactive compounds that alkylate DNA most efficiently at sequences directed by topoisomerase II. The epoxydihydrofuran ring of psorospermin was used as a DNA alkylating moiety, and this was fused to the pyridobenzophenoxazine ring of A-62176. The chlorohydrin ring opened form of the epoxide was also prepared and tested. These hybrid compounds showed enhanced DNA alkylating activity in the presence of topoisomerase II, exhibited significant activity against all the cancer cells tested at submicromolar concentrations, and were more potent than both parent compounds. However, the biochemical assays indicated that they lost some of the topoisomerase II and Mg²⁺ dependency for reaction with DNA that is associated with psorospermin and A-62176, respectively.

Abstract:

It is generally accepted that DNA predominantly exists in duplex form in cells. However, under torsional stress imposed by active transcription, DNA can assume nonduplex structures. The BCL2 promoter region forms two different secondary DNA structures on opposite strands called the G-quadruplex and the i-motif. The i-motif is a highly dynamic structure that exists in equilibrium with a flexible hairpin species. Here we identify a pregnanol derivative and a class of piperidine derivatives that differentially modulate gene expression by stabilizing either the i-motif or the flexible hairpin species. Stabilization of the i-motif structure results in significant upregulation of the BCL2 gene and associated protein expression; in contrast, stabilization of the flexible hairpin species lowers BCL2 levels. The BCL2 levels reduced by the hairpin-binding compound led to chemosensitization to etoposide in both in vitro and in vivo models. Furthermore, we show antagonism between the two classes of compounds in solution and in cells. For the first time, our results demonstrate the principle of small molecule targeting of i-motif structures in vitro and in vivo to modulate gene expression. © 2014 American Chemical Society.