Department of Chemical Sciences, University of Naples Federico II, 80126 Naples, Italy.
Institut des Biomolécules Max Mousseron, Université de Montpellier, CNRS, ENSCM, 34095 Montpellier, France.
Bioorg Chem. 2023 Dec;141:106917. doi: 10.1016/j.bioorg.2023.106917. Epub 2023 Oct 14.
The limitations associated with the in vivo use of the thrombin binding aptamer (TBA or TBA) have dramatically stimulated the search of suitable chemically modified analogues in order to discover effective and reversible inhibitors of thrombin activity. In this context, we previously proposed cyclic and pseudo-cyclic TBA analogues with improved stability that proved to be more active than the parent aptamer. Herein, we have investigated a novel library of TBA derivatives carrying naphthalene diimide (NDI) moieties at the 3'- or 5'-end. In a subset of the investigated oligonucleotides, additional 3-hydroxypropylphosphate (HPP) groups were introduced at one or both ends of the TBA sequence. Evaluation of the G-quadruplex thermal stability, serum nuclease resistance and in vitro anticoagulant activity of the new TBA analogues allowed rationalizing the effect of these appendages on the activity of the aptamer on the basis of their relative position. Notably, most of the different TBA analogues tested were more potent thrombin inhibitors than unmodified TBA. Particularly, the analogue carrying an NDI group at the 5'-end and an HPP group at the 3'-end, named N-TBA-p, exhibited enhanced G-quadruplex thermal stability (ΔT + 14° C) and ca. 10-fold improved nuclease resistance in serum compared to the native aptamer. N-TBA-p also induced prolonged and dose-dependent clotting times, showing a ca. 11-fold higher anticoagulant activity compared to unmodified TBA, as determined by spectroscopic methods. Overall, N-TBA-p proved to be in vitro a more efficient thrombin inhibitor than all the best ones previously investigated in our group. Its interesting features, associated with its easy preparation, make it a very promising candidate for future in vivo studies.
与体内使用凝血酶结合适体（TBA 或 TBA）相关的局限性极大地刺激了对合适的化学修饰类似物的搜索，以发现有效的和可逆的凝血酶活性抑制剂。在这种情况下，我们之前提出了具有改进稳定性的环状和拟环状 TBA 类似物，这些类似物被证明比母体适体更具活性。在此，我们研究了一种带有萘二酰亚胺（NDI）部分的新型 TBA 衍生物文库，这些部分位于 3'-或 5'-末端。在所研究的寡核苷酸中，在 TBA 序列的一个或两个末端引入了额外的 3-羟丙基磷酸酯（HPP）基团。评估新 TBA 类似物的 G-四链体热稳定性、血清核酸酶抗性和体外抗凝活性，根据它们的相对位置，合理推断这些附属物对适体活性的影响。值得注意的是，大多数测试的不同 TBA 类似物都比未修饰的 TBA 更有效地抑制凝血酶。特别是，在 5'-末端带有 NDI 基团且在 3'-末端带有 HPP 基团的类似物，命名为 N-TBA-p，与天然适体相比，表现出增强的 G-四链体热稳定性（ΔT +14°C）和在血清中约 10 倍的核酸酶抗性增强。N-TBA-p 还诱导了延长且剂量依赖性的凝固时间，与未修饰的 TBA 相比，显示出约 11 倍的更高抗凝活性，如通过光谱方法确定的。总体而言，与我们之前在组内研究的最佳抑制剂相比，N-TBA-p 被证明在体外是一种更有效的凝血酶抑制剂。其有趣的特性，与其易于制备相关，使其成为未来体内研究的非常有前途的候选物。
