Garcia Lia C, Donadío Lucia Gandolfi, Mann Ella, Kolusheva Sofiya, Kedei Noemi, Lewin Nancy E, Hill Colin S, Kelsey Jessica S, Yang Jing, Esch Timothy E, Santos Marina, Peach Megan L, Kelley James A, Blumberg Peter M, Jelinek Raz, Marquez Victor E, Comin Maria J
Laboratory of Organic Synthesis, Center of Research and Development in Chemistry, National Institute of Industrial Technology, Buenos Aires, Argentina.
Department of Chemistry, Ben Gurion University, Beer Sheva 84105, Israel.
Bioorg Med Chem. 2014 Jun 15;22(12):3123-40. doi: 10.1016/j.bmc.2014.04.024. Epub 2014 Apr 20.
The development of selective agents capable of discriminating between protein kinase C (PKC) isoforms and other diacylglycerol (DAG)-responsive C1 domain-containing proteins represents an important challenge. Recent studies have highlighted the role that Ras guanine nucleotide-releasing protein (RasGRP) isoforms play both in immune responses as well as in the development of prostate cancer and melanoma, suggesting that the discovery of selective ligands could have potential therapeutic value. Thus far, the N-methyl-substituted indololactone 1 is the agonist with the highest reported potency and selectivity for RasGRP relative to PKC. Here we present the synthesis, binding studies, cellular assays and biophysical analysis of interactions with model membranes of a family of regioisomers of 1 (compounds 2-5) that differ in the position of the linkage between the indole ring and the lactone moiety. These structural variations were studied to explore the interaction of the active complex (C1 domain-ligand) with cellular membranes, which is believed to be an important factor for selectivity in the activation of DAG-responsive C1 domain containing signaling proteins. All compounds were potent and selective activators of RasGRP when compared to PKCα with selectivities ranging from 6 to 65 fold. However, the parent compound 1 was appreciably more selective than any of the other isomers. In intact cells, modest differences in the patterns of translocation of the C1 domain targets were observed. Biophysical studies using giant vesicles as model membranes did show substantial differences in terms of molecular interactions impacting lipid organization, dynamics and membrane insertion. However, these differences did not yield correspondingly large changes in patterns of biological response, at least for the parameters examined.
开发能够区分蛋白激酶C（PKC）亚型与其他二酰基甘油（DAG）响应性含C1结构域蛋白的选择性试剂是一项重大挑战。最近的研究突出了Ras鸟嘌呤核苷酸释放蛋白（RasGRP）亚型在免疫反应以及前列腺癌和黑色素瘤发展中所起的作用，这表明发现选择性配体可能具有潜在的治疗价值。到目前为止，N-甲基取代的吲哚内酯1是相对于PKC而言，对RasGRP报道的效力和选择性最高的激动剂。在此，我们展示了1的一族区域异构体（化合物2 - 5）的合成、结合研究、细胞分析以及与模型膜相互作用的生物物理分析，这些异构体在吲哚环与内酯部分之间的连接位置上有所不同。研究这些结构变化是为了探索活性复合物（C1结构域 - 配体）与细胞膜的相互作用，据信这是激活含DAG响应性C1结构域的信号蛋白时选择性的一个重要因素。与PKCα相比，所有化合物都是RasGRP的强效和选择性激活剂，选择性范围为6至65倍。然而，母体化合物1比任何其他异构体的选择性明显更高。在完整细胞中，观察到C1结构域靶点转位模式存在适度差异。使用巨型囊泡作为模型膜的生物物理研究确实表明，在影响脂质组织、动力学和膜插入的分子相互作用方面存在显著差异。然而，至少对于所检查的参数而言，这些差异并未导致生物反应模式产生相应的巨大变化。
