Watanabe Daiya, Yamamoto Atsushi, Tomoo Koji, Matsumoto Keita, Murata Mitsuo, Kitamura Kunihiro, Ishida Toshimasa
Department of Physical Chemistry, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara,Takatsuki, Osaka 569-1094, Japan.
J Mol Biol. 2006 Oct 6;362(5):979-93. doi: 10.1016/j.jmb.2006.07.070. Epub 2006 Aug 1.
To quantitatively estimate the inhibitory effect of each substrate-binding subsite of cathepsin B (CB), a series of epoxysuccinyl derivatives with different functional groups bound to both carbon atoms of the epoxy ring were synthesized, and the relationship between their inhibitory activities and binding modes at CB subsites was evaluated by the X-ray crystal structure analyses of eight complexes. With the common reaction in which the epoxy ring of inhibitor was opened to form a covalent bond with the SgammaH group of the active center Cys29, the observed binding modes of the substituents of inhibitors at the binding subsites of CB enabled the quantitative assessment of the inhibitory effect of each subsite. Although the single blockage of S1' or S2' subsite exerts only the inhibitory effect of IC50 = approximately 24 microM (k2 = approximately 1250 M(-1) s(-1)) or approximately 15 microM (k2 = approximately 1800 M(-1) s(-1)), respectively, the synchronous block of both subsites leads to IC50 = approximately 23 nM (k2 = 153,000 - 185,000 M(-1) s(-1)), under the condition that (i) the inhibitor possesses a P1' hydrophobic residue such as Ile and a P2' hydrophobic residue such as Ala, Ile or Pro, and (ii) the C-terminal carboxyl group of a P2' residue is able to form paired hydrogen bonds with the imidazole NH of His110 and the imidazole N of His111 of CB. The inhibitor of a Pn' > or = 3' substituent was not potentiated by collision with the occluding loop. On the other hand, it was suggested that the inhibitory effects of Sn subsites are independent of those of Sn' subsites, and the simultaneous blockage of the funnel-like arrangement of S2 and S3 subsites leads to the inhibition of IC50 = approximately 40 nM (k2 = approximately 66,600 M(-1) s(-1)) regardless of the lack of Pn' substituents. Here we present a systematic X-ray structure-based evaluation of structure-inhibitory activity relationship of each binding subsite of CB, and the results provide the structural basis for designing a more potent CB-specific inhibitor.
为了定量评估组织蛋白酶B（CB）每个底物结合亚位点的抑制作用，合成了一系列环氧琥珀酰衍生物，这些衍生物的环氧环的两个碳原子上连接有不同的官能团，并通过八个复合物的X射线晶体结构分析评估了它们在CB亚位点的抑制活性与结合模式之间的关系。在抑制剂的环氧环打开并与活性中心Cys29的SγH基团形成共价键的常见反应中，观察到的抑制剂取代基在CB结合亚位点的结合模式使得能够对每个亚位点的抑制作用进行定量评估。尽管单独阻断S1'或S2'亚位点仅分别产生IC50约为24μM（k2约为1250 M-1 s-1）或约15μM（k2约为1800 M-1 s-1）的抑制作用，但在以下条件下，两个亚位点的同步阻断导致IC50约为23 nM（k2 = 153,000 - 185,000 M-1 s-1）：（i）抑制剂具有P1'疏水残基如Ile和P2'疏水残基如Ala、Ile或Pro，以及（ii）P2'残基的C末端羧基能够与CB的His110的咪唑NH和His111的咪唑N形成配对氢键。Pn'≥3'取代基的抑制剂不会因与封闭环碰撞而增强抑制作用。另一方面，表明Sn亚位点的抑制作用与Sn'亚位点的抑制作用无关，并且无论是否缺乏Pn'取代基，同时阻断S2和S3亚位点的漏斗状排列都会导致IC50约为40 nM（k2约为66,600 M-1 s-1）的抑制作用。在此，我们展示了基于X射线结构的对CB每个结合亚位点的结构-抑制活性关系的系统评估，结果为设计更有效的CB特异性抑制剂提供了结构基础。
