Novel nAChR-Targeted Peptides

新型 nAChR 靶向肽

• 批准号: 8183568
• 负责人: J MICHAEL MCINTOSH
• 金额: $ 51.96万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-30 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8183568
• 关键词: Absence of pain sensation; Acetylcholine; Affinity; Agonist; Analgesics; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Assay; Biological Factors; Cells; Chronic; Cloning; Collection; Communities; Complex; Conotoxin; Conus genus; Development; Disease; Diversity Library; Dopamine; Drosophila acetylcholine receptor alpha-subunit; Gated Ion Channel; Generations; Genes; Health; Immune; Knock-out; Knockout Mice; Knowledge; Laboratories; Letters; Ligands; Lymphocyte; Marines; Minority; Modeling; Mood Disorders; Mus; Mutagenesis; Mutant Strains Mice; Nervous system structure; Neurocognitive; Neurotransmitters; Nicotine Dependence; Nicotinic Receptors; Norepinephrine; Pain; Parkinson Disease; Peptides; Process; Property; Resource Sharing; Resources; Retinal Cone; Roentgen Rays; Role; Series; Snails; Source; Structure; Techniques; Testing; Therapeutic Agents; Tissues; Toxin; Variant; Venoms; Vertebrates; Work; addiction; alpha-Conotoxin; analog; base; constriction; desensitization; extracellular; gene cloning; insight; macrophage; meetings; mutant; nerve injury; neuropsychiatry; next generation; nicotinic receptor alpha4beta2; novel; novel strategies; pressure; protein aminoacid sequence; public health relevance; receptor; receptor binding; small molecule; stoichiometry

DESCRIPTION (provided by applicant): The ability to selectively manipulate the function of nicotinic acetylcholine receptors (nAChRs) will have wide ranging impact on the ability to understand the role of these receptors normal and pathological states. On the other hand, probes with which to characterize nAChRs are very limited. We have recently developed defining antagonists (alpha-conotoxins) for nAChRs. The project will exploit these recently developed probes to assess nAChR function and structure (Aims 1 & 2). In separate, but complementary aims we will develop new antagonists for critical nAChR subtypes for which there are no selective ligands. Specifically we propose to: Aim 1) Test the hypothesis that blocking alpha9alpha10 nAChRs is mechanistically responsible for analgesia and/or reduction of immune cell accumulation associated with nerve injury. Verification would establish a novel approach for the treatment of nerve injury and associated pain. We have developed a panel of subtype selective alpha-conotoxins that will be used together with receptor subunit knockout mice to achieve this aim. Aim 2) Use toxin-receptor mutagenesis studies to elucidate the critical toxin/receptor interactions that underlie subtype selectivity. nAChR subtypes containing closely related alpha6- and alpha4-subunits presynaptically modulate the release of dopamine and norepinephrine that are implicated in addiction, Parkinson's disease and mood disorders. Existing small molecules cannot distinguish well among these subunits. In contrast, an existing set of alpha-conotoxins (different than those in "Aim 1" above) are the most selective ligands known. We will use these toxins to provide mechanistic insight into selective action at alpha6- vs. alpha4-containing subunit interfaces. Aims 3-4) Develop and characterize new antagonists of nAChRs from Conus. Currently, selective ligands are only available for a minority of known nAChR subtypes, severely limiting our understanding. We will develop antagonists of nAChR subtypes for which there are no selective antagonists; we will focus on alpha4betaalpha5, alpha3beta4alpha5, alpha6beta4 and alternate stoichiometries of alpha4beta nAChRs, subtypes believed important in addiction and neuropsychiatric disorders. Conus is the richest natural source of nAChR antagonists that have been refined through 50 million years of evolutionary pressure. Development of this cornucopia will be achieved through biochemical characterization of venom compounds (Aim 3) as well as a gene cloning and iterative synthesis approach (Aim 4). Hundreds of alpha-conotoxins genes have been cloned and the encoded peptides will be synthesized. These peptides will then be fully characterized with respect to receptor subtype selectivity. Subsequently, synthetic strategies will be utilized to develop second-generation ligands with refined selectivity. Development of these antagonists will enable us to functionally knock out the critical nAChR subtypes including those that contain alpha4, alpha5 and alpha6 subunits. This will not only provide us with a platform for further mechanistic insight into nAChR function, but also will provide the wider scientific community probes to meet vital, but currently unfulfilled, needs (see Letters of Support, Significance, Progress, and Resource Sharing).

描述（由申请人提供）：选择性操纵烟碱乙酰胆碱受体（nAChR）功能的能力将对理解这些受体在正常和病理状态下的作用的能力产生广泛影响。另一方面，用于表征nAChR的探针非常有限。我们最近开发了nAChR的定义拮抗剂（α-芋螺毒素）。该项目将利用这些最近开发的探针来评估nAChR的功能和结构（目标1和2）。在单独的，但互补的目标，我们将开发新的拮抗剂的关键nAChR亚型，没有选择性的配体。具体而言，我们建议：目的1）测试阻断α 9 α 10 nAChR在机制上负责镇痛和/或减少与神经损伤相关的免疫细胞积累的假设。验证将建立一种治疗神经损伤和相关疼痛的新方法。我们已经开发了一组亚型选择性α-芋螺毒素，将与受体亚基敲除小鼠一起使用，以实现这一目标。目的2）利用毒素-受体诱变研究阐明亚型选择性的关键毒素/受体相互作用。nAChR亚型含有密切相关的α 6-和α 4-亚基，它们在突触前调节多巴胺和去甲肾上腺素的释放，而多巴胺和去甲肾上腺素与成瘾、帕金森病和情绪障碍有关。现有的小分子不能很好地区分这些亚基。相比之下，现有的α-芋螺毒素组（不同于上述“目标1”中的那些）是已知的最具选择性的配体。我们将使用这些毒素提供机制的见解，在α 6-与α 4-含有亚基接口的选择性行动。目的3-4）开发和表征来自芋螺的nAChR的新拮抗剂。目前，选择性配体仅可用于少数已知的nAChR亚型，严重限制了我们的理解。我们将开发无选择性拮抗剂的nAChR亚型拮抗剂;我们将重点关注α 4 β α 5、α 3 β 4 α 5、α 6 β 4和α 4 β nAChR的替代化学计量，这些亚型被认为在成瘾和神经精神疾病中很重要。芋螺是nAChR拮抗剂最丰富的天然来源，经过5000万年的进化压力已经得到了完善。这个聚宝盆的发展将通过毒液化合物的生物化学表征（目标3）以及基因克隆和迭代合成方法（目标4）来实现。已经克隆了数百个α-芋螺毒素基因，并将合成编码肽。然后，这些肽将在受体亚型选择性方面进行充分表征。随后，将利用合成策略开发具有精细选择性的第二代配体。这些拮抗剂的开发将使我们能够功能性敲除关键的nAChR亚型，包括含有α 4，α 5和α 6亚基的那些。这不仅将为我们提供一个平台，进一步深入了解nAChR功能的机制，而且还将提供更广泛的科学界探索，以满足重要的，但目前尚未满足的需求（见支持信，意义，进展和资源共享）。