Screening for AMPA receptor auxiliary subunit specific modulators

AMPA receptors (AMPAR) are ligand gated ion channels critical for synaptic transmission and plasticity. Their dysfunction is implicated in a variety of psychiatric and neurological diseases ranging from major depressive disorder to amyotrophic lateral sclerosis. Attempting to potentiate or depress AMPAR activity is an inherently difficult balancing act between effective treatments and debilitating side effects. A newly explored strategy to target subsets of AMPARs in the central nervous system is to identify compounds that affect specific AMPAR-auxiliary subunit complexes. This exploits diverse spatio-temporal expression patterns of known AMPAR auxiliary subunits, providing means for designing brain region-selective compounds. Here we report a high-throughput screening-based pipeline that can identify compounds that are selective for GluA2-CNIH3 and GluA2-stargazin complexes. These compounds will help us build upon the growing library of AMPAR-auxiliary subunit specific inhibitors, which have thus far all been targeted to TARP γ-8. We used a cell-based assay combined with a voltage-sensitive dye (VSD) to identify changes in glutamate-gated cation flow across the membranes of HEK cells co-expressing GluA2 and an auxiliary subunit. We then used a calcium flux assay to further validate hits picked from the VSD assay. VU0612951 and VU0627849 are candidate compounds from the initial screen that were identified as negative and positive allosteric modulators (NAM and PAM), respectively. They both have lower IC50/EC50s on complexes containing stargazin and CNIH3 than GSG1L or the AMPAR alone. We have also identified a candidate compound, VU0539491, that has NAM activity in GluA2(R)-CNIH3 and GluA2(Q) complexes and PAM activity in GluA2(Q)-GSG1L complexes.α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体（AMPA receptors, AMPAR）是一类对突触传递与突触可塑性至关重要的配体门控离子通道。其功能异常与多种精神及神经疾病相关，涵盖重度抑郁症至肌萎缩侧索硬化症等多种病症。试图增强或抑制AMPA受体的活性，本质上是一场在有效治疗与致残性副作用之间艰难权衡的博弈。目前一种新兴的中枢神经系统AMPA受体亚群靶向策略，是筛选可作用于特定AMPA受体辅助亚基复合物的化合物。该策略利用已知AMPA受体辅助亚基多样化的时空表达模式，为设计脑区选择性化合物提供了可行路径。本研究报道了一种基于高通量筛选的筛选流程，可筛选出对GluA2-CNIH3与GluA2-stargazin复合物具有选择性的化合物。这类化合物将助力我们扩充AMPA受体辅助亚基特异性抑制剂的现有文库——迄今为止，该类抑制剂均以TARP γ-8为靶点。本研究采用结合电压敏感染料（voltage-sensitive dye, VSD）的细胞水平检测方法，检测共表达GluA2与辅助亚基的HEK细胞膜上谷氨酸门控阳离子流的变化。随后通过钙流检测对电压敏感染料检测中筛选得到的命中化合物进行进一步验证。VU0612951与VU0627849为初始筛选得到的候选化合物，分别被鉴定为负向变构调节剂（negative allosteric modulator, NAM）与正向变构调节剂（positive allosteric modulator, PAM）。相较于GSG1L或单独的AMPA受体，这两种化合物对含有stargazin与CNIH3的复合物的半数抑制浓度（IC50）/半数有效浓度（EC50）均更低。本研究还筛选得到另一候选化合物VU0539491，其在GluA2(R)-CNIH3与GluA2(Q)复合物中表现出负向变构调节活性，而在GluA2(Q)-GSG1L复合物中则表现为正向变构调节活性。