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.
Excitatory synaptic imbalance may lead to various neurological disorders including epilepsy. The proposed research is aimed at identifying small molecules that can control excitatory synapse formation in the brain - we will focus on excitatory synapse formation mediated by fibroblast growth factor 22 (FGF22) and identify FGF22 inhibitors. Since genetic inactivation of FGF22 prevents epilepsy in mice, identification of FGF22 inhibitors will help design strategies for the treatment and prevention of human epilepsy.