Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands.
Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands.
复制标题
DOI：
10.7554/elife.74648
复制
发表时间：
2022-01-04
期刊：
影响因子：
7.7
通讯作者：
Lester HA
中科院分区：
文献类型：
--
作者：
Nichols AL;Blumenfeld Z;Fan C;Luebbert L;Blom AEM;Cohen BN;Marvin JS;Borden PM;Kim CH;Muthusamy AK;Shivange AV;Knox HJ;Campello HR;Wang JH;Dougherty DA;Looger LL;Gallagher T;Rees DC;Lester HA
烟碱部分激动剂为戒烟提供了一种公认的帮助，因此有助于减少烟草相关疾病。改进药物是一个持续的研究领域。然而，仍然没有还原的方法来检查这些化合物在活细胞中的细胞和亚细胞药代动力学特性。在这里，我们开发了新的基于强度的药物传感荧光报告（iDrugSnFR）的烟碱部分激动剂dianicline，野靛碱，和两个野靛碱衍生物- 10-氟野靛碱和9-溴-10-乙基野靛碱。我们报告了基于配体周质结合蛋白的生物传感器的第一个原子级结构，加速了iDrugSnFR的发展，并解释了激活机制。烟碱iDrugSnFR检测其在溶液中以及在细胞系和小鼠海马神经元的质膜（PM）和内质网（ER）中的药物伴侣。在PM，溶液变化的速度限制了几乎所有情况下荧光响应的增长和衰减速率。相比之下，我们发现这些烟碱类药物的跨膜速率相差>30倍。新的烟碱iDrugSnFR提供了对烟碱激动剂的实时药代动力学特性的深入了解，并提供了一种方法，使iDrugSnFR能够为药物神经科学和成瘾神经科学提供信息。
Nicotinic partial agonists provide an accepted aid for smoking cessation and thus contribute to decreasing tobacco-related disease. Improved drugs constitute a continued area of study. However, there remains no reductionist method to examine the cellular and subcellular pharmacokinetic properties of these compounds in living cells. Here, we developed new intensity-based drug-sensing fluorescent reporters (iDrugSnFRs) for the nicotinic partial agonists dianicline, cytisine, and two cytisine derivatives – 10-fluorocytisine and 9-bromo-10-ethylcytisine. We report the first atomic-scale structures of liganded periplasmic binding protein-based biosensors, accelerating development of iDrugSnFRs and also explaining the activation mechanism. The nicotinic iDrugSnFRs detect their drug partners in solution, as well as at the plasma membrane (PM) and in the endoplasmic reticulum (ER) of cell lines and mouse hippocampal neurons. At the PM, the speed of solution changes limits the growth and decay rates of the fluorescence response in almost all cases. In contrast, we found that rates of membrane crossing differ among these nicotinic drugs by >30-fold. The new nicotinic iDrugSnFRs provide insight into the real-time pharmacokinetic properties of nicotinic agonists and provide a methodology whereby iDrugSnFRs can inform both pharmaceutical neuroscience and addiction neuroscience.