Interaction of designed sulfated flavanoids with antithrombin: Lessons on the design of organic activators
Interaction of designed sulfated flavanoids with antithrombin: Lessons on the design of organic activators
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DOI：
10.1021/jm020132y
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发表时间：
2002-09-26
影响因子：
7.3
通讯作者：
Desai, UR
中科院分区：
文献类型：
--
作者：
Gunnarsson, GT;Desai, UR
最近，我们设计了 (-)-表儿茶素硫酸盐 (ECS)，这是第一个小非糖分子，作为抗凝血酶的激活剂，用于加速抑制因子 Xa，因子 Xa 是凝血级联的关键蛋白酶 (Gunnarsson, G. T.; Desai, U. R. J. Med. Chem. 2002,45, 1233-1243)。尽管发现硫酸化类黄酮 ECS 与抗凝血酶的结合亲和力（类似于 10.7 μM）与参考三糖 DEF（类似于 4.5 μM）相当，但与使用 DEF 观察到的大约 300 倍相比，它仅加速了 Xa 因子的抑制作用 10 倍。为了确定抑制剂的构象激活是否依赖于有机激活剂的结构并探究激活缺陷的基础，我们研究了类似的硫酸化黄酮类化合物与抗凝血酶的相互作用。 (+)-硫酸儿茶素 (CS) 是 ECS 的手性立体异构体，与血浆抗凝血酶结合的亲和力高出 3 倍 (K-D = 3.5 muM)，对 Xa 因子抑制的二阶速率常数高出 2 倍 (k(ACT) = 6750 M-1 s(-1)）。相反，发现其外消旋对应物（+/-）-硫酸儿茶素的 K-D 和 k(ACT) 分别低至 7.4 倍和 2.4 倍。平衡解离常数对 pH 6.0 和 7.4 时介质离子强度的依赖性表明，非离子相互作用贡献了总结合能的主要部分（大约 55-73%），并且在相互作用中形成了 1-2 个离子对（与参考三糖的预期类似 4 个离子对相比）。竞争性结合实验表明，激活剂CS不与在五糖结合位点结合抗凝血酶的糖配体竞争，但它与全长低亲和力肝素竞争。分子对接研究表明，CS 在扩展的肝素结合位点上似乎有可能结合，该位点与参考三糖 DEF 的结合域相邻。组合起来，结果表明，尽管具有小硫酸化黄酮的抗凝血酶的构象激活取决于激活剂的结构，但设计的激活剂不结合在抗凝血酶中的五糖结合位点，导致弱激活。机理研究强调了合理设计特定高亲和力有机抗凝血酶激活剂的合理方向。
Recently, we designed (-)-epicatechin sulfate (ECS), the first small nonsaccharide molecule, as an activator of antithrombin for the accelerated inhibition of factor Xa, a key proteinase of the coagulation cascade (Gunnarsson, G. T.; Desai, U. R. J. Med. Chem. 2002,45, 1233-1243). Although sulfated flavanoid ECS was found to bind antithrombin with an affinity (similar to10.7 muM) comparable to the reference trisaccharide DEF (similar to4.5 muM), it accelerated the inhibition of factor Xa only 10-fold as compared to the similar to300-fold observed with DEF. To determine whether this conformational activation of the inhibitor is dependent on the structure of the organic activator and to probe the basis for the deficiency in activation, we studied the interaction of similar sulfated flavanoids with antithrombin. (+)-Catechin sulfate (CS), a chiral stereoisomer of ECS, bound plasma antithrombin with a 3-fold higher affinity (K-D = 3.5 muM) and a 2-fold higher second-order rate constant for factor Xa inhibition (k(ACT) = 6750 M-1 s(-1)). On the contrary, the K-D and k(ACT) were found to be lower similar to7.4- and similar to2.4-fold, respectively, for its racemic counterpart, (+/-)-catechin sulfate. Dependence of the equilibrium dissociation constant on the ionic strength of the medium at pH 6.0 and 7.4 suggests that nonionic interactions contribute a major proportion (similar to55-73%) of the total binding energy, and only 1-2 ion pairs, in comparison to the expected similar to4 ion pairs for the reference trisaccharide, are formed in the interaction. Competitive binding experiments indicate that activator CS does not compete with a saccharide ligand that binds antithrombin in the pentasaccharide binding site, while it competes with full-length low-affinity heparin. A molecular docking study suggests plausible binding of CS in the extended heparin binding site, which is adjacent to the binding domain for the reference trisaccharide DEF. In combination, the results demonstrate that although conformational activation of antithrombin with small sulfated flavanoids is dependent on the structure of the activator, the designed activators do not bind in the pentasaccharide binding site, in antithrombin resulting in weak activation. The mechanistic investigation highlights plausible directions to take in the rational design of specific high-affinity organic antithrombin activators.