Polyarginines are potent furin inhibitors
Polyarginines are potent furin inhibitors
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DOI：
10.1074/jbc.m003848200
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发表时间：
2000-11-24
影响因子：
4.8
通讯作者：
Lindberg, I
中科院分区：
文献类型：
--
作者：
Cameron, A;Appel, J;Lindberg, I
普遍存在的丝氨酸内切蛋白酶弗林蛋白酶与细菌毒素和病毒糖蛋白的激活以及某些肿瘤的转移进展有关。尽管高分子质量生物工程丝氨酸蛋白酶抑制剂已得到充分表征，但迄今为止尚未报道小型无毒纳摩尔抑制剂。在这里，我们描述了使用位置扫描酰胺化和乙酰化合成的L-和D六肽组合文库来鉴定此类抑制剂，结果表明所有位置的L-Arg或L-Lys都产生最有效的抑制剂。然而，进一步的研究表明肽终止基团阻碍了抑制。因此，合成了一系列非酰胺化和乙酰化的聚精氨酸。已确定的最有效的抑制剂是九钠-L-精氨酸，其对弗林蛋白酶的 K-i 为 40 nM。相关转化酶 PACE4 和激素原转化酶-1 (PC1) 的 K-i 值分别为 110 nM 和 2.5 muM。虽然九-L-精氨酸被弗林蛋白酶裂解，但在 37°C 下孵育 6 小时后的主要产物是六-和七-L-精氨酸，两者都保留了针对弗林蛋白酶的大部分效力和特异性，六-D-精氨酸是与六-L-精氨酸一样有效和特异性的弗林蛋白酶抑制剂（六-D-精氨酸的 K-i 值：106 nM，弗林蛋白酶、PACE4 和 PC1 分别为 580 nM 和 13.2 muM）。 PC2 不受任何测试的聚精氨酸抑制；事实上，在 L-聚精氨酸存在的情况下，PC2 的活性增加了对照的 140%，还提供了数据，显示弗林蛋白酶和 PC2 扩展了亚位点识别，而发现 N 端乙酰化降低了 L-六肽 LLRVKR 对弗林蛋白酶的抑制效力 8 倍，C 端酰胺化降低了效力
The ubiquitous serine endoprotease furin has been implicated in the activation of bacterial toxins and viral glycoproteins as well as in the metastatic progression of certain tumors, Although high molecular mass bioengineered serpin inhibitors have been well characterized, no small nontoxic nanomolar inhibitors have been reported to date. Here we describe the identification of such inhibitors using positional scanning amidated and acetylated synthetic L- and D hexapeptide combinatorial libraries, The results indicated that L-Arg or L-Lys in all positions generated the most potent inhibitors. However, further investigation revealed that the peptide terminating groups hindered inhibition. Consequently, a series of non-amidated and acetylated polyarginines was synthesized. The most potent inhibitor identified, nona-L-arginine, had a K-i for furin of 40 nM. The K-i, values for the related convertases PACE4 and prohormone con vertase-1 (PC1) were 110 nM and 2.5 muM respectively. Although nona-L-arginine was cleaved by furin, the major products after a 6-h incubation at 37 degreesC were hexa-and hepta-L-arginines, both of which retained the great majority of their potency and specificity against furin, Hexa-D-arginine was as potent and specific a furin inhibitor as hexa-L-arginine (K-i values of hexa-D-arginine: 106 nM, 580 nM, and 13.2 muM for furin, PACE4, and PC1, respectively). PC2 was not inhibited by any polyarginine tested; indeed, PC2 showed an increase in activity of up to 140% of the control in the presence of L-polyarginines, Data are also presented that show extended subsite recognition by furin and PC2, Whereas N-terminal acetylation was found to reduce the inhibitory potency of the L-hexapeptide LLRVKR against furin 8-fold, C-terminal amidation reduced the potency