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

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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.