C-Lysine Conjugates: pH-Controlled Light-Activated Reagents for Efficient Double-Stranded DNA Cleavage with Implications for Cancer Therapy

Abstract

Double-stranded DNA cleavage of light-activated lysine conjugates is strongly enhanced at the slightly acidic pH (<7) suitable for selective targeting of cancer cells. This enhancement stems from the presence of two amino groups of different basicities. The first amino group plays an auxiliary role by enhancing solubility and affinity to DNA whereas the second amino group which is positioned next to the light-activated DNA-cleaver undergoes protonation at the desired pH threshold. This protonation results in two synergetic effects which account for the increased DNA-cleaving ability at the lower pH. First, lysine conjugates show tighter binding to DNA at the lower pH, which is consistent with the anticipated higher degree of interaction between two positively charged ammonium groups with the negatively charged phosphate backbone of DNA. Second, the unproductive pathway which quenches the excited state of the photocleaver through intramolecular electron transfer is eliminated once the donor amino group next to the chromophore is protonated. Experiments in the presence of traps for diffusing radicals show that reactive oxygen species do not contribute significantly to the mechanism of DNA cleavage at the lower pH, which is indicative of tighter binding to DNA under these conditions. This feature is valuable not only because many solid tumors are hypoxic but also because cleavage which does not depend on diffusing species is more localized and efficient. Sequence-selectivity experiments suggest combination of PET and base alkylation as the chemical basis for the observed DNA-damage. The utility of these molecules for phototherapy of cancer is confirmed by the drastic increase in toxicity of five conjugates against cancer cell lines upon photoactivation.

Opening Enediyne Scissors Wider: pH‐Dependent DNA Photocleavage by meta‐Diyne Lysine Conjugates

“…In the efforts to better understand the photochemistry and photophysics responsible for the highly efficient DNA cleavage, Yang et al . discover surprising DNA photocleavage activity of the mono acetylene‐lysine conjugates (Fig. ).…”

“…Because the latter process leads to four formal H-transfers to the enediyne, it increases the DNA-damaging ability of this functional group even further (9). We had further amplified the potential of the photochemically activated enediynes by equipping them with pH-regulated amino acid functionality with variable protonation states (10). Although several amino acid/ enediyne hybrids (11,12) have been reported in the literature (13,14) and the list is growing (15)(16)(17), we found the unique advantage of lysine groups for the control of reactivity and biological utility of such hybrids.…”

Intracellular DNA Damage by Lysine‐Acetylene Conjugates

“…This difference is not limited to the comet assay—our earlier experiments with pure DNA also required micromolar concentrations of the conjugate to observe the cleavage [18, 19]. The observation has two consequences.…”

Rescue of the activity of HNH nuclease mutants - towards controlled enzymes for gene therapy

“…Alternative nuclease domains have been applied in few cases in mainly monomeric chimera [53][54][55][56][57][58] and non-FokI-based zinc finger nucleases have also been developed by introducing mutations or small modifications into the zinc finger array, such as the exchange of the metal ion-binding cysteines to histidines [59][60][61]. Trials to establish controlled artificial nucleases through lightactivation [62,63], DNA binding [64], cold-shock technique [65], protein-protein interaction [66], DNA modification [67] or metal ion dependent DNA binding [68] were also executed. Bioinformatics was utilized to predict ZFN and TALEN off-target cleavage [69].…”