物理化学学报 >> 2012, Vol. 28 >> Issue (10): 2418-2422.doi: 10.3866/PKU.WHXB201209143

生物物理化学 上一篇    下一篇

靛红类双功能抑制剂: 调控SARS-3CL蛋白酶聚集状态与活性

周璐1,3, 金凤1, 刘莹1,2, 尚尔昌1, 魏平1, 李春梅1, 来鲁华1,2   

  1. 1 北京大学化学与分子工程学院物理化学研究所, 分子动态与稳态结构国家重点实验室, 北京分子科学国家实验室;
    2 北京大学定量生物学中心, 北京 100871;
    3 复旦大学药学院, 上海 201203
  • 收稿日期:2012-07-25 修回日期:2012-09-10 发布日期:2012-09-26
  • 通讯作者: 刘莹, 来鲁华 E-mail:liuying@pku.edu.cn; lhlai@pku.edu.cn
  • 基金资助:

    国家自然科学基金(90913021, 20473001, 11021463)和国家重点基础研究发展规划项目(973) (2009CB9185003)资助

Isatin Dual Functional Inhibitors: Modulating the Aggregation State and Enzyme Activity of SARS-3CL Proteinase

ZHOU Lu1,3, JIN Feng1,LIU Ying1,2, SHANG Er-Chang1, WEI Ping1, LI Chun-Mei1, LAI Lu-Hua1,2   

  1. 1 Beijing National Laboratory for Molecular Sciences, State Key Laboratory of Structural Chemistry of Unstable and Stable Species, Institute of Physical Chemistry, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, P. R. China;
    2 Center for Quantitative Biology, Peking University, Beijing 100871, P. R. China;
    3 School of Pharmacy, Fudan University, Shanghai 201203, P. R. China
  • Received:2012-07-25 Revised:2012-09-10 Published:2012-09-26
  • Supported by:

    The project was supported by the National Natural Science Foundation of China (90913021, 20473001, 11021463) and National Key Basic Research Program of China (973) (2009CB9185003).

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摘要：

1-(2-萘甲基)靛红-5-甲酰胺类化合物通过与底物口袋结合来抑制SARS-3CL 蛋白酶的活性, 而SARS-3CL蛋白酶自身的N端8 肽是作用于蛋白二聚界面的抑制剂. 本文设计同时占据SARS-3CL蛋白酶底物口袋和二聚界面的双功能抑制剂, 通过固相多肽合成方法制备由1-(2-萘甲基)靛红-5-甲酸和N端8肽组成的化合物, 得到不同长度连接链的6 个目标产物. 用显色底物方法测定化合物对SARS-3CL蛋白酶的抑制活性,其中化合物3的活性最高, IC50值(半抑制率)为3.8 μmol·L-1, 连接偶数甘氨酸的活性明显要好于连接奇数甘氨酸的化合物. 用超速离心沉降速率方法研究了化合物3对SARS-3CL蛋白酶聚集状态与活性的调控作用, 其同时具有诱导与抑制二聚的双重能力, 综合调控结果是抑制SARS-3CL蛋白酶的二聚. 这项研究给应用合成的化合物研究酶活性调节机制提供了一个示例.

关键词: 靛红类衍生物, 调控, SARS-3CL蛋白酶, 二聚, 双功能抑制剂

Abstract:

The 1-(2-naphthlmethyl) isatin-5-formamide compounds can inhibit SARS-3CL proteinase by binding to its substrate pocket, while the N-terminal octapeptide of SARS-3CL proteinase was found to act as a dimerization inhibitor. In this work, the dual functional inhibitors which can occupy both substrate pocket of SARS-3CL proteinase and its dimer interface were designed. Six title compounds were gotten by linking 1-(2-naphthlmethyl) isatin-5-formic acid and N-terminal octapeptides using a polyglycine linker through solid-phase peptide synthesis method. The in vitro inhibition activity against SARS-3CL proteinase was measured by continuous colorimetric assay using colorimetric substrate. Compound 3 showed the highest inhibition activity with an IC50 (half maximal inhibitory concentration of a substance) of 3.8 μmol·L-1. The change of inhibition activity with the linker length was studied. Inhibitors with the even spacers were showed better activity than the odd ones, which could be explained by the angle restriction of peptide bonds. The modulating of the aggregation state and enzyme activity towards SARS-3CL proteinase were studied using sedimentation velocity experiments. Compound 3 was found to not only inhibit the enzyme activity of SARS-3CL proteinase, but also shift the monomer-dimer equilibrium of the enzyme. The integrated control result is inhibiting SARS-3CL proteinase dimer formation. This work provides an example of using synthesized compounds to study enzyme activity regulation mechanism.

Key words: Isatin derivative, Modulation, SARS-3CL proteinase, Dimerization, Dual functional inhibitor