Lobell Robert B, Davide Joseph P, Kohl Nancy E, Burns H Donald, Eng Wai-Si, Gibson Raymond E
Department of Cancer Research, West Point, PA 19486, USA.
J Biomol Screen. 2003 Aug;8(4):430-8. doi: 10.1177/1087057103256153.
Farnesyl:protein transferase (FPTase) catalyzes the covalent addition of the isoprenyl moiety of farnesylpyrophosphate to the C-terminus of the Ras oncoprotein and other cellular proteins. Inhibitors of FPTase (FTIs) have been developed as potential anticancer agents, and several compounds have been evaluated in clinical trials. To facilitate the identification of cell-active FTIs with high potency, the authors developed a method that uses a radiolabeled FTI that serves as a ligand in competitive displacement assays. Using high-affinity [(3)H]-labeled or [(125)I]-labeled FTI radioligands, they show that specific binding to FPTase can be detected in intact cells. Binding of these labeled FTI radioligands can be competed with a variety of structurally diverse FTIs, and the authors show that inhibition of FTI radioligand binding correlates well with inhibition of FPTase substrate prenylation in cells. This method provides a rapid and quantitative means of assessing FTI potency in cells and is useful for guiding the discovery of potent, novel inhibitors of FPTase. Similar methods could be employed in the optimization of inhibitors for other intracellular drug targets.
蛋白质转移酶（FPTase）催化法尼基焦磷酸的异戊二烯基部分共价连接到Ras癌蛋白和其他细胞蛋白的C末端。FPTase抑制剂（FTIs）已被开发为潜在的抗癌药物，并且几种化合物已在临床试验中进行了评估。为了便于鉴定具有高效力的细胞活性FTIs，作者开发了一种方法，该方法使用放射性标记的FTI作为竞争性置换测定中的配体。使用高亲和力的[³H]标记或[¹²⁵I]标记的FTI放射性配体，他们表明在完整细胞中可以检测到与FPTase的特异性结合。这些标记的FTI放射性配体的结合可以被多种结构不同的FTIs竞争，并且作者表明FTI放射性配体结合的抑制与细胞中FPTase底物异戊二烯化的抑制密切相关。该方法提供了一种快速且定量的方法来评估细胞中FTI的效力，并且对于指导发现强效、新型的FPTase抑制剂很有用。类似的方法可用于优化其他细胞内药物靶点的抑制剂。
