Intracellular DNA Damage by Lysine-Acetylene Conjugates

SAGE-Hindawi Access to Research Journal of Nucleic Acids Volume 2010, Article ID 931394, 6 pages doi:10.4061/2010/931394 Research Article Intracellular DNA Damage by Lysine-Acetylene Conjugates Wang-Yong Yang, Qiang Cao, Catherine Callahan, Catalina Galvis, Qing-Xiang Sang, and Igor V. Alabugin Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306-4390, USA Correspondence should be addressed to Igor V. Alabugin, Received 25 April 2010; Revised 26 June 2010; Accepted 6 July 2010 Academic Editor: Ashis Basu Copyright © 2010 Wang-Yong Yang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Previously, we reported the design and properties of alkyne C-lysine conjugates, a powerful and tunable family of DNA cleaving reagents. We also reported that, upon photoactivation, these molecules are capable of inducing cancer cells death. To prove that the cell death stems from DNA cleavage by the conjugates, we investigated intracellular DNA damage induced by these molecules in LNCap cancer cells using single cell gel electrophoresis (SCGE) assays. The observation of highly efficient DNA damage confirmed that lysine acetylene conjugate is capable of cleaving the densely compacted intracellular DNA. This result provides a key mechanistic link between efficient DNA cleavage and cytotoxicity towards cancer cells for this family of light-activated anticancer agents. 1. Introduction Because double stranded (ds) DNA cleavage is much harder to repair than single stranded (ss) DNA cleavage, ds damage is particularly efficient in inducing self-programmed cell death or apoptosis [1]. A particularly striking example of this efficiency is provided by natural enediyne antibiotics [2]. These compounds, often hailed as the most potent family of anticancer agents [3], produce cleavage of both strands of DNA duplex via two hydrogen abstractions from two opposite strands of DNA backbone by a reactive biradical, p-benzyne, generated from the enediyne core via a process, called the Bergman cyclization [4–6]. However, natural enediynes not only lack selectivity towards cancer cells, but also do not cause the ds cleavage with 100% efficiency. Even the best of them, calicheamicin leads to only 25% cleavage [7]. Thus, design of compounds which are capable of more efficient ds DNA cleavage and combine this efficiency with selectivity towards cancer cells remains the focal point of the anticancer therapeutic agents targeting DNA. We have found that DNA damaging potential of enediynes can be increased if their reactivity is tuned towards C1–C5 photocyclizations, a new reaction discovered in our lab which leads to incorporation of four rather than two hydrogen atoms from the environment [8, 9]. Because C1–C5 cyclization proceeds under photochemical conditions for thermal C1–C5 cyclization, see [10, 11], it takes advantage of the high degree of spatial and temporal controls over reactivity inherent to the photochemical activation. The use of tissue-penetrating light allows for efficient, and selective, spatial and temporal control over prodrug activation as light can be delivered directly to the tumor when it contains a high concentration of the prodrug. Skin cancer is the most obvious target for this therapy and, in 2006, the UK National Institute of Health and Clinical Excellence (NICE) recommended PDT for basal cell carcinoma. However, PDT can be also used to treat tumors on the lining of internal organs or cavities. Other tumors can be targeted with low-energy tissue penetrating photons, especially if the three-dimensional control of activation is provided by the two-photon excitation mode. For two photon excitation of enediynes, see [12–14]. In addition, this radical-anionic C1–C5 cyclization of enediynes is triggered by photoinduced electron transfer (PET). This mechanistic feature increases cellular selectivity because activation is possible only in the direct vicinity of a suitable electron 2 Journal of Nucleic Acids F F F N F N D + F F F R H R R H H R R = tetrafluoropyridyl (TFP) N F F H H F R R H H R R H H − H H R R R F N F F H R H+ F N − F F F F F F D hν F N F F − ∗ F H H R H R R D + D H R H H H Scheme 1: C1–C5 photocyclization of bis-TFP-enediyne and proposed mechanism in the proximity to DNA (four abstracted hydrogens are shown in red, TFP = tetrafluoropyridine). donor such as DNA to occur. In the absence of such a donor, TFP-substituted enediynes (Scheme 1) are unreactive, both thermally and photochemically. We have also found that related TFP-substituted monoacetylenes are capable of photochemical alkylation of electron rich π-systems [15–17] and investigated whether this reaction can be also used for controlled DNA-modification. A priori, efficient DNA-cleavage by monoalkynes incapable of the Bergman or C1–C5 cyclizations can involve several possible mechanisms like base alkylation, hydrogen abstraction, generation of reactive oxygen species as well as PET. In order to increase solubility of TFP-warheads in water and their affinity to DNA, we combined them with lysine via carboxyl moiety of the amino acid, Figure 1 [18]. Importantly, this mode of attachment leaves both amino groups of lysine available for an acid-base reaction which converts them into cationic ammonium groups. We found that DNA-damaging ability of such hybrid molecules can be fine-tuned in the narrow range of physiological pH conditions which results in a dramatic increase in reactivity at the lower pH of hypoxic tumor cells [19]. Less basic α-amino group is protonated at the lower pH than 7 and this protonation not only prevents quenching the excited state of the chromophore but also provides tighter binding to negatively charged DNA. Remarkably, the change in reactivity occurs at a relatively narrow and predefined pH point (∼pH 6). These DNA-photocleavers provide the DNA cleavage ratios of up to the 1 : 2 ds : ss at pH 5.5 at concentrations and irradiation times where almost no ds cleavage is observed at the pH of healthy cells. This dramatic increase of ds DNA cleavage at the lower pH renders these molecules more efficient ds DNA cleavers than calicheamicine under the conditions suitable for selective targeting of acidic cancer tissues (Figure 2(a)). We also found that the C-lysine conjugates bind selectively to nicks and gaps in a DNA duplex and, upon photochemical activation, transform the easily repairable ss-DNA damage into much more therapeutically important ds-DNA damage [20] (Figure 2(b)). The medicinal potential of these molecules has been illustrated by a >90% LNCap cancer cell death induced by photochemically activated TFP-acetylene-lysine conjugate 3 in one treatment at concentrations as low as 10 nM. Notably, at these concentrations, toxicity without light is negligible (...truncated)