Fluorescent biosensors for subcellular pharmacokinetics

用于中枢神经系统(CNS)的药物开发，特别是用于精神病学的药物开发已经部分放缓 因为我们不知道某些药物发挥治疗或有害作用的机制。这个 项目提供了数据来检验这一假设，即几种中枢神经系统药物除了急性作用外，还在 更慢的，“由内而外”的时尚。这些药物一开始会与它们的经典分子靶点结合，但在 细胞器。 通过测量神经性药物和它们在活细胞细胞器内的靶向相互作用，这个项目 有助于测试由内而外的药理学。这些实验发明，然后利用基因编码的荧光 用于测量细胞器中药物的生物传感器。生物传感器是细菌和古生菌的周质结合。 蛋白质(PBPs)，融合成环状排列的绿色荧光蛋白(cp-GFP)。子方法A是一种 基于溶液的药物筛选x现有生物传感器。该图书馆有92种化合物，其中包括许多口头化合物 已有的药物被批准用于各种适应症，但强调精神病学。60件精品精品的收藏 生物传感器蛋白由现有的五个家族组成，它们现在可以感知谷氨酸、多巴胺、GABA和 5-羟色胺能药物子方法B利用“定向进化”来提高“命中率”，以达到 在药理上合适的亚微摩尔浓度下检测药物。主要工具-站点- 饱和突变、原子级结构、计算对接和高通荧光 筛选--预计将汇聚到适当的生物传感器上。子方法C表示精致的 当药物在细胞外应用时，ER中的生物传感器可以执行活细胞、时间分辨成像。我们 从简单的问题开始，“药物进入急诊室了吗？有多快？”然后我们分析信号 在细胞器中也表达药物的经典靶标。我们预计会有一组丰富的数据来说明 通过与内质网中的靶点结合来缓冲扩散，从而揭示内质网中药物与受体的相互作用 活细胞的细胞器。然后，子方法转向使用病毒载体脑部的小鼠制剂 切片，在完整的动物身上进行双光子成像。子方法D和E相辅相成 其他的。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。我们将保留PBP 部分生物传感器，但使用额外的cp-荧光蛋白，已知在低pH值下起作用，还 修改链接器。结果将成为荧光生物传感器平台的集合，每个平台专门用于 在一类细胞器内表现最好，并以此为目标。子方法E扩展药物生物传感器 战略，以适应新类别的多溴联苯和新的药物类别。我们将保留cp-荧光蛋白部分 生物传感器，但优化了新的多溴联苯和连接物。 变革性的总体结果将产生至少10个，甚至多达100个生物传感器来检测 细胞器内的药物，以及作为强有力的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、成瘾和神经退化提供变革性的治疗策略。

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.