Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, Russia.
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia.
Proteins. 2024 Mar;92(3):329-342. doi: 10.1002/prot.26616. Epub 2023 Oct 20.
Thrombin is one of the key enzymes of the blood coagulation system and a promising target for the development of anticoagulants. One of the most specific natural thrombin inhibitors is hirudin, contained in the salivary glands of medicinal leeches. The medicinal use of recombinant hirudin is limited because of the lack of sulfation on Tyr63, resulting in a 10-fold decrease in activity compared to native (sulfated) hirudin. In the present work, a set of hirudin derivatives was tested for affinity to thrombin: phospho-Tyr63, Tyr63(carboxymethyl)Phe, and Tyr63Glu mutants, which mimic Tyr63 sulfation and Gln65Glu mutant and lysine-succinylated hirudin, which enhance the overall negative charge of hirudin, as well as sulfo-hirudin and desulfo-hirudin as references. Using steered molecular dynamics simulations with subsequent umbrella sampling, phospho-hirudin was shown to exhibit the highest affinity to thrombin among all hirudin analogs, including native sulfo-hirudin; succinylated hirudin was also prospective. Phospho-hirudin exhibited the highest antithrombotic activity in in vitro assay in human plasma. Taking into account the modern methods for obtaining phospho-hirudin and succinylated hirudin, they are prospective as anticoagulants in clinical practice.
凝血酶是血液凝血系统的关键酶之一，也是开发抗凝剂的有前途的靶点。天然凝血酶抑制剂中最特异的一种是水蛭素，它存在于医用水蛭的唾液腺中。由于缺乏 Tyr63 的硫酸化，重组水蛭素的药用受到限制，与天然（硫酸化）水蛭素相比，其活性降低了 10 倍。在本工作中，一组水蛭素衍生物被测试其与凝血酶的亲和力：磷酸化 Tyr63、Tyr63（羧甲基）Phe 和 Tyr63Glu 突变体，模拟 Tyr63 的硫酸化和 Gln65Glu 突变体以及赖氨酸琥珀酰化水蛭素，增强水蛭素的整体负电荷，以及磺基水蛭素和去磺基水蛭素作为参考。使用导向分子动力学模拟和随后的伞状采样，结果表明磷酸化水蛭素在所有水蛭素类似物中表现出最高的与凝血酶的亲和力，包括天然的磺基水蛭素；琥珀酰化水蛭素也很有前景。在人血浆中的体外试验中，磷酸化水蛭素表现出最高的抗血栓形成活性。考虑到获得磷酸化水蛭素和琥珀酰化水蛭素的现代方法，它们在临床实践中作为抗凝剂很有前景。
