Catecholamine Uptake, Micro-fluorometry/Drug Screening

描述（由申请人提供）：去甲肾上腺素（hNET）、多巴胺（hDAT）和血清素（hSERT）的人类单胺转运蛋白（hMATs）是治疗药物（抗抑郁药）和成瘾性精神兴奋剂（可卡因、安非他明）的靶标。与MATs相互作用的药物信息主要来自放射性配体底物测定。随着I期CEBRA的申请，我们将开发一种新的检测hMAT药物相互作用的方法，特别是儿茶酚胺，利用荧光底物和实时单细胞荧光显微镜和光谱学。我们计划用一种新的儿茶酚胺底物ASP[4-（4-（二甲胺）苯基）- n -甲基吡啶]分析转染细胞中的hNET和hDAT活性。ASP在结构上与MPP相似，但ASP荧光在可见光范围内具有较大的Stokes位移（4751 = bbb10 610）。我们已经证明ASP是na依赖的底物，对NET和DAT具有uM亲和力。在目的1中，我们将测量表达hNET和MAT的人胚胎肾细胞（HEK）中ASP的积累，并测量转运体对抗抑郁药和精神兴奋剂的活性。ASP荧光的各向异性和猝灭可以用来区分结合和运输，我们将测试抗抑郁药、可卡因和安非他明对hNET和hDAT的作用，以检查哪些过程受到影响。在Aim 1中，我们将首先在原生组织培养系统（颈上神经节神经元）中研究NET，并在DAT中进行类似的研究。原代组织培养也提供了一种检测内源性相关蛋白影响的方法，这些蛋白可能是特定的神经元治疗靶点，避免了系统性并发症。目标1和目标2的总体目标是开发ASP测定法，用于高通量筛选抑制或刺激单胺运输和结合的假定药物，特别是儿茶酚胺、去甲肾上腺素和多巴胺。

DESCRIPTION (provided by applicant): The human monoamine transporters (hMATs) for norepinephrine (hNET), dopamine (hDAT), and serotonin (hSERT) are targets for therapeutic drugs (antidepressants) and addictive psycho-stimulants (cocaine, amphetamines). Information about drugs that interact with MATs comes largely from radio-ligand substrate assays. With the Phase I CEBRA application, we will develop a new assay for hMAT drug interactions, in particular the catecholamines, utilizing fluorescent substrates and real-time, single cell fluorescence microscopy and spectroscopy. We plan to analyze hNET and hDAT activity in transfected cells with a novel substrate for the catecholamines, ASP [4-(4-(dimethylamino)styrl)-N-methyl-pyridinium]. ASP is structurally similar to MPP, but ASP fluoresces in the visible range with large Stokes shift (4751 => 610 1). We have already demonstrated that ASP is a Na-dependent substrate with uM affinity for NET and DAT. In Aim I we will measure ASP accumulation in human embryonic kidney cells (HEK) expressing hNET and MAT and measure transporter activity against antidepressants and psycho-stimulants. Anisotropy and quenching of ASP fluorescence can be exploited to distinguish binding from transport and we will test antidepressants, cocaine, and amphetamines against hNET and hDAT to examine which processes is affected. In Aim 1 we will first study NET in a native tissue culture system (superior cervical ganglion neurons) and initiate similar studies with DAT. Primary tissue culture also provides a means of examining the impact of endogenous associated proteins, which may be specific neuronal therapeutic targets, avoiding systemic complications. The overarching goal of Aims I and II is to develop the ASP assay for high-throughput screening of putative drugs that inhibit or stimulate monoamine transport and binding, in particular the catecholamines, norepinephrine and dopamine.