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: 8792428
• 负责人: Hisashi Umemori
• 金额: $ 3.22万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 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 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8792428
• 关键词: Small; Molecule; Inhibitors; FGF22; Mediated

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）：大脑正常运作的关键步骤是兴奋性和抑制性突触的不同形成。这些突触的失衡会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 et al., Nature, 2010）。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特别受损。值得注意的是，缺乏fgf22的小鼠对癫痫发作有抵抗力，而缺乏fgf7的小鼠更容易发生癫痫发作。这些结果表明，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治疗策略。在这里，我们将重点关注FGF22抑制剂的鉴定，FGF22是促进大脑兴奋性突触形成的突触组织者。由于FGF22和FGF7分别选择性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结合但不与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，采用一种新的无标记热移测定方法来鉴定FGF22的小分子结合物。在主筛选之后，我们将重新测试FGF22的结合，并使用FGF7进行反筛选，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化合物对原代神经元中FGF22介导的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，我们将确认与FGF22的直接结合，并使用表面等离子体共振（SPR）确定Kd。这些分析的数据将用于为探针优化的结构-活性关系研究提供信息。为了评估我们的筛选，我们对35000种化合物进行了试点筛选。初步的FGF22-热转移试验鉴定出278种FGF22结合物。重新测试了它们与FGF22的结合，并对FGF7的结合进行了反筛选，鉴定出13种化合物与FGF22结合，但与FGF7不结合。然后通过基于细胞的实验测试它们的细胞毒性和对fgf介导的突触形成的影响，最终鉴定出两种化合物，它们特异性地抑制fgf22依赖性兴奋性突触的形成，而没有神经毒性。这些初步数据有力地支持了我们筛选整个Molecular Libraries Small Molecule Repository （MLSMR）收集（约365,000个化合物）的目标，并有望成功鉴定fgf22介导的兴奋性突触形成的特异性抑制剂。已确定的FGF22抑制剂将在未来的研究中评估其作为癫痫治疗药物的适用性。因此，我们的研究将为治疗癫痫提供新的策略，并将对公共卫生产生重大影响。