Developing nanobody immune libraries against native neuronal nicotinic receptor complexes

开发针对天然神经元烟碱受体复合物的纳米抗体免疫文库

基本信息

批准号：
10591889
负责人：
Christian Peters
金额：
$ 8万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-05-01 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10591889
关键词：
Acetylcholine Acute Affinity Chromatography Arousal Attention Behavior Behavioral Binding Binding Sites Biological Biological Assay Biological Availability Brain Cells Chronic Complex Dependence Development Diagnostic Discrimination Disease Drug Addiction Drug Exposure Electrophysiology (science)Exhibits Family Fluorescence Future Generations Genes Goals Grant Habenula Health Heterogeneity Human Immune Immunotherapy In Vitro Individual Ion Channel Gating Knock-out Lead Libraries Ligand Binding Ligands Link Magnetism Mammalian Cell Medial Methodology Methods Midbrain structure Molecular Molecular Conformation Molecular Target Nervous System Neurobiology Neurons Neurotransmitters Nicotine Nicotinic Agonists Nicotinic Receptors Output Pathway interactions Pattern Pharmaceutical Preparations Pharmacologic Substance Phase Phenotype Physiological Property Proteins Psychological reinforcement Public Health Reagent Regulation Research Rewards Rodent Signal Transduction System Tegmentum Mesencephali Therapeutic Transgenic Model United States United States National Institutes of Health Ventral Tegmental Area Yeasts addiction cell type cholinergic cholinergic neuron combat counterscreen drug of abuse effectiveness testing human disease improved in vivo innovation interpeduncular nucleus molecular diagnostics nanobodies novel novel strategies protein distribution protein expression receptor receptor-activity-modifying protein screening smoking cessation stoichiometry therapeutic target tool

项目摘要

ABSTRACT Nicotinic acetylcholine receptors are ubiquitous in the brain and underlie regulation of attention and arousal by cholinergic neurons. nAChRs are highly expressed in neurons of the reinforcement and reward pathways, and maladaptive effects on nAChRs resulting from drug exposure contribute to drug addiction. These receptors are crucial in addiction both to nicotine (a direct orthosteric agonist of nAChRs) and to other drugs of abuse resulting from drug induced plastic changes in cholinergic activity in reward centers. A persistent challenge facing any nAChR-targeted pharmaceutical approach against addiction is the broad diversity in assembly and distribution of the brain-expressed forms of the protein. The long-term goal of this project is to develop an accurate and comprehensive methodology to identify and therapeutically target the full family of native nAChR conformations. The short-term goal of this R03 proposal is to demonstrate that a yeast-display nanobody library, screened against two distinct, widely expressed and physiologically critical conformations of nAChRs, will demonstrate the ability to robustly parse two separate stoichiometries of the nAChR in in vitro fluorescence and electrophysiology assays. The rationale is to deliver proof-of-concept that this approach can specifically target individual receptor types, to stimulate further characterization of successfully identified reagents for their possible functionality in modifying nAChR activity, and ultimately to establish the approach as a feasible method to study the native expression and assembly properties of the entire family of native nAChRs. This study includes two related objectives: 1) to develop a nanobody library enriched for yeast-mounted variable domain modules that potently bind purified 42 nAChRs and 2) to establish an effective counter-screening assay to deplete stoichiometrically non-specific nanobodies to purify interactors that selectively target two physiologically relevant, structurally distinct assemblies of 42. This represents an innovative application of the cutting-edge yeast nanobody screening method against a persistent roadblock in understanding the maladaptive changes to nicotinic receptor protein expression and distribution. Development and delivery of a biologic toolbox to study and manipulate all native nAChRs in vivo, as is the overall goal of this research, would represent a significant biomedical advance to investigate and combat addiction and other human ailments associated with the cholinergic nervous system.

抽象的烟碱乙酰胆碱受体在大脑中普遍存在，是注意力和唤醒调节的基础胆碱能神经元在强化和奖赏通路的神经元中高度表达，并且药物暴露对 nAChR 的适应不良影响会导致药物成瘾。对尼古丁（nAChR 的直接正构激动剂）和其他药物滥用成瘾至关重要药物诱导的奖赏中心胆碱能活动的可塑性变化是一个持续的挑战。任何以 nAChR 为靶点的抗成瘾药物方法都面临着组装和组装的广泛多样性。该项目的长期目标是开发一种蛋白质的大脑表达形式的分布。准确和全面的方法来识别和治疗靶向天然 nAChR 全家族该 R03 提案的短期目标是证明酵母展示纳米抗体。文库，针对两种不同的、广泛表达的和生理学关键的 nAChR 构象进行筛选，将展示稳健解析 nAChR 体外荧光的两个独立化学计量的能力其基本原理是提供这种方法可以特异性地进行的概念验证。针对个体受体类型，以刺激成功识别的试剂的进一步表征修改 nAChR 活性的可能功能，并最终将该方法建立为可行的方法研究整个天然 nAChR 家族的天然表达和组装特性的方法。研究包括两个相关目标：1）开发一个富含酵母装载变量的纳米抗体库有效结合纯化的 α4β2 nAChR 的结构域模块和 2) 建立有效的反筛选消耗化学计量非特异性纳米抗体的测定，以纯化选择性靶向两个的相互作用物具有生理相关性、结构独特的 42 组装体，这代表了 42 的创新应用。尖端酵母纳米抗体筛选方法克服了理解酵母纳米抗体的持续障碍烟碱受体蛋白表达和分布的适应不良变化。作为本研究的总体目标，研究和操纵体内所有天然 nAChR 的生物工具箱将研究和对抗成瘾和其他人类疾病的重大生物医学进展与胆碱能神经系统有关。