Bogusky M J, Culberson J C, Pitzenberger S M, Garsky V M, Wallace A, Pessi A, Koblan K S
Department of Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486, USA.
J Pept Res. 1999 Jul;54(1):66-73. doi: 10.1034/j.1399-3011.1999.00085.x.
Farnesyl-protein transferase (FPTase) catalyzes the posttranslational farnesylation of the cysteine residue located in the C-terminal tetrapeptide of the Ras oncoprotein. Prenylation of this residue is essential for membrane association and cell-transforming activities of ras. Inhibitors of FPTase have been demonstrated to display antitumor activity in both tissue culture and animal models, and thus represent a potential therapeutic strategy for the treatment of human cancers. A synthetic tetrapeptide library, which included an expanded set of 68 L-, D- and noncoded amino acids, has been screened for inhibitors of FPTase activity. The tetrapeptide, NH2-D-Trp-D-Met-L-Phe(pCl)-L-Gla-NH2 was shown to be competitive with the isoprenyl cosubstrate, farnesyl diphosphate (FPP) but not with the peptide substrate, the C-terminal tetrapeptide of the Ras protein. The FPTase-bound conformation of the inhibitor, NH2-D-Trp-D-Met-L-Phe(pCl)-L-Gla-NH2 was determined by NMR spectroscopy. Distance constraints were derived from two-dimensional transferred nuclear Overhauser effect (TRNOE) experiments. Ligand competition experiments identified the NOEs that originate from the active-site conformation of the inhibitor. Structures were calculated using a combination of distance geometry and restrained energy minimization. The peptide backbone is shown to adopt a reverse-turn conformation most closely approximating a type II' beta-turn. The resolved conformation of the inhibitor represents a distinctly different structural motif from that determined for Ras-competitive inhibitors. Knowledge of the bound conformation of this novel inhibitor provides a template and future direction for the design of new classes of FPTase antagonists.
法尼基蛋白转移酶（FPTase）催化位于Ras癌蛋白C末端四肽中的半胱氨酸残基的翻译后法尼基化。该残基的异戊二烯化对于ras的膜结合和细胞转化活性至关重要。FPTase抑制剂已在组织培养和动物模型中显示出抗肿瘤活性，因此代表了一种治疗人类癌症的潜在治疗策略。已经筛选了一个合成四肽文库，其中包括一组扩展的68种L-、D-和非编码氨基酸，以寻找FPTase活性的抑制剂。四肽NH2-D-Trp-D-Met-L-Phe(pCl)-L-Gla-NH2被证明与异戊二烯基共底物法尼基二磷酸（FPP）具有竞争性，但与肽底物Ras蛋白的C末端四肽不具有竞争性。通过核磁共振光谱确定了抑制剂NH2-D-Trp-D-Met-L-Phe(pCl)-L-Gla-NH2与FPTase结合的构象。距离限制来自二维转移核Overhauser效应（TRNOE）实验。配体竞争实验确定了源自抑制剂活性位点构象的核Overhauser效应。使用距离几何和受限能量最小化相结合的方法计算结构。肽主链显示出采用最接近II'型β-转角的反向转角构象。该抑制剂的解析构象代表了与Ras竞争性抑制剂所确定的明显不同的结构基序。这种新型抑制剂结合构象的知识为设计新型FPTase拮抗剂提供了模板和未来方向。
