Laboratory of Physical Biochemistry, National Research Center for Hematology, Russian Academy of Medical Sciences, Moscow, Russia.
PLoS One. 2011;6(5):e19969. doi: 10.1371/journal.pone.0019969. Epub 2011 May 16.
The development of new anticoagulants is an important goal for the improvement of thromboses treatments.
The design, synthesis and experimental testing of new safe and effective small molecule direct thrombin inhibitors for intravenous administration.
Computer-aided molecular design of new thrombin inhibitors was performed using our original docking program SOL, which is based on the genetic algorithm of global energy minimization in the framework of a Merck Molecular Force Field. This program takes into account the effects of solvent. The designed molecules with the best scoring functions (calculated binding energies) were synthesized and their thrombin inhibitory activity evaluated experimentally in vitro using a chromogenic substrate in a buffer system and using a thrombin generation test in isolated plasma and in vivo using the newly developed model of hemodilution-induced hypercoagulation in rats. The acute toxicities of the most promising new thrombin inhibitors were evaluated in mice, and their stabilities in aqueous solutions were measured.
New compounds that are both effective direct thrombin inhibitors (the best K(I) was <1 nM) and strong anticoagulants in plasma (an IC(50) in the thrombin generation assay of approximately 100 nM) were discovered. These compounds contain one of the following new residues as the basic fragment: isothiuronium, 4-aminopyridinium, or 2-aminothiazolinium. LD(50) values for the best new inhibitors ranged from 166.7 to >1111.1 mg/kg. A plasma-substituting solution supplemented with one of the new inhibitors prevented hypercoagulation in the rat model of hemodilution-induced hypercoagulation. Activities of the best new inhibitors in physiological saline (1 µM solutions) were stable after sterilization by autoclaving, and the inhibitors remained stable at long-term storage over more than 1.5 years at room temperature and at 4°C.
The high efficacy, stability and low acute toxicity reveal that the inhibitors that were developed may be promising for potential medical applications.
开发新型抗凝剂是改善血栓治疗的重要目标。
设计、合成和实验测试新型安全有效的小分子直接凝血酶抑制剂，用于静脉给药。
使用我们原创的基于遗传算法的全局能量最小化的 SOL 计算机辅助分子设计程序，设计新的凝血酶抑制剂。该程序考虑了溶剂的影响。根据评分函数（计算的结合能）设计出最佳的分子，并在缓冲体系中使用显色底物、在分离的血浆中使用凝血酶生成试验以及在体内使用新开发的大鼠血液稀释诱导高凝模型，对其体外的凝血酶抑制活性进行实验评估。在小鼠中评估最有前途的新型凝血酶抑制剂的急性毒性，并测量其在水溶液中的稳定性。
发现了新型化合物，它们既是有效的直接凝血酶抑制剂（最佳 K(i) <1 nM），也是血浆中的强抗凝剂（凝血酶生成试验的 IC(50)约为 100 nM）。这些化合物的基本片段包含以下新残基之一：异硫脲、4-氨基吡啶鎓或 2-氨基噻唑鎓。最佳新型抑制剂的 LD(50)值范围为 166.7 至 >1111.1 mg/kg。在血液稀释诱导高凝大鼠模型中，添加一种新型抑制剂的血浆替代溶液可防止高凝。在生理盐水中（1 µM 溶液），最佳新型抑制剂的活性在高压灭菌的消毒后稳定，并且抑制剂在室温下和 4°C 下长期储存超过 1.5 年保持稳定。
高效、稳定和低急性毒性表明，所开发的抑制剂可能具有潜在的医学应用前景。
