Yamamoto Atsushi, Tomoo Koji, Matsugi Ken-ichi, Hara Tadaoki, In Yasuko, Murata Mitsuo, Kitamura Kunihiro, Ishida Toshimasa
Department of Physical Chemistry, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan.
Biochim Biophys Acta. 2002 Jun 3;1597(2):244-51. doi: 10.1016/s0167-4838(02)00284-4.
In order to elucidate the substrate specificity of the Sn subsites (n=1-3) of cathepsin B, its crystal structure inhibited by E64c [(+)-(2S,3S)-3-(1-[N-(3-methylbutyl)amino]-leucylcarbonyl)oxirane-2-carboxylic acid] was analyzed by the X-ray diffraction method. Iterative manual rebuilding and convenient conjugate refinement of structure decreased R- and free R-factors to 19.7% and to 23.9%, respectively, where 130 water molecules were included for the refinement using 14,759 independent reflections from 10 to 2.3 A resolution. The epoxy carbonyl carbon of E64c was covalently bonded to the Cys(29) S(gamma) atom and the remaining parts were located at Sn subsites (n=1-3). The substrate specificity of these subsites was characterized based on their interactions with the inhibitor. Base on these structural data, we developed a novel cathepsin B-specific noncovalent-type inhibitor, which may bind to S2'-S3. The molecular design of possessing structural elements of both CA074 and E64c, assisted by energy minimization and molecular dynamics (MD) simulation, may lead to a new lead noncovalent-type inhibitor.
为了阐明组织蛋白酶B的Sn亚位点（n = 1 - 3）的底物特异性，采用X射线衍射法分析了其被E64c [（+） - （2S，3S） - 3 - （1 - [N - （3 - 甲基丁基）氨基] - 亮氨酰羰基）环氧乙烷 - 2 - 羧酸]抑制后的晶体结构。通过结构的迭代人工重建和便捷的共轭精修，R因子和自由R因子分别降至19.7%和23.9%，其中在精修过程中纳入了130个水分子，使用了10至2.3 Å分辨率下的14,759个独立反射。E64c的环氧羰基碳与半胱氨酸（29）的S（γ）原子共价结合，其余部分位于Sn亚位点（n = 1 - 3）。基于这些亚位点与抑制剂的相互作用对其底物特异性进行了表征。基于这些结构数据，我们开发了一种新型的组织蛋白酶B特异性非共价型抑制剂，它可能与S2'-S3结合。在能量最小化和分子动力学（MD）模拟的辅助下，兼具CA074和E64c结构元件进行分子设计，可能会产生一种新的先导非共价型抑制剂。
