DNA Cleavage by Copper−ATCUN Complexes. Factors Influencing Cleavage Mechanism and Linearization of dsDNA

Abstract

The reactivity of two [peptide-Cu] complexes ([GGH-Cu](-) and [KGHK-Cu](+)) toward DNA cleavage has been quantitatively investigated. Neither complex promoted hydrolytic cleavage, but efficient oxidative cleavage was observed in the presence of a mild reducing agent (ascorbate) and dioxygen. Studies with scavengers of ROS confirmed hydrogen peroxide to be an obligatory diffusible intermediate. While oxidative cleavage of DNA was observed for Cu(2+)(aq) under the conditions used, the kinetics of cleavage and reaction products/pathway were distinct from those displayed by [peptide-Cu] complexes. DNA cleavage chemistry is mediated by the H(2)O-dependent pathway following C-4'H abstraction from the minor groove. Such a cleavage path also provides a ready explanation for the linearization reaction promoted by [KGHK-Cu](+). Kinetic activities and reaction pathways are compared to published results on other chemical nucleases. Both [peptide-Cu] complexes were found to display second-order kinetics, with rate constants k(2) approximately 39 and 93 M(-1) s(-1) for [GGH-Cu](-) and [KGHK-Cu](+), respectively. Neither complex displayed enzyme-like saturation behavior, consistent with the relatively low binding affinity and residence time expected for association with dsDNA, and the absence of a prereaction complex. However, the intrinsic activity of each is superior to other catalyst systems, as determined from relative k(2) or k(cat)/K(m) values. Linearization of DNA was observed for [KGHK-Cu](+) relative to [GGH-Cu](-), consistent with the increased positive charge and longer residency time on dsDNA.

Catalytic metallodrugs

“…Formation of diffusible hydroxyl radicals has been observed by use of the common rhodamine B assay, and prior studies of RNA and DNA cleavage revealed a significant reduction in diffusible hydroxyl radical concentration when substoichiometric catalyst was used to mediate damage to a target protein or nucleic acid [55]. This is consistent with formation and immediate reaction of a metal-associated ROS.…”

“…A key design problem that we had identified several years ago stems from the absence of significant binding affinity of the catalyst for the nucleic acid substrate [14,27,28]. A significant improvement in catalyst efficiency is demonstrated when the catalytic metal center was coupled to a targeting site with at least modest affinity for the reactant [14,22,23,29,30]. These studies also demonstrated the positioning of the metal-associated reactive oxygen species (ROS), relative to the scissile bond on the ribose ring, to be the most important factor regulating oxidative cleavage activity [5,22,23].…”

Tuning the coordination properties of multi-histidine peptides by using a tripodal scaffold: solution chemical study and catechol oxidase mimicking

“…[8][9][10][11][12] Previously, we also studied some histidine-rich peptides in order to create similar metal ion environment to native enzymes. 1,6,[8][9][10][11] Such metallopeptides may possess notable catalytic activity to promote either hydrolytic 8,14 or redox [9][10][11][12][13] reactions. However, from structural point of view, linear peptides may only be useful to mimic metal binding sites of native enzymes, if the residues involved in the coordination are part of a relatively short sequence.…”

Recognition mechanism of p63 by the E3 ligase Itch

“…37,38 The ATCUN motif has a well-defined structure of coordinated ligands around the metal center and is an optimal center for the design synthetic metal drugs. ATCUN metal peptides complexes have been studied for their ability to induce localized oxidative damage to nucleic acids or proteins, generating not diffusible radical species, 39,41,54 and, for this property, their potential application in biotechnological and pharmaceutical field has been proposed. [55][56][57] The biological effects that we describe here for both forms of the pep63 on the WW domain of peptide provides a titration curve.…”