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: 9163507
• 负责人: Henry A. Lester
• 金额: $ 84.59万
• 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/9163507
• 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; Life; 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; 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; gamma-Aminobutyric Acid; high reward; high risk; improved; innovation; neuronal cell body; neuropsychiatric disorder; novel therapeutics; receptor binding; receptor upregulation; relating to nervous system; research study; screening; second messenger; 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.

针对中枢神经系统（CNS）的药物开发，尤其是针对精神病学的药物开发，已经部分放缓