The Institute for Drug Research, The School of Pharmacy, The Hebrew University of Jerusalem, Hadassah Ein-Karem, Jerusalem, Israel.
Bioconjug Chem. 2010 Aug 18;21(8):1425-31. doi: 10.1021/bc900543b.
The ability to cleave DNA with the aid of chemical nucleases has been a challenge in the scientific community, particularly in the absence of a redox active metal ion. Inspired by structural characterization of the active site found in Staphylococcal nuclease, we have designed a series of organic molecule comprising cyclic pentapeptides conjugated to a DNA intercalator (e.g., anthraquinone). The cyclic peptide is designed to cleave the phosphodiester backbone, whereas the intercalator is expected to improve binding affinity to the substrate (DNA). Our lead compound (1-AQ), composed of the cyclic peptide cyc-d-Lys-Gly-Arg-Ser-Arg conjugated to anthraquinone, degrades DNA into small fragments at physiologically relevant conditions (i.e., 37 degrees C, pH = 7.4). We find that 1-AQ is highly effective in degrading duplex DNA at micromolar concentrations as corroborated by agarose and polyacrylamide gel electrophoresis. Changing the DNA intercalator to acridine (1-Ac) renders the compound comparable in nuclease activity to 1-AQ. In comparison to control compounds (Lin-1 and 1) that lack either the cyclic scaffold or the DNA intercalator, our lead compound (1-AQ) is found to be significantly more active as a DNA chemical nuclease. We have studied the importance of the triad (Arg-Ser-Arg) as the designed module for DNA cleavage. Changing l-Ser to l-Glu (cyc-d-Lys-Gly-Arg-Glu-Arg, Glu-AQ) results in an inactive compound, whereas the cyclic peptide Gly-AQ (cyc-d-Lys-Gly-Arg-Gly-Arg, where glycine replaces l-serine) has similar DNA nuclease activity to 1-AQ. In addition, changing the stereochemistry from d-lysine to l-lysine results in a cyclic peptide (1-L-AQ) exerting weak DNA nuclease activity, highlighting the importance of the cyclic backbone conformation for efficient DNA nuclease activity. The addition of ROS scavengers does not reduce DNA nuclease activity; an observation that supports a hydrolytic cleavage mechanism. Finally, we have estimated the kinetics of DNA cleavage of a 15-mer duplex DNA substrate by compound 1-AQ. By monitoring DNA duplex degradation by following the change in absorbance (hyperchromicity) at various 1-AQ concentrations, we report a maximal k(obs) value (as an underestimation of k(max)) of 1.62 h(-1) at a 7.5-fold of 1-AQ. We have also compared the other two active peptide conjugates, namely, 1-Ac and Gly-AQ to that of 1-AQ. Both compounds exert similar nuclease activity to that of 1-AQ. To the best of our knowledge, this is the most active metal-free DNA nuclease reported to date that exerts its DNA nuclease activity at biologically relevant conditions.
在缺乏氧化还原活性金属离子的情况下，用化学核酸酶切割 DNA 的能力一直是科学界面临的挑战。受葡萄球菌核酸酶活性位点结构特征的启发，我们设计了一系列包含与 DNA 嵌入剂（例如蒽醌）偶联的环状五肽的有机分子。环状肽被设计用于切割磷酸二酯骨架，而嵌入剂有望提高与底物（DNA）的结合亲和力。我们的先导化合物（1-AQ）由与蒽醌偶联的环状肽 cyc-d-Lys-Gly-Arg-Ser-Arg 组成，可在生理相关条件（即 37°C，pH = 7.4）下将 DNA 降解为小片段。我们发现，1-AQ 在微摩尔浓度下非常有效地降解双链 DNA，这得到琼脂糖和聚丙烯酰胺凝胶电泳的证实。将 DNA 嵌入剂改为吖啶（1-Ac）可使该化合物的核酸酶活性与 1-AQ 相当。与缺乏环状支架或 DNA 嵌入剂的对照化合物（Lin-1 和 1）相比，我们的先导化合物（1-AQ）被发现作为化学核酸酶的 DNA 具有显著更高的活性。我们研究了三肽（Arg-Ser-Arg）作为 DNA 切割设计模块的重要性。将 l-Ser 替换为 l-Glu（cyc-d-Lys-Gly-Arg-Glu-Arg，Glu-AQ）会导致化合物失活，而环状肽 Gly-AQ（cyc-d-Lys-Gly-Arg-Gly-Arg，其中甘氨酸取代 l-丝氨酸）对 DNA 核酸酶的活性与 1-AQ 相似。此外，将立体化学从 d-赖氨酸变为 l-赖氨酸会导致环状肽（1-L-AQ）表现出较弱的 DNA 核酸酶活性，这突出了环状骨架构象对于有效 DNA 核酸酶活性的重要性。添加 ROS 清除剂不会降低 DNA 核酸酶活性；这一观察结果支持水解切割机制。最后，我们估计了化合物 1-AQ 对 15 -mer 双链 DNA 底物的 DNA 切割动力学。通过在各种 1-AQ 浓度下监测 DNA 双链体降解，根据吸光度（增色）的变化来监测，我们报告在 7.5 倍的 1-AQ 时，最大 k(obs)值（作为 k(max)的低估值）为 1.62 h(-1)。我们还比较了另外两种活性肽缀合物，即 1-Ac 和 Gly-AQ 与 1-AQ 的比较。这两种化合物都表现出与 1-AQ 相似的核酸酶活性。据我们所知，这是迄今为止报道的最活跃的无金属 DNA 核酸酶，它在生物相关条件下发挥其 DNA 核酸酶活性。
