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

基本信息

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
项目类别：
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

项目摘要

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（由申请人提供）：大脑正确功能的关键步骤是兴奋性和抑制性突触的差异形成。这些突触的失衡导致包括癫痫在内的各种神经系统疾病。我们最近发现，成纤维细胞生长因子（FGF）家族的两个成员FGF22和FGF7分别在大脑中组织兴奋性和抑制性突触（Terauchi等，Nature，Nature，2010）。在缺乏FGF22或FGF7的突变体中，兴奋性或抑制性神经末端的区分特异性受损。值得注意的是，FGF22缺乏的小鼠对癫痫发作具有抗性，而FGF7缺乏的小鼠容易发生。这些结果表明，可以鉴定可以抑制FGF22介导的兴奋性突触形成或可以激活FGF7介导的抑制性突触形成的小分子可能导致癫痫的新治疗策略。在这里，我们将重点介绍FGF22抑制剂的鉴定，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分别选择性地参与了兴奋性或抑制性突触形成，因此我们的策略是筛选与FGF22结合但不与FGF7结合的小分子，而不是通过结合测定法与FGF7结合，然后鉴定FGF22-FGF22-介导的介导的 - 介导的介导的兴奋性兴奋性突触突触形成突触synapse sematesseant。在这里，我们建议与分子文库探测生产中心网络（MLPCN）合作，以采用一种新型的无标签热偏移测定法，以识别FGF22的小分子粘合剂。在主屏幕之后，我们将重新测试FGF22绑定，并使用FGF7执行反屏幕，以识别与FGF22结合但不与FGF7结合的化合物。然后，我们将确定HIT化合物对原代神经元中FGF22介导的兴奋性突触形成的功能作用，以鉴定FGF22的细胞活性抑制剂。最后，我们将确认与FGF22的直接结合，并使用表面等离子体共振（SPR）确定KD。这些测定法的数据将用于为探针优化的结构活性关系研究提供信息。为了评估我们的屏幕，我们执行了35,000种化合物的试验屏幕。主要的FGF22-热移测定法确定了278 FGF22粘合剂。对FGF22结合进行了重新测试，并筛选了FGF7结合，并鉴定出13种与FGF22结合但不与FGF7结合的化合物。然后，通过基于细胞的测定法对它们进行了细胞毒性和对FGF介导的突触形成的影响，从而鉴定出2种特定抑制FGF22依赖性兴奋性突触形成而没有神经毒性的化合物。这些初步数据强烈支持我们旨在筛选整个分子库小分子存储库（MLSMR）收集（〜365,000种化合物），并有望成功鉴定FGF22介导的兴奋性突触形成的特定抑制剂。确定的FGF22抑制剂将被评估为在未来研究中作为癫痫的疗法的适用性。因此，我们的研究将提出治疗癫痫的新策略，并将对公共卫生产生重大影响。