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

为了达到效果，大多数药物需要进入人体细胞并达到特定的目标：化合物的功效在很大程度上取决于它能在多大程度上达到目标。这一过程的效率越低，即因为药物被隔离在“错误的”细胞隔室中，需要施用的剂量就越高，从而导致潜在的副作用和与其他靶标的不必要的相互作用。在这个项目中，学生将专注于研究单个药物分子的荧光类似物的亚细胞动力学。根据我的导师的记录，这些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、化学偶联技术和新型荧光显微镜方法的改进，现在可以在单分子、单细胞水平上标记和可视化药物。导师最近展示了一种被标记的第二信使cAMP的细胞内扩散的微尺度成像，该信使被标记为与其自身大小相当的荧光团。因此，在这个项目中，我们希望建立在这个概念的基础上，以可视化的亚细胞动力学的原型药物对cAMP水平的影响，例如，通过结合被称为磷酸二酯酶的抑制酶，其在细胞内的分布仍然不清楚。为此，我们将建立一个显微镜，可以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，通过发射荧光的各向异性增加来报告药物结合。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，这将有助于设计和优化现有或新的药物。该项目目前与一家大型制药公司进行了工业合作，并得到了皇家学会的标准资助。

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.