Molecular design of cathepsin L-specific inhibitor based on the teritiary structure.

基于三级结构的组织蛋白酶L特异性抑制剂的分子设计。

• 批准号: 06453194
• 负责人: ISHIDA Toshimasa
• 金额: $ 4.1万
• 依托单位: Osaka University of Pharmaceutical Sciences
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06453194/
• 关键词: cathepsin L; procathepsin L; recombinant; expression; X-ray analysis; NMR; inhibitor; molecular design

In the term of 1994-1996, we went ahead with the research project according to the following research plans :(1) Large scale expression of rat cathepsin L in Escherichia coli.(2) Isolation and purification of recombinant rat cathepsin L from Escherichia coli.(3) Identification of active conformation of recombinant rat cathepsin L by the comparison of native one.(4) X-Ray crystal structure determination of recombinant rat cathepsin L.(5) Preparation and physicochemical characterization of recombinant rat cathepsin L-inhibitor complex.(6) X-Ray crystal structure determination of recombinant rat cathepsin L and its complex with inhibitor.(7) NMR solution conformation of recombinant rat cathepsin L and its complex with inhibitor.(8) Model building and characterization of cathepsin L active site and elucidation of the catalytic mechanism at atomic level.(9) Molecular design and inhibitory measurement of cathepsin L-specific inhibitor based on the results of (4) - (8).We succeeded already in … More the research plans of (1) - (3), and are now vigorously studying the projects of (4) - (7) at the same time, with hoping these accomplishments in 1997. The main reason why we could not complete the research project within 1994-1996 is due to the instability of recombinant cathepsin L.Although structure determinations by X-ray diffraction and NMR methods require the stability of sample for a long time, rat cathepsin L itself decomposes to peptide fragments within few days due to the high autocatalytic activity. In order to overcome such trouble, we prepared (i) two kinds of recombinant procathepsin L from E.coli., which are resistant against the autocatalysis, and (ii) cathepsin L-inhibitor complex, where the inhibitor was complexed with recombinant cathepsin L at its expression state. Although the tertiary structure of mature cathepsin L itself could not be analyzed, we believe that the structure analyzes of the procathepsin Ls and cathepsin L-inhibor complex would not affect the accomplishment of this research project, seriously, i.e., the molecular design of cathepsin L-specific inhibitor. At present, the tertiary structures of a procathepsin L and cathepsin L-inhibitor complex are being analyzed by X-ray diffraction and various 2D NMR spectroscopy methods, and we expect the completion in 1997. Less

1994-1996年，我们按以下研究计划进行了研究：（1）大鼠组织蛋白酶L在大肠杆菌中的大量表达。(2)大肠杆菌重组大鼠组织蛋白酶L的分离纯化。(3)重组大鼠组织蛋白酶L活性构象的鉴定。(4)重组大鼠组织蛋白酶L的X-射线晶体结构测定。(5)重组大鼠组织蛋白酶L-抑制剂复合物的制备及理化性质研究。(6)重组大鼠组织蛋白酶L及其抑制剂复合物的X-射线晶体结构测定。(7)重组大鼠组织蛋白酶L及其与抑制剂复合物的NMR溶液构象。(8)组织蛋白酶L活性中心模型的建立、表征及催化机理的原子水平解析。(9)基于（4）-（8）的结果进行组织蛋白酶L特异性抑制剂的分子设计和抑制测量。 ...更多信息 （1）-（3）的研究计划，同时正在积极研究（4）-（7）的项目，希望在1997年取得成果。1994-1996年未能完成本课题的主要原因是重组组织蛋白酶L的不稳定性。虽然X射线衍射和核磁共振方法的结构测定需要样品长时间的稳定性，但大鼠组织蛋白酶L本身由于具有高的自催化活性，在几天内就分解成肽段。为了克服这一困难，我们制备了（i）两种来自大肠杆菌的重组组织蛋白原L，其对自催化具有抗性，和（ii）组织蛋白酶L-抑制剂复合物，其中抑制剂在其表达状态下与重组组织蛋白酶L复合。虽然成熟的组织蛋白酶L本身的三级结构无法分析，但我们相信组织蛋白酶L原和组织蛋白酶L-β或复合物的结构分析不会严重影响本研究项目的完成，即，组织蛋白酶L特异性抑制剂的分子设计。目前，通过X-射线衍射和各种2D NMR光谱方法分析了组织蛋白酶L原和组织蛋白酶L-抑制剂复合物的三级结构，我们预计将于1997年完成。少