Laurence Hurley

Abstract:

(-)-CC-1065, the unnatural enantiomer of the potent and sequence-selective, DNA-reactive antibiotic, (+)-CC-1065, was prepared by synthesis and its covalent reaction with DNA was studied and compared to that of the natural product. Although (-)-CC-1065 also formed covalent adducts in which the cyclopropyl carbon was bonded to the N3 atom of adenine, and the thermal strand breakage that it produced paralleled that seen for (+)-CC-1065, it lay in the opposite direction along the minor groove and exhibited a markedly different sequence requirement for the covalently modified adenine. While (-)-CC-1065 and its full carbon framework analogue, (-)-AB'C', reacted readily at adenines near to, but generally distinct from, (+)CC-1065-reactive adenines and exhibited potent cytotoxicity, their simpler analogues did not alkylate DNA under the conditions employed and were biologically nonpotent At relatively high concentrations, the smallest such analogue, (-)-A, reacted detectably only at the same sites selected by (+)-CC-1065. An analysis of the reactivity patterns of (+)- and (-)-CC-1065 and their analogues with DNA restriction fragments supported the conclusion that the mode of sequence recognition for (-)-CC-1065 adduct formation is fundamentally different from that of (+)-CC-1065 and is primarily controlled by specific minor groove, AT-selective binding interactions, rather than by sequence requirements of the covalent step, as occurs for (+)-CC-1065 and the (+)-CPI analogues. Models are proposed comparing the interactions of the enantiomeric alkylating moieties variously oriented in the minor groove at potential reaction sites. The evolutionary significance of both the alkylating moiety and the minor groove binding segments of the natural product is discussed.

PMID: 11764956;Abstract:

Ecteinascidin 743 (Et 743), a natural product derived from the Caribbean tunicate Eteinascidia turbinata, is a potent antitumor agent currently in phase II clinical trials. Et 743 binds in the minor groove of DNA, forming covalent adducts by reacting with N2 of guanine. Although DNA is considered to be the macromolecular receptor for Et 743, the precise mechanism by which Et 743 exerts its remarkable antitumor activity has not yet been elucidated. The aim of this study is to provide a rationale for the antitumor activity of Et 743 by studying its fundamental interactions with DNA at the molecular level. First, DNA structural distortions induced by Et 743 were characterized using gel electrophoresis. Surprisingly, Et 743 bends DNA toward the major groove, a unique feature among DNA-interactive agents that occupy the minor groove. Second, in order to gain further insight into the molecular basis behind the apparent sequence selectivity of Et 743, the stability and structure of Et 743 adducts at different target sequences were determined. On the basis of this data, the overall stability of the Et 743-DNA adducts was found to be governed by the DNA target sequence, where the inability of Et 743 to form optimum bonding networks with its optimum recognition sites leads to the formation of an unstable adduct. Consequently, the reaction of Et 743 with DNA is reversible, and the rate of the reverse reaction is a function of the target and flanking sequences. The results from this study demonstrate that Et 743 differs from other DNA alkylating agents by its effects on DNA structure and sequence-dependent chemical stability. This information provides important insight into the underlying mechanisms for its unique profile of antitumor activity.

A polypurine (guanine)/polypyrimidine (cytosine)-rich sequence within the proximal promoter region of the human RET oncogene has been shown to be essential for RET basal transcription. Specifically, the G-rich strand within this region consists of five consecutive runs of guanines, which is consistent with the general motif capable of forming intramolecular G-quadruplexes. Here we demonstrate that, in the presence of 100 mM K+, this G-rich strand has the ability to adopt two intramolecular G-quadruplex structures in vitro. Moreover, comparative circular dichroism (CD) and DMS footprinting studies have revealed that the 3'-G-quadruplex structure is a parallel-type intramolecular structure containing three G-tetrads. The G-quadruplex-interactive agents TMPyP4 and telomestatin further stabilize this G-quadruplex structure. In addition, we demonstrate that the complementary C-rich strand forms an i-motif structure in vitro, as shown by CD spectroscopy and chemical footprinting. This 19-mer duplex sequence is predicted to form stable intramolecular G-quadruplex and i-motif species having minimum symmetrical loop sizes of 1:3:1 and 2:3:2, respectively. Together, our results indicate that stable G-quadruplex and i-motif structures can form within the proximal promoter region of the human RET oncogene, suggesting that these secondary structures play an important role in transcriptional regulation of this gene.

Abstract:

AIM: To search for the potent telomerase inhibitors with structures of cationic porphyrins to improve the interactions between G-quadruplex and porphyrins by systematically varying the meso substituents. METHOD: Porphyrins bearing mixed 3-quinolyl/3-pyridyl meso groups were synthesized using the Adler-Longo method by condensation of aldehydes with pyrrole, followed by methylation and ion-exchange. The compounds were tested for the telomerase inhibitory activity and c-Myc inhibitory activity. RESULT: All compounds were found to be potent and approximately equivalent in terms of their ability to inhibit the action of telomerase in a cell-free assay. Compound 4 had the best inhibitory activity on c-Myc.

PMID: 8117929;Abstract:

Bizelesin, an intrahelical DNA-DNA interstrand cross-linker related to (+)-CC-1065, has been shown to alkylate DNA through guanine in restriction enzyme sequences in which there is a suitably positioned adenine contained in a highly reactive monoalkylation sequence on the opposite strand. Oligomers containing the sequence 5′-TTTTTN*, in which "N" was either G, C, or T, were synthesized to evaluate the cross-linking potential of bizelesin at nonadenine bases. Kinetic analysis of monoalkylation and cross-linking events demonstrates that it is the reaction at "N" (guanine or cytosine) that results in the cross-link which is the slow step. On the basis of this analysis and the normal unreactivity of guanine and cytosine to alkylation by the cyclopropapyrroloindole alkylating moiety of (+)-CC-1065, we propose that the molecular mechanism for this type of cross-linking reaction most likely involves a covalent immobilization of the second alkylating arm, resulting in a "proximity-driven" reaction. © 1993 American Chemical Society.