Small Molecule Inhibitors of FGF22-Mediated Excitatory Synaptogenesis & Epilepsy

FGF22 介导的兴奋性突触发生的小分子抑制剂

• 批准号: 8325818
• 负责人: Hisashi Umemori
• 金额: $ 3.89万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8325818
• 关键词: Binding; Biological Assay; Brain; Cell Differentiation process; Cells; Chemicals; Collection; Data; Development; Epilepsy; Epileptogenesis; Excitatory Synapse; FGF7 gene; Family; Fibroblast Growth Factor; Fluorescent Dyes; Future; Genetic; Hippocampus (Brain); Human; Inhibitory Synapse; Institutes; Label; Lead; Libraries; Ligand Binding; Ligands; Measures; Mediating; Molecular Bank; Mus; Nature; Nerve; Neurons; Pharmacological Treatment; Prevention; Prevention strategy; Production; Proteins; Public Health; Relative (related person); Research; Resistance; Role; Structure-Activity Relationship; Surface Plasmon Resonance; Synapses; Testing; Therapeutic; Thermodynamics; Toxic effect; base; bindin; cytotoxicity; cytotoxicity test; design; effective therapy; follow-up; high throughput screening; improved; inhibitor/antagonist; member; mutant; nervous system disorder; neurotoxicity; novel; novel strategies; prevent; repository; small molecule; synaptogenesis; therapeutic target; treatment strategy

DESCRIPTION (provided by applicant): A critical step for the proper functioning of the brain is the differential formation of excitatory and inhibitory synapses. An imbalance in these synapses leads to various neurological disorders including epilepsy. We have recently found that two members of the fibroblast growth factor (FGF) family, FGF22 and FGF7, organize excitatory and inhibitory synapses, respectively, in the brain (Terauchi et al., Nature, 2010). The differentiatio of excitatory or inhibitory nerve terminals is specifically impaired in mutants lacking FGF22 or FGF7. Remarkably, FGF22-deficient mice are resistant to epileptic seizures, and FGF7-deficient mice are prone to them. These results indicate that the identification of small molecules that can inhibit FGF22-mediated excitatory synapse formation or those that can activate FGF7-mediated inhibitory synapse formation may lead to novel treatment strategies for epilepsy. Here, we will focus on the identification of inhibitors for FGF22, the synaptic organizer that promotes excitatory synapse formation in the brain. Since FGF22 and FGF7 are selectively involved in excitatory or inhibitory synapse formation, respectively, our strategy is to screen small molecules that bind to FGF22 but not to FGF7 by the binding assay and then identify inhibitors of FGF22- mediated excitatory synapse formation by the synapse formation assay. Here, we propose to collaborate with the Molecular Libraries Probe Production Centers Network (MLPCN) to employ a novel label-free thermal shift assay to identify small molecule binders to FGF22. Following the primary screen, we will retest the FGF22 binding and perform a counter screen with FGF7 to identify compounds that bind to FGF22 but not to FGF7. We will then determine the functional effects of hit compounds on FGF22-mediated excitatory synapse formation in primary neurons to identify cell-active inhibitors of FGF22. Finally, we will confirm direct bindin to FGF22 and determine the Kd using surface plasmon resonance (SPR). Data from these assays will be used to inform structure-activity relationship studies for probe optimization. To evaluate our screen, we have performed a pilot screen of 35,000 compounds. The primary FGF22- thermal shift assay identified 278 FGF22 binders. They were retested for FGF22 binding and counter screened for FGF7 binding, and 13 compounds were identified that showed binding to FGF22 but not to FGF7. They were then tested for cytotoxicity and effects on FGF-mediated synapse formation by the cell-based assay, resulting in the identification of 2 compounds that specifically inhibit FGF22-dependent excitatory synapse formation without neurotoxicity. These preliminary data strongly support our aims to screen the entire Molecular Libraries Small Molecule Repository (MLSMR) collection (~365,000 compounds) and promise successful identification of specific inhibitors of FGF22-mediated excitatory synapse formation. Identified FGF22 inhibitors will be assessed for their suitability as therapeutics for epilepsy in future studies. Thus, our study will suggest novel strategies for treating epilepsy and will have significant impact on public health.

描述(申请人提供)：大脑正常运作的关键一步是兴奋性和抑制性突触的不同形成。这些突触的不平衡会导致包括癫痫在内的各种神经疾病。我们最近发现成纤维细胞生长因子家族中的两个成员，FGF22和FGF7，分别在大脑中组织兴奋性和抑制性突触(Teruchi等人，自然，2010年)。在缺乏FGF22或FGF7的突变体中，兴奋性或抑制性神经末梢的分化尤其受损。值得注意的是，FGF22基因缺陷的小鼠对癫痫发作具有抵抗力，而FGF7基因缺陷的小鼠更容易出现癫痫发作。这些结果表明，识别能够抑制FGF22介导的兴奋性突触形成的小分子或能够激活FGF7介导的抑制性突触形成的小分子可能会导致癫痫的新的治疗策略。在这里，我们将集中于FGF22的抑制物的识别，FGF22是促进大脑中兴奋性突触形成的突触组织者。由于FGF22和FGF7分别选择性地参与兴奋性或抑制性突触的形成，我们的策略是通过结合实验筛选与FGF22结合而不与FGF7结合的小分子，然后通过突触形成实验确定FGF22介导的兴奋性突触形成的抑制剂。在这里，我们建议与分子文库探针生产中心网络(MLPCN)合作，使用一种新的无标记热位移分析来鉴定FGF22的小分子结合。在第一次筛选之后，我们将重新测试FGF22结合，并用Fgf7进行计数器筛选，以确定与FGF22结合但不与Fgf7结合的化合物。然后，我们将确定HIT化合物对FGF22介导的原代神经元兴奋性突触形成的功能影响，以确定FGF22的细胞活性抑制剂。最后，我们将确认FGF22的直接结合，并使用表面等离子体共振(SPR)确定Kd。来自这些检测的数据将被用于为探针优化的结构-活性关系研究提供信息。为了评估我们的筛选，我们对35,000种化合物进行了试点筛选。初步的FGF22-热位移实验鉴定出278个FGF22结合子。对它们进行了FGF22结合的重新测试和Fgf7结合的反筛选，鉴定出13个化合物显示与FGF22结合，但不与Fgf7结合。然后通过基于细胞的分析测试它们的细胞毒性和对成纤维细胞生长因子介导的突触形成的影响，结果鉴定出两种化合物，它们特异性地抑制FGF22依赖的兴奋性突触形成，而没有神经毒性。这些初步数据有力地支持了我们的目标，即筛选整个分子库小分子储存库(MLSMR)集合(约365,000种化合物)，并有望成功识别FGF22介导的兴奋性突触形成的特定抑制剂。已确定的FGF22抑制剂将在未来的研究中评估它们作为癫痫治疗药物的适宜性。因此，我们的研究将提出治疗癫痫的新策略，并将对公众健康产生重大影响。