Laurence Hurley

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.

PMID: 18252774;PMCID: PMC2330228;Abstract:

The c-myb promoter contains multiple GGA repeats beginning 17 bp downstream of the transcription initiation site. GGA repeats have been previously shown to form unusual DNA structures in solution. Results from chemical footprinting, circular dichroism and RNA and DNA polymerase arrest assays on oligonucleotides representing the GGA repeat region of the c-myb promoter demonstrate that the element is able to form tetrad:heptad:heptad:tetrad (T:H:H:T) G-quadruplex structures by stacking two tetrad:heptad G-quadruplexes formed by two of the three (GGA)4 repeats. Deletion of one or two (GGA)4 motifs destabilizes this secondary structure and increases c-myb promoter activity, indicating that the G-quadruplexes formed in the c-myb GGA repeat region may act as a negative regulator of the c-myb promoter. Complete deletion of the c-myb GGA repeat region abolishes c-myb promoter activity, indicating dual roles of the c-myb GGA repeat element as both a transcriptional repressor and an activator. Furthermore, we demonstrated that Myc-associated zinc finger protein (MAZ) represses c-myb promoter activity and binds to the c-myb T:H:H:T G-quadruplexes. Our findings show that the T:H:H:T G-quadruplex-forming region in the c-myb promoter is a critical cis-acting element and may repress c-myb promoter activity through MAZ interaction with G-quadruplexes in the c-myb promoter. © 2008 The Author(s).

The nuclease hypersensitivity element III1 (NHE III1) in the c-MYC promoter controls up to 80-90% of the transcriptional activity of this gene. We have demonstrated that the guanine-rich strand of the NHE III1 forms a G-quadruplex consisting of a mixture of four biologically relevant loop isomers that function as a silencer element. NMR studies have shown that these G-quadruplexes are propeller-type parallel structures consisting of three stacked G-tetrads and three double-chain reversal loops. An NMR-derived solution structure for this quadruplex provides insight into the unusual stability of the structure. This structure is a target for small molecule inhibitors of c-MYC gene expression.

Sequences with the potential to form RNA G-quadruplexes (G4s) are common in mammalian introns, especially in the proximity of the 5' splice site (5'SS). However, the difficulty of demonstrating that G4s form in pre-mRNA in functional conditions has meant that little is known about their effects or mechanisms of action. We have shown previously that two G4s form in Bcl-X pre-mRNA, one close to each of the two alternative 5'SS. If these G4s affect splicing but are in competition with other RNA structures or RNA binding proteins, then ligands that stabilize them would increase the proportion of Bcl-X pre-mRNA molecules in which either or both G4s had formed, shifting Bcl-X splicing. We show here that a restricted set of G4 ligands do affect splicing, that their activity and specificity are strongly dependent on their structures and that they act independently at the two splice sites. One of the ligands, the ellipticine GQC-05, antagonizes the major 5'SS that expresses the anti-apoptotic isoform of Bcl-X and activates the alternative 5'SS that expresses a pro-apoptotic isoform. We propose mechanisms that would account for these see-saw effects and suggest that these effects contribute to the ability of GQC-05 to induce apoptosis.