Fluorescent biosensors for subcellular pharmacokinetics

用于中枢神经系统（CNS），特别是精神病学的药物开发已经放缓，部分原因是 因为我们不知道某些药物发挥其治疗或有害作用的机制。的 该项目提供了数据，以检验这一假设，即几种中枢神经系统药物的行为，除了他们的急性作用，在一个 慢一点的"由内而外"的方式这些药物将首先与它们的经典分子靶点结合， 细胞器 通过测量神经药物及其在活细胞细胞器内的靶向相互作用，该项目 有助于测试由内而外的药理学。实验发明，然后利用，基因编码的荧光 生物传感器来测量细胞器中的药物。生物传感器是细菌和古细菌的周质结合 蛋白质（PBP），与环状排列的绿色荧光蛋白（cp-GFP）融合。子方法A是 基于溶液药物筛选x现有生物传感器。92种化合物的库包括许多口服 现有的药物批准用于各种适应症，但强调精神病学。收集60个纯化的 生物传感器蛋白质包括五个现有的家族，它们现在感测谷氨酸、多巴胺、GABA和 肾上腺素能药物。子方法B利用"定向进化"来改进"命中率"，以实现 检测在合适的亚微摩尔浓度下的药物。主要工具-网站- 饱和诱变、原子尺度结构、计算对接和高穿透荧光 筛选-预计将集中在适当的生物传感器上。子方法C表达了改进的 ER中的生物传感器，并执行活细胞，时间分辨成像，而药物是细胞外应用。我们 开始与简单的问题，"药物进入急诊室，多快？"然后我们分析信号 在细胞器中也表达药物的经典靶点。我们期待着一组丰富的"动力学"数据 通过与ER内的靶点结合来缓冲扩散，从而揭示ER内的药物-受体相互作用 活细胞的细胞器。该子方法然后毕业到小鼠制备，使用病毒载体，脑 切片和完整动物的双光子成像。次级办法D和E互为补充 其他. D将亚细胞药代动力学扩展到酸性细胞器，包括分泌颗粒和 神经递质囊泡已经被怀疑通过“酸捕获”积累药物。我们会保留PBP 部分生物传感器，但采用额外的cp-荧光蛋白，已知在低pH值下发挥作用， 修饰接头。其结果将成为一个荧光生物传感器平台的集合，每个平台专门用于 在一类细胞器中表现最好，并且针对一类细胞器。子方法E扩展了药物生物传感器 新的PBPs类别和新的药物类别的战略。我们将保留cp-荧光蛋白部分， 生物传感器，但优化新的PBPs和连接器。 变革性的整体结果将产生至少10个，多达100个生物传感器来检测 细胞器内的药物，以及作为强大研究工具的亚细胞药代动力学的明确路线图。数据 可以为精神病学、成瘾和神经变性提出变革性的治疗策略。

Drug development for the central nervous system (CNS), especially for psychiatry, has slowed, partially because we do not know the mechanisms by which some drugs exert their therapeutic or harmful effects. The project provides data to test the hypothesis that several CNS drugs act, in addition to their acute effects, in a slower, “inside-out” fashion. The drugs would start by binding to their classical molecular targets, but in organelles. By measuring neural drugs, and their target interactions, within organelles of living cells, this project helps to test inside-out pharmacology. The experiments invent, then exploit, genetically encoded fluorescent biosensors to measure drugs in organelles. The biosensors are bacterial and archaeal periplasmic binding proteins (PBPs), fused to circularly permuted green fluorescent protein (cp-GFP). Sub-Approach A is a solution-based screen of drugs x existing biosensors. The library of 92 compounds includes many orally available drugs approved for various indications, but emphasizing psychiatry. The collection of 60 purified biosensor proteins comprises five existing families, which now sense glutamate, dopamine, GABA, and serotonergic drugs. Sub-Approach B utilizes “directed evolution” to improve the “hits”, toward the goal of detecting the drugs at pharmacologically appropriate sub-micromolar concentrations. The major tools—site- saturation mutagenesis, atomic-scale structure, computational docking, and high-through fluorescence screening--are expected to converge on appropriate biosensors. Sub-Approach C expresses the refined biosensors in ER and performs live-cell, time-resolved imaging while the drugs are applied extracellularly. We begin with the simple questions, “does the drug enter the ER, and how quickly?” We then analyze signals within organelles that also express the classical targets for the drugs. We expect a rich set of data on “kinetic buffering” of diffusion by binding to the targets within ER, thus revealing drug-receptor interaction within organelles of live cells. The sub-approach then graduates to mouse preparations, using viral vectors, brain slices, and two-photon imaging in intact animals will be employed. Sub-Approaches D and E complement each other. D extends subcellular pharmacokinetics to acidic organelles, including secretory granules and neurotransmitter vesicles already suspected of accumulating drugs via “acid trapping”. We'll retain the PBP portions of the biosensors, but employ additional cp-fluorescent proteins, known to function at low pH, and also modify linkers. The result will become a collection of fluorescent biosensor platforms, each specialized to perform best within, and targeted to, a class of organelles. Sub-approach E extends the drug biosensor strategy to new classes of PBPs, and to new classes of drugs. We will retain the cp-fluorescent protein part of the biosensors, but optimize the new PBPs and linkers. The transformative overall results will produce at least ten, and as many as 100, biosensors to detect drugs within organelles, and a clear roadmap for subcellular pharmacokinetics as a robust research tool. Data could suggest transformative therapeutic strategies for psychiatry, addiction, and neurodegeneration.