Selective inhibition of fibroblast activation protein protease based on dipeptide substrate specificity
Selective inhibition of fibroblast activation protein protease based on dipeptide substrate specificity
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DOI：
10.1074/jbc.m511112200
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发表时间：
2006-03-17
影响因子：
4.8
通讯作者：
Wolf, BB
中科院分区：
文献类型：
--
作者：
Edosada, CY;Quan, C;Wolf, BB
成纤维细胞活化蛋白（FAP）是属于脯氨酰肽酶家族的跨膜丝氨酸肽酶。FAP与癌症有关;然而，其具体作用仍然难以捉摸，因为尚未开发出将FAP与其他脯氨酰肽酶如二肽基肽酶-4（DPP-4）区分开的抑制剂。为了鉴定用于FAP选择性抑制剂设计的肽基序，我们使用P-2-Pro（1）和乙酰基（Ac）-P-2-Pro（1）二肽底物文库，其中P-2是变化的，底物水解发生在Pro（1）和荧光离去基团之间。对于P-2-Pro（1）文库，FAP偏好P-2残基的Ile、Pro或Arg;然而，DPP-4显示出对该文库的广泛反应性，排除了选择性。相比之下，对于Ac-P-2-Pro（1）文库，FAP仅切割Ac-Gly-Pro，而DPP-4显示与所有底物几乎没有反应性。FAP还裂解甲酰基-、苄氧羰基-、生物素基-和肽基-Gly-Pro底物，DPP-4裂解这些底物很差，表明抑制剂设计的N-酰基-Gly-Pro基序。因此，我们合成并测试了化合物Ac-Gly-脯氨酸硼酸，其抑制FAP的Ki为23 +/- 3 nM。这比其他脯氨酰肽酶（包括DPP-4、DPP-7、DPP-8、DPP-9、脯氨酰寡肽酶和酰基肽水解酶）的Ki值低9至5400倍。这些结果将Ac-Gly-BoroPro鉴定为FAP选择性抑制剂，并表明基于N-酰基-Gly-Pro的抑制剂将允许测试FAP作为治疗靶标。
Fibroblast activation protein (FAP) is a transmembrane serine peptidase that belongs to the prolyl peptidase family. FAP has been implicated in cancer; however, its specific role remains elusive because inhibitors that distinguish FAP from other prolyl peptidases like dipeptidyl peptidase-4 (DPP-4) have not been developed. To identify peptide motifs for FAP-selective inhibitor design, we used P-2-Pro(1) and acetyl (Ac)-P-2-Pro(1) dipeptide substrate libraries, where P-2 was varied and substrate hydrolysis occurs between Pro(1) and a fluorescent leaving group. With the P-2-Pro(1) library, FAP preferred Ile, Pro, or Arg at the P-2 residue; however, DPP-4 showed broad reactivity against this library, precluding selectivity. By contrast, with the Ac-P-2-Pro(1) library, FAP cleaved only Ac-Gly-Pro, whereas DPP-4 showed little reactivity with all substrates. FAP also cleaved formyl-, benzyloxycarbonyl-, biotinyl-, and peptidyl-Gly-Pro substrates, which DPP-4 cleaved poorly, suggesting an N-acyl-Gly- Pro motif for inhibitor design. Therefore, we synthesized and tested the compound Ac-Gly-prolineboronic acid, which inhibited FAP with a K-i of 23 +/- 3 nM. This was similar to 9- to similar to 5400-fold lower than the Ki values for other prolyl peptidases, including DPP-4, DPP-7, DPP-8, DPP-9, prolyl oligopeptidase, and acylpeptide hydrolase. These results identify Ac-Gly-BoroPro as a FAP-selective inhibitor and suggest that N-acyl-Gly- Pro-based inhibitors will allow testing of FAP as a therapeutic target.