Laurence Hurley

PMID: 455297;Abstract:

Anthramycin-DNA adducts, produced in vitro by reaction of anthramycin with calf thymus DNA, have been shown to be stable only as long as the secondary structure of DNA is maintained. Denaturation either by heat or enzymatic degradation of the DNA adduct, with DNase I and snake venom phosphodiesterase, leads to the release of significant amounts of the bound drug as unchanged anthramycin. These observations led us to suspect that the DNA adduct might be a suitable prodrug system for anthramycin, which might be more efficacious and less toxic than the administration of the free drug. In order to test this hypothesis, the ability of the adduct versus free drug to inhibit DNA synthesis and induce unshceduled DNA synthesis in a human cell line was evaluated. The results demonstrated that the anthramycin-DNA adduct was less potent than the free drug in these systems in both respects. The anthramycin and anthramycin-DNA conjugate were compared in mice for lethality, tisue levels, alteration of hexobarbital sleeping times, and efficacy against a mouse ascites tumor model. These results showed that the DNA adduct was three times more lethal and produced similar increases in sleeping times at equitoxic doses. The increase in lethality of the anthramycin-DNA adduct could be explained by elevated and more prolonged blood and tissue levels following administration of the DNA conjugate as compared to free anthramycin. When tested for efficacy against a mouse ascites tumor line, the anthramycin-DNA adduct was found to be less efficacious than the free drug.

PMID: 18355457;PMCID: PMC2585383;Abstract:

In its simplest form, a DNA G-quadruplex is a four-stranded DNA structure that is composed of stacked guanine tetrads. G-quadruplex-forming sequences have been identified in eukaryotic telomeres, as well as in non-telomeric genomic regions, such as gene promoters, recombination sites, and DNA tandem repeats. Of particular interest are the G-quadruplex structures that form in gene promoter regions, which have emerged as potential targets for anticancer drug development. Evidence for the formation of G-quadruplex structures in living cells continues to grow. In this review, we examine recent studies on intramolecular G-quadruplex structures that form in the promoter regions of some human genes in living cells and discuss the biological implications of these structures. The identification of G-quadruplex structures in promoter regions provides us with new insights into the fundamental aspects of G-quadruplex topology and DNA sequence-structure relationships. Progress in G-quadruplex structural studies and the validation of the biological role of these structures in cells will further encourage the development of small molecules that target these structures to specifically modulate gene transcription. © 2008 Elsevier Masson SAS. All rights reserved.

Abstract:

As expected, Bizelesin, which is a biscyclopropa[c]pyrrolo[3,2-e]indol-4(5H)-one [(+)-CPI]-derived DNA-DNA cross-linker, has a high interstrand cross-linking reactivity with the palindromic sequence 5'-d(CGTAATTȦCG)2. Contrary to expectations, the target duplex is rearranged to yield two products: one (major product) contains an AT step wherein both adenines are syn-oriented and hydrogen bonded to thymines forming a stable Hoogsteen base-paired region flanked by Watson-Crick base-paired regions (5HG); the other (minor product) contains anti-oriented AT-step adenines that show no evidence of hydrogen bonding with pairing thymines (5OP) in an otherwise normally base-paired duplex. In another unexpected outcome, the reaction of two 'uncoupled' monoalkylating (+)-CPI 'halves' of Bizelesin with the same duplex alkylates same-strand adenines three base pairs apart [5'-d(CGTAȦTTȦCG)2] rather than the anticipated opposite-strand adenines six base pairs apart (which would mimic Bizelesin). To probe the molecular mechanism that leads to Bizelesin's unusual DNA rearrangement, which appears to be a requirement for DNA-DNA interstrand cross-linking, we have carried out conformational exchange analyses (NOESY and ROESY) and restrained molecular dynamics simulations of these adducts. These studies suggest that Bizelesin controls the rearrangement of the six-base-pair target prior to cross-linkage and restricts the cross-linked DNA adduct's range of motion, freezing-out adduct conformers defined by alternative drug-DNA hydrogen-bond regimes. The two competing cross-linkage pathways share a common first step, the opening of the central AT-step base pairs, an event that is facilitated by the energetics of monoadduct-induced DNA bending distortion. One pathway (to 5HG) stabilizes these open bases by reorganizing the AT-step region into two Hoogsteen base pairs, the thymine bases of which also hydrogen bond with Bizelesin's ureadiyl subunit. A second pathway (to 5OP) directly stabilizes the open bases by forming a hydrogen-bonding complex between the AT-step thymines and Bizelesin's ureadiyl subunit. Cross-linked DNA motion drives both of the 5HG and 5OP adducts from one ephemeral hydrogen-bonding regime to another, a process documented in the NOESY conformational exchange data and simulated in restrained molecular dynamics trajectories. These results, together with the analysis of other six-base-pair Bizelesin cross-linked species, suggest a novel mechanism for sequence recognition by this cross-linker where monoalkylation distortive stress associated with a bent DNA conformation must be dissipated by a cooperative interaction between drug and duplex to produce a straight B-form-like structure before cross-linking can proceed. This example provides a new mechanism for DNA sequence recognition involving a 'drug-induced rearrangement' of DNA that critically depends upon the interplay of drug and sequence recognition elements.