Machida Shinnosuke, Usuba Kakeru, Blaskovich Michelle A, Yano Akiko, Harada Kazuo, Sebti Saïd M, Kato Nobuo, Ohkanda Junko
The Institute of Scientific and Industrial Research (ISIR), Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka, Japan.
Chemistry. 2008;14(5):1392-401. doi: 10.1002/chem.200701634.
Synthetic chemical probes designed to simultaneously targeting multiple sites of protein surfaces are of interest owing to their potential application as site specific modulators of protein-protein interactions. A new approach toward bivalent inhibitors of mammalian type I geranylgeranyltransferase (GGTase I) based on module assembly for simultaneous recognition of both interior and exterior protein surfaces is reported. The inhibitors synthesized in this study consist of two modules linked by an alkyl spacer; one is the tetrapeptide CVIL module for binding to the interior protein surface (active pocket) and the other is a 3,4,5-alkoxy substituted benzoyl motif that contains three aminoalkyl groups designed to bind to the negatively charged protein exterior surface near the active site. The compounds were screened by two distinct enzyme inhibition assays based on fluorescence spectroscopy and incorporation of a [(3)H]-labeled prenyl group onto a protein substrate. The bivalent inhibitors block GGTase I enzymatic activity with K(i) values in the submicromolar range and are approximately one order of magnitude and more than 150 times more effective than the tetrapeptide CVIL and the methyl benzoate derivatives, respectively. The bivalent compounds 6 and 8 were shown to be competitive inhibitors, suggesting that the CVIL module anchors the whole molecule to the GGTase I active site and delivers the other module to the targeting protein surface. Thus, our module-assembly approach resulted in simultaneous multiple-site recognition, and as a consequence, synergetic inhibition of GGTase I activity, thereby providing a new approach in designing protein-surface-directed inhibitors for targeting protein-protein interactions.
设计用于同时靶向蛋白质表面多个位点的合成化学探针因其作为蛋白质 - 蛋白质相互作用的位点特异性调节剂的潜在应用而备受关注。本文报道了一种基于模块组装的新型二价哺乳动物I型香叶基香叶基转移酶（GGTase I）抑制剂，用于同时识别蛋白质的内部和外部表面。本研究中合成的抑制剂由通过烷基间隔基连接的两个模块组成；一个是用于结合蛋白质内部表面（活性口袋）的四肽CVIL模块，另一个是3,4,5 - 烷氧基取代的苯甲酰基基序，其含有三个设计用于结合活性位点附近带负电荷的蛋白质外部表面的氨基烷基。通过基于荧光光谱的两种不同的酶抑制试验以及将[（3）H]标记的异戊二烯基掺入蛋白质底物来筛选这些化合物。二价抑制剂以亚微摩尔范围内的K（i）值阻断GGTase I的酶活性，分别比四肽CVIL和苯甲酸甲酯衍生物有效约一个数量级和超过150倍。二价化合物6和8被证明是竞争性抑制剂，这表明CVIL模块将整个分子锚定到GGTase I活性位点，并将另一个模块递送至靶向蛋白质表面。因此，我们的模块组装方法导致了同时的多位点识别，结果是对GGTase I活性的协同抑制，从而为设计用于靶向蛋白质 - 蛋白质相互作用的蛋白质表面导向抑制剂提供了一种新方法。
