De Filippis V, Quarzago D, Vindigni A, Di Cera E, Fontana A
CRIBI Biotechnology Centre, University of Padua, Italy.
Biochemistry. 1998 Sep 29;37(39):13507-15. doi: 10.1021/bi980717n.
Hirudin is the most potent and specific inhibitor of thrombin, a key enzyme in the coagulation process existing in equilibrium between its procoagulant (fast) and anticoagulant (slow) form. In a previous study, we described the solid-phase synthesis of a Trp3 analogue of fragment 1-47 of hirudin HM2, which displayed approximately 5-fold higher thrombin inhibitory potency relative to that of the natural product [De Filippis, V., et al. (1995) Biochemistry 34, 9552-9564]. By combining automated and manual peptide synthesis, here we have produced in high yields seven analogues of fragment 1-47 containing natural and non-natural amino acids. In particular, we have replaced Val1 with tert-butylglycine (tBug), Ser2 with Arg, and Tyr3 with Phe, cyclohexylalanine (Cha), Trp, alpha-naphthylalanine (alphaNal), and beta-naphthylalanine (betaNal). The crude reduced peptides are able to fold almost quantitatively into the disulfide-cross-linked species, whose unique alignment (Cys6-Cys14, Cys16-Cys28, and Cys22-Cys37) has been shown to be identical to that of the natural fragment. The results of conformational characterization provide evidence that synthetic peptides retain the structural features of the natural species, whereas thrombin inhibition data indicate that the synthetic analogues are all more potent inhibitors of thrombin. In particular, Val --> tBug exchange leads to a 3-fold increase in binding, interpreted as arising from a favorable reduction of the entropy of binding, due to the presence of the more symmetric side chain of tBug relative to that of Val. The S2R analogue binds 24- and 125-fold more tightly than the natural fragment to the fast or slow form of thrombin. These results are explained by considering that Arg2 may favorably couple to Glu192, a key residue involved in the slow to fast transition, thus stabilizing the slow form. Replacement of Tyr3 with more hydrophobic residues having different side chain orientations and electronic structures improves binding by 2-40-fold, suggesting that nonpolar interactions and shape-dependent packing effects strongly influence binding at this position. Overall, these results provide new insights for elucidating the mechanism of hirudin-thrombin recognition at the molecular level and highlight new strategies for designing more potent and selective inhibitors of thrombin.
水蛭素是凝血酶最有效且最具特异性的抑制剂，凝血酶是凝血过程中的关键酶，以促凝血（快速）形式和抗凝血（缓慢）形式处于平衡状态。在先前的一项研究中，我们描述了水蛭素HM2 1-47片段的Trp3类似物的固相合成，该类似物相对于天然产物显示出约5倍更高的凝血酶抑制效力[De Filippis, V., 等人（1995年）《生物化学》34卷，9552 - 9564页]。通过结合自动化和手动肽合成，我们在此高产率地制备了七种含有天然和非天然氨基酸的1-47片段类似物。具体而言，我们用叔丁基甘氨酸（tBug）取代了Val1，用Arg取代了Ser2，并用Phe、环己基丙氨酸（Cha）、Trp、α-萘基丙氨酸（αNal）和β-萘基丙氨酸（βNal）取代了Tyr3。粗制的还原肽几乎能定量折叠成二硫键交联的物种，其独特的排列方式（Cys6 - Cys14、Cys16 - Cys28和Cys22 - Cys37）已被证明与天然片段相同。构象表征结果提供了证据，表明合成肽保留了天然物种的结构特征，而凝血酶抑制数据表明合成类似物都是更有效的凝血酶抑制剂。特别是，Val→tBug的交换导致结合力增加3倍，这被解释为由于tBug相对于Val具有更对称的侧链，导致结合熵有利地降低。S2R类似物与凝血酶的快速或缓慢形式结合比天然片段紧密24倍和125倍。考虑到Arg2可能与Glu192（参与缓慢到快速转变的关键残基）形成有利的偶联，从而稳定缓慢形式，这些结果得到了解释。用具有不同侧链取向和电子结构的更多疏水残基取代Tyr3可使结合力提高2 - 40倍，这表明非极性相互作用和形状依赖性堆积效应在该位置对结合有强烈影响。总体而言，这些结果为在分子水平阐明水蛭素 - 凝血酶识别机制提供了新的见解，并突出了设计更有效和选择性更高的凝血酶抑制剂的新策略。
