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 (mGluR) 是 C 类 G 蛋白偶联受体，以二聚体的形式发挥作用。 虽然已知 mGluR 会形成同二聚体，但最近的研究表明它们也可以形成异二聚体， 但不是乱七八糟的。因为 mGluR 在大脑中广泛表达并调节兴奋性 和可塑性，它们已成为多种病理学药物靶标的候选者。迄今为止 然而，临床前数据带来的兴奋并没有导致临床上 mGluR 靶向疗法的出现， 尽管有大量可用的配体对这些受体具有良好的选择性。我们最近的工作审查 mGluR2/4 异二聚体提供了一种可能的解释：靶向时非常有效的配体 当同一受体表达为异二聚体时，同二聚体受体通常没有作用 另一个 mGluR。使问题更加复杂的是，观察到的药理反应的这些变化 mGluR2/4 异二聚体不能推广到所有 mGluR 异二聚体，甚至不能推广到所有含有 mGluR2 的 异二聚体。因此，为了了解任何 mGluR 配体如何在大脑中发挥作用，我们必须检查 每个可能的异二聚体对单独的药理学反应。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 都将具有未知的倾向 同二聚体和异二聚体。为了解决这个问题，我们设计了一种新型的二聚体组成控制系统 使用内质网保留序列与正交、分裂内含肽、自切除蛋白配对的组合 序列，这将允许表达已知的近野生型 mGluR 二聚体的纯群体 作品。我们计划生成一个全面的配体与 mGluR 二聚体图谱，不仅用于帮助 解释实验数据，还可以改进针对一系列 mGluR 的治疗策略 病理学。为了实现这些目标，我们将追求以下具体目标： 1、建立和 使用分裂内含肽-ER 保留策略表征标记 mGluR 的完整补充，2，To 采用适应的 CODA-RET 方法来获得平行异二聚体特异性 G 蛋白招募数据， 3、系统评估每个可能的 mGluR 二聚体对的药理学反应 选择性激动剂、竞争性拮抗剂、PAM 和 NAM。