Fluorescent biosensors for subcellular pharmacokinetics

用于亚细胞药代动力学的荧光生物传感器

• 批准号: 9353864
• 负责人: Henry A. Lester
• 金额: $ 83.12万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-16 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9353864
• 关键词: Acids; Acute; Address; Adverse effects; Affinity; Animals; Antidepressive Agents; Antipsychotic Agents; Arrestins; Binding; Biosensor; Blood; Brain; Brain Diseases; Buffers; Cell membrane; Cells; Cellular biology; Central Nervous System Agents; Cholinergic Agents; Chronic; Collection; Complement; Data; Data Set; Dendrites; Development; Diffusion; Directed Molecular Evolution; Discipline; Docking; Dopamine; Drug Kinetics; Drug Receptors; Drug Targeting; Drug effect disorder; Endoplasmic Reticulum; Event; Family; Fluorescence; G-Protein-Coupled Receptors; Glutamates; Goals; Green Fluorescent Proteins; Image; Ion Channel; Ions; Kinetics; Knowledge; Libraries; Ligands; Liver; Major Depressive Disorder; Measures; Mediating; Mental disorders; Metabolic Biotransformation; Molecular Chaperones; Molecular Target; Mus; Muscle; Mutagenesis; Nerve Degeneration; Neuraxis; Neurosciences; Neurotransmitters; Nicotine; Nicotinic Receptors; Oral; Organelles; Periplasmic Binding Proteins; Pharmaceutical Preparations; Pharmacology; Preclinical Drug Evaluation; Preparation; Proteins; Psychiatry; Research; Research Personnel; Schizophrenia; Second Messenger Systems; Secretory Vesicles; Serotonin Agents; Signal Transduction; Signal Transduction Pathway; Site; Slice; Structure; Suggestion; Testing; Therapeutic; Therapeutic Effect; Time; Tissues; Vesicle; Viral Vector; World Health Organization; addiction; base; burden of illness; disability-adjusted life years; drug development; experience; experimental study; extracellular; gamma-Aminobutyric Acid; high reward; high risk; improved; innovation; neuronal cell body; neuropsychiatric disorder; novel drug class; novel therapeutics; receptor binding; receptor upregulation; relating to nervous system; screening; small molecule; success; tool; trafficking; two-photon; uptake

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.

中枢神经系统（CNS）的药物开发，尤其是精神病学药物的开发已经部分放缓 因为我们不知道某些药物发挥治疗或有害作用的机制。这 项目提供数据来检验以下假设：几种中枢神经系统药物除了其急性作用外，还具有以下作用： 较慢的、“由内而外”的时尚。这些药物首先会与它们的经典分子靶标结合，但在 细胞器。 通过测量活细胞细胞器内的神经药物及其目标相互作用，该项目 有助于测试由内而外的药理学。这些实验发明并利用了基因编码的荧光 测量细胞器中药物的生物传感器。生物传感器是细菌和古菌周质结合 蛋白质 (PBP)，与循环排列的绿色荧光蛋白 (cp-GFP) 融合。子方法 A 是 基于溶液的药物筛选 x 现有生物传感器。 92 种化合物库包括许多口服化合物 批准用于各种适应症的可用药物，但强调精神病学。 60个纯化的集合 生物传感器蛋白由五个现有家族组成，现在可感知谷氨酸、多巴胺、GABA 和 血清素能药物。子方法B利用“定向进化”来提高“命中率”，以达到 检测药理学适当的亚微摩尔浓度的药物。主要工具—网站— 饱和诱变、原子尺度结构、计算对接和高通量荧光 筛选——预计将集中在适当的生物传感器上。子方法C表达了精炼的 ER 中的生物传感器并在药物应用于细胞外时进行活细胞、时间分辨成像。我们 从简单的问题开始，“药物是否进入急诊室，多快？”然后我们分析信号 细胞器内也表达了药物的经典靶标。我们期望获得有关“动力学”的丰富数据 通过与内质网内的靶标结合来缓冲“扩散”，从而揭示内质网内的药物-受体相互作用 活细胞的细胞器。然后，子方法逐渐发展到小鼠制剂，使用病毒载体、大脑 将采用完整动物的切片和双光子成像。子方法 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 神经递质囊泡已被怀疑通过“酸捕获”积聚药物。我们将保留 PBP 生物传感器的一部分，但采用了额外的 cp 荧光蛋白，已知在低 pH 下起作用，并且还 修改链接器。结果将成为荧光生物传感器平台的集合，每个平台都专门用于 在一类细胞器内并针对一类细胞器表现最佳。子方法E扩展了药物生物传感器 新类别 PBP 和新类别药物的策略。我们将保留 cp 荧光蛋白部分 生物传感器，但优化新的 PBP 和连接器。 变革性的总体结果将产生至少 10 个、最多 100 个生物传感器来检测 细胞器内的药物，以及亚细胞药代动力学作为强大研究工具的清晰路线图。数据 可以为精神病学、成瘾和神经退行性疾病提出变革性的治疗策略。