A novel system for controlling dimeric receptor composition to discover unique heterodimer pharmacology

控制二聚体受体组成的新系统，以发现独特的异二聚体药理学

• 批准号: 10735066
• 负责人: Paul J. Kammermeier
• 金额: $ 41.55万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10735066
• 关键词: Agonist; Amino Acid Sequence; Atlases; Biological Assay; Brain; Cataloging; Cell membrane; Cells; Chimera organism; Cicatrix; Clinic; Complement; Data; Electrophysiology (science); Exhibits; Family; Family member; Fluorescence Resonance Energy Transfer; G-Protein-Coupled Receptors; GTP-Binding Proteins; Glutamates; Goals; Heterodimerization; Homo; Ion Channel; Label; Length; Ligands; Link; Measurement; Measures; Membrane Proteins; Metabotropic Glutamate Receptors; Methods; Molecular Conformation; Mutagens; Neurons; Neurotransmitters; Pathology; Pharmacology; Physiology; Population; Positioning Attribute; Probability; RNA Splicing; Signal Transduction; Surface; System; Tail; Testing; Therapeutic; Titrations; Work; antagonist; design; dimer; druggable target; fluorophore; improved; intein; interest; metabotropic glutamate receptor 2; metabotropic glutamate receptor 4; novel; patch clamp; pharmacologic; pre-clinical; receptor; receptor expression; receptor function; recruit; response; retention rate; targeted treatment; tool

Metabotropic glutamate receptors (mGluRs) are class C G protein coupled receptors that function as dimers. While mGluRs are known to form homodimers, more recent work has shown that they can also heterodimerize, but not promiscuously. Because mGluRs exhibit widespread expression in the brain and regulate excitability and plasticity, they have become candidates as druggable targets for a variety of pathologies. To date however, excitement generated by preclinical data has not resulted in mGluR-targeting therapies in the clinic, despite a wealth of available ligands with good selectivity targeting these receptors. Our recent work examining mGluR2/4 heterodimers provides a possible explanation: ligands that are highly efficacious when targeting homodimeric receptors are often without effect when the same receptor is expressed as a heterodimer with another mGluR. Further complicating matters, these changes in pharmacological responses observed in mGluR2/4 heterodimers are not generalizable to all mGluR heterodimers, or even all mGluR2 containing heterodimers. Thus, to understand how any mGluR ligand will function in the brain, we must examine the pharmacological responses of each possible heterodimer pair in isolation. But this is complicated because every mGluR can also form homodimers, so any pair of expressed mGluR will have an unknown propensity to homo- and heterodimerize. To solve this problem, we have designed a novel dimer composition control system using a combination of ER retention sequences paired with orthogonal, split inteins, self-excising protein sequences, that will allow expression of pure populations of nearly wild type mGluR dimers of known composition. We plan to generate a comprehensive ligand vs. mGluR dimer atlas to be used to not only aid in interpretation of experimental data but also to improve therapeutic strategies targeting mGluRs for a range of pathologies. To accomplish these goals, we will pursue the following Specific Aims: 1, T To build and characterize the full complement of tagged mGluRs using the split intein-ER retention strategy, 2, To employ an adapted CODA-RET approach to obtain parallel heterodimer specific G protein recruitment data, and 3, To systematically assess the pharmacological responses of each probable mGluR dimer pair to selective agonists, competitive antagonists, PAMs and NAMs.

代谢型谷氨酸受体（MGLURS）是C蛋白偶联受体，可作为二聚体。 尽管已知MGLUR会形成同二聚体，但最近的工作表明它们也可以异二聚体化，但 但不是滥交。因为mglurs在大脑中表现出广泛的表达并调节兴奋性 以及可塑性，它们已成为候选人作为多种病理的可药靶标。迄今为止 但是，临床前数据产生的兴奋并未导致诊所中MGLUR靶向疗法， 尽管有大量可用的配体针对这些受体具有良好的选择性。我们最近的工作 MGLUR2/4异二聚体提供了一种可能的解释：靶向时高效的配体 当相同的受体表示为异二聚体时，同二聚体受体通常无效 另一个mglur。进一步使事情变得复杂，在 MGLUR2/4异二聚体不能推广到所有mglur异二聚体，甚至所有含有所有含有所有MGLUR2 异二聚体。因此，要了解任何mglur配体如何在大脑中起作用，我们必须检查 每个可能的异二聚体对的药理反应分离。但这很复杂，因为 每个mglur也可以形成同型二聚体，因此任何一对表达的mglur都会有未知的倾向 同性恋和异二聚体。为了解决这个问题，我们设计了一个新颖的二聚体组成控制系统 结合与正交，分裂的Inteins，自审查蛋白配对的ER保留序列的组合 序列，这将允许表达已知的几乎野生型Mglur二聚体的纯种群 作品。我们计划生成一种全面的配体与Mglur二聚体图集，不仅用于帮助 解释实验数据，同时还要改善针对MGLUR的治疗策略 病理。为了实现这些目标，我们将追求以下具体目标：1，t建立和 使用拆分内部内部保留策略（2）表征标记的mglurs的完整补充 采用改编的尾巴ret方法获得平行异二聚体特异性G蛋白募集数据， 和3，系统地评估每个可能的mglur二聚体对的药理反应 选择性激动剂，竞争性对手，PAM和NAMS。