High spatial-temporal resolution imaging of drug uptake at the single cell level using fluorescence spectroscopy

为了达到它们的效果，大多数药物需要进入人体细胞并达到特定的目标：化合物的疗效在很大程度上取决于它能达到的程度。这一过程的效率越低，即因为药物被隔离在“错误的”细胞室，需要给药的剂量就越高，从而导致潜在的副作用和与其他靶点的不必要的相互作用。因此，在这个项目中，学生将专注于研究荧光类似物单个药物分子的亚细胞动力学。根据我的主管的记录，这类药物的广泛作用领域将与G蛋白偶联受体信号级联有关。荧光染料、化学偶联技术和新的荧光显微镜方法的改进，现在允许在单分子、单细胞水平上标记和可视化药物。这位主管最近可以展示标记的第二信使cAMP细胞内扩散的微观成像，cAMP用与其自身大小相当的荧光团标记。因此，在这个项目中，我们想要在这个概念的基础上可视化典型药物对cAMP水平的影响的亚细胞动力学，例如，通过与称为磷酸二酯酶的抑制酶结合，其在细胞内的分布尚不清楚。为此，我们将安装一台显微镜，允许测量单个活细胞内的药物结合，结合单分子成像、荧光波动光谱和荧光偏振，通过发射荧光的增加的各向异性来报告药物结合。这项研究将解决一个重要的生物学问题，同时为非侵入性、单细胞药理学建立一个新的方法学工具箱，帮助设计和优化现有或新药。该项目目前整合在与一家大型制药公司的工业合作中，并得到皇家学会的标准拨款支持。

To achieve their effects most drugs, need to enter within a human cell and reach a specific target: the efficacy of a compound will largely depend how well it can reach it. The less efficient this process, i.e. because the drug is sequestered in the 'wrong' cellular compartment, the higher the dose that needs to be administered, leading to potential side effects and unwanted interactions with other targets. In this project the student will therefore focus on the study of the subcellular dynamics of fluorescent analogues individual drug molecules. In line with my supervisor's track record, the broad area of action of such drugs will relate to the G protein-coupled Receptor signalling cascade. Improvements in fluorescent dyes, chemical conjugation techniques and novel fluorescence microscopy approaches, allow now to tag and visualize a drug at the single molecule, single cell level. The supervisor could recently demonstrate micro-scale imaging of the intracellular diffusion of a labelled second messenger, cAMP, tagged with a fluorophore comparable to its own size. In this project we therefore want to build on this concept to visualize the subcellular dynamics of prototypical drug used impact on cAMP levels, e.g., by binding to inhibiting enzymes called Phosphodiesterase's, whose intracellular distribution is still unclear. To do so, we will setup a microscope that allows measuring drug binding within single, living cells, combining single molecule imaging, fluorescence fluctuation spectroscopy to fluorescent polarization, reporting on drug binding by the increased anisotropy of the emitted fluorescence. This research will solve an important biological question, while establishing a new methodological toolbox for non-invasive, single cell pharmacology that will help design and optimize current or new drugs.The project is currently integrated within an Industrial collaboration with a major Pharmaceutical Company and supported by a standard grant of the Royal Society.