Bifunctional peptide boronate inhibitors of thrombin: Crystallographic analysis of inhibition enhanced by linkage to an exosite 1 binding peptide
Bifunctional peptide boronate inhibitors of thrombin: Crystallographic analysis of inhibition enhanced by linkage to an exosite 1 binding peptide
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DOI：
10.1021/bi980225a
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发表时间：
1998-10-13
期刊：
影响因子：
2.9
通讯作者：
Deadman, JJ
中科院分区：
文献类型：
--
作者：
Skordalakes, E;Elgendy, S;Deadman, JJ
水蛭素（49-64）片段对凝血酶的外部位点1的亲和力先前已用于增强简单竞争性抑制剂的效力[DiMaio，J.，吉布斯，B.，Munn，D.，列费夫尔，J.，Ni，F.，Konishi，Y.等人（1990）J. Biol. Chern. 265，21698-21703.，和Maraganore，J.M.，Bourdon，P.，Jablonski，J.，Ramachandran，K. L.，和芬顿，J.W.，II（1990）Biochemistry 29，7095-7087.]。使用类似的方法，我们已经增强了活性位点导向的凝血酶抑制剂的活性，通过连接这个片段通过一种新的反向定向连接器的两个三肽硼酸酯抑制剂。在P-1处，化合物1含有一个类奎宁的异硫脲鎓侧链，而化合物2含有一个不带电荷的溴丙基残基。化合物1对人α-凝血酶的抑制显示出缓慢的紧密结合竞争动力学（最终Ki为2.2 pM，k（1）为3.51 × 10（7）M-1 s（-1），k（-1）为1.81 × 10（-4）s（-1））。加入水蛭肽（20 μ M）竞争外位点1的结合，并将k（1）和k（-1）恢复到类似三肽的水平，分别为0.29 × 10（7）M-1 s（-1）和0.13 × 10（-4）s（-1）。与类似的三肽（K-iTry/K-iThr类似于4）相比，化合物1对凝血酶的特异性增强，K-iTry/K-iThr类似于900。化合物2充当竞争性抑制剂（0.6nM的K-iThr），并且是高度选择性的，对胰蛋白酶没有影响。人α-凝血酶与化合物1（1.8埃）和化合物2（1.85埃）的复合物的晶体学分析显示抑制剂的双链与Ser（195）之间的共价键（键长B-O分别为1.55和1.61埃）。化合物1的异硫脲鎓基团与Asp（189）形成双齿相互作用。抑制剂的P-2和P-3残基与凝血酶的S-2和S-3位点形成相互作用，类似于其它基于D-Phe-Pro的抑制剂[Bode，W.，Turk，D.，和Karshikov，A.（1992）Protein Sci. 1，426-471.]。接头离开凝血酶的活性位点裂缝，不形成相互作用，而Hir（49-64）区段与外位点I的结合类似于先前对水蛭素的描述[Rydel，T. J.，Tulinsky，A.，和Bode，W.（1991）J. 221，583-601.]。由于在这些位点中的每一个处的结合与单独添加的类似肽的结合的相似性，该方法可用于改善所有类型的活性位点定向的凝血酶抑制剂的抑制活性，并且也可适用于其它蛋白酶的抑制剂的设计。
The affinity of the hirudin(49-64) segment for exosite 1 of thrombin has been used previously to enhance the potency of simple competitive inhibitors [DiMaio, J., Gibbs, B., Munn, D., Lefebvre, J., Ni, F., Konishi, Y. (1990) J. Biol. Chern. 265, 21698-21703., and Maraganore, J. M., Bourdon, P., Jablonski, J., Ramachandran, K. L., and Fenton, J. W., II (1990) Biochemistry 29, 7095-7087.]. Using a similar approach, we have enhanced the activity of two active site directed thrombin inhibitors by attaching this segment via a novel reverse oriented Linker to each of two tripeptide boronate inhibitors. At P-1, compound 1 contains an arginine-like, isothiouronium, side chain, while compound 2 contains an uncharged, bromopropyl residue. Inhibition of human alpha-thrombin by compound 1 shows slow, tight-binding competitive kinetics (final K-i of 2.2 pM, k(1) of 3.51 x 10(7) M-1 s(-1), and k(-1) of 1.81 x 10(-4) s(-1)). The addition of hirugen peptide (20 mu M) competes for exosite 1 binding and restores the k(1) and k(-1) to that of the analogous tripeptide, 0.29 x 10(7) M-1 s(-1) and 0.13 x 10(-4) s(-1), respectively. Compound 1 has enhanced specificity for thrombin over trypsin with K-iTry/K-iThr of similar to 900 compared to the analogous tripeptide, with K-iTry/K-iThr of similar to 4. Compound 2 acts as a competitive inhibitor (K-iThr of 0.6 nM) and is highly selective with no effect on trypsin. Crystallographic analysis of complexes of human alpha-thrombin with compound 1 (1.8 Angstrom) and compound 2 (1.85 Angstrom) shows a covalent bond between the baron of the inhibitor and Ser(195) (bond lengths B-O of 1.55 and 1.61 Angstrom, respectively). The isothiouronium group of compound 1 forms bidentate interactions with Asp(189). The P-2 and P-3 residues of the inhibitors form interactions with the S-2 and S-3 sites of thrombin similar to other D-Phe-Pro based inhibitors [Bode, W., Turk, D., and Karshikov, A. (1992) Protein Sci. 1, 426-471.]. The linker exits the active site cleft of thrombin forming no interactions, while the binding of Hir(49-64) segment to exosite I is similar to that previously described for hirudin [Rydel, T. J., Tulinsky, A., and Bode, W. (1991) J. Mel. Biol. 221, 583-601.]. Because of the similarity of binding at each of these sites to that of the analogous peptides added alone, this approach may be used to improve the inhibitory activity of all types of active site directed thrombin inhibitors and may also be applicable to the design of inhibitors of other proteases.