Substrate-based design of reversible Pin1 inhibitors.
Substrate-based design of reversible Pin1 inhibitors.
复制标题
基于底物的可逆 Pin1 抑制剂设计。
DOI：
10.1021/bi0262395
复制
发表时间：
2002
期刊：
影响因子：
2.9
通讯作者：
G. Fischer
中科院分区：
文献类型：
--
作者：
Yixin Zhang;S. Füssel;U. Reimer;M. Schutkowski;G. Fischer
人Pin 1是一种对-Ser/Thr（PO（3）H（2））-Pro-基序具有高度特异性的肽基脯氨酰顺/反异构酶，是细胞周期进程所必需的。为了设计可逆的Pin 1抑制剂，通过使用基于底物结构的方法，一组肽被应用于系统地分析Pin 1底物识别的最小结构要求。Pin 1对Ala-Pro、Ser-Pro和Ser（PO（3）H（2））-Pro的催化作用（k（cat）/K（m）< 5 mM（-1）s（-1））通过脯氨酰异构化的直接紫外-可见分光光度检测来检测，同时观察到这些二肽对Pin 1的弱竞争性抑制（K（i）> 1 mM）。链长从P2延伸到P1'或从P1延伸到P2'亚位点的底物，对于Ala-Ser（PO（3）H（2））-Pro，k（cat）/K（m）值为100 mM（-1）s（-1），对于Ser（PO（3）H（2））-Pro-Arg，k（cat）/K（m）值为38 mM（-1）s（-1）。对于Pin 1及其酵母同源物PIN 1，最佳亚位点识别元件包括5个氨基酸残基，中间位置是必需的Ser（PO（3）H（2））。在Pin 1或Pin 2 1存在下，Ac-Ala-Ala-Ser（PO（3）H（2））-Pro-Arg-NH 4-硝基苯胺的顺反互变势垒很低，k（cat）/K（m）分别为9300 mM（-1）s（-1）和12000 mM（-1）s（-1）。脯氨酸前的D-Ser（PO（3）H（2））残基可以作为底物失活决定簇而不损害基态亲和力。类似地，用硫代酰胺键取代脯氨酸之前的酰胺键产生有效的抑制剂。Pin 1被这种底物类似物抑制剂可逆地抑制，IC（50）值在低微摩尔范围内。含有D-氨基酸的抑制剂还表现出对细胞裂解物中磷酸酶活性的显著稳定性。
Human Pin1, a peptidyl-prolyl cis/trans isomerase with high specificity to -Ser/Thr(PO(3)H(2))-Pro- motifs, is required for cell cycle progression. In an effort to design reversible Pin1 inhibitors by using a substrate structure based approach, a panel of peptides were applied to systematically analyze the minimal structural requirements for Pin1 substrate recognition. Pin1 catalysis (k(cat)/K(m) < 5 mM(-1) s(-1)) for Ala-Pro, Ser-Pro, and Ser(PO(3)H(2))-Pro was detected using direct UV-visible spectrophotometric detection of prolyl isomerization, while weak competitive inhibition of Pin1 by these dipeptides was observed (K(i) > 1 mM). Substrates with chain lengths extending from either the P2 to P1' or the P1 to P2' subsite gave k(cat)/K(m) values of 100 mM(-1) s(-1) for Ala-Ser(PO(3)H(2))-Pro and 38 mM(-1) s(-1) for Ser(PO(3)H(2))-Pro-Arg. For both Pin1 and its yeast homologue Ess1, the optimal subsite recognition elements comprise five amino acid residues with the essential Ser(PO(3)H(2)) in the middle position. The resulting substrate Ac-Ala-Ala-Ser(PO(3)H(2))-Pro-Arg-NH-4-nitroanilide possesses a very low cis/trans interconversion barrier in the presence of either Pin1 or Ess1, with k(cat)/K(m) = 9300 mM(-1) s(-1) and 12000 mM(-1) s(-1), respectively. The D-Ser(PO(3)H(2)) residue preceding proline could serve as a substrate-deactivating determinant without compromising ground state affinity. Similarly, substitution of the amide bond preceding proline with a thioxo amide bond produces a potent inhibitor. Pin1 is reversibly inhibited by such substrate analogue inhibitors with IC(50) values in the low micromolar range. The D-amino acid containing inhibitor also exhibits remarkable stability against phosphatase activity in cell lysate.