DRUG-CONJUGATED NANOCRYSTALS AS FLUORESCENT LABELS

细胞表面受体、离子通道和转运体是神经系统和靶部位信号传导和兴奋性的重要组成部分。因此，它们代表了目前制药工业中探索的大多数药物靶点。目前，膜蛋白的检测、定量和定位主要是利用放射性标记配体或间接利用抗体技术实现的。由于放射性示踪剂研究的空间分辨率较差，以及膜蛋白表面结构域选择性抗体探针的可用性有限，这些方法受到限制。本文提出了一种利用药物共轭纳米晶体的新型荧光标记策略。这种方法将使使用荧光显微镜实时监测蛋白质运输成为可能。该策略将使用多巴胺、去甲肾上腺素和血清素转运体以及血清素（5HT2）受体进行测试。苯tropane-conjugated纳米晶体将被合成，它们对转运体的亲和力将通过转运体转染细胞的氚化多巴胺、去甲肾上腺素和血清素通量测定来确定。将合成苯异丙胺-纳米晶体偶联物，并在受体转染的细胞中使用竞争结合和磷酸肌肽水解测定其标记5HT2受体的能力。聚光和共聚焦成像将用于确定纳米缀合物是否为检测非渗透细胞中的转运体和受体提供合适的信号。为了用荧光显微镜跟踪转运体或受体的运输，纳米晶体标记的细胞将暴露于已知会引起细胞表面转运体和受体的内化和重新分配的外源性调节剂。

Cell surface receptors, ion channels and transporters are critical components of signaling and excitability in the nervous system and at target sites. As such they represent the majority of drug targets currently explored in the pharmaceutical industry. Currently the detection, quantitation and localization of membrane proteins is achieved largely using radiolabeled ligands or indirectly with antibody techniques. These approaches are limited due to the poor spatial resolution of radiotracer studies and the limited availability of surface domain-selective antibody probes for membrane proteins. In the proposed work a novel fluorescence labeling strategy using drug-conjugated nanocrystals will be developed. This method will enable the monitoring of protein trafficking in real time using fluorescence microscopy. This strategy will be tested using dopamine, norepinephrine and serotonin transporters as well as serotonin (5HT2) receptors. Phenyltropane-conjugated nanocrystals will be synthesized and their affinity for the transporters will be determined by tritiated dopamine, norepinephrine, and serotonin flux assays on transporter transfected cells. Phenylisopropylamine-nanocrystal conjugates will be synthesized and their ability to label 5HT2 receptors will be determined using competition binding and phosphoinositide hydrolysis assays in receptor transfected cells. Epifluorescence and confocal imaging will be used to determine whether the nanoconjugates provide suitable signal for detection of the transporters and receptors in nonpermeabilized cells. To follow transporter or receptor trafficking with fluorescence microscopy nanocrystal-labeled cells will be exposed to exogenus modulators known to cause internalizatioin and redistribution of cell surface transporters and receptors.