Screening for AMPA receptor auxiliary subunit specific modulators
Screening for AMPA receptor auxiliary subunit specific modulators
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DOI：
10.1371/journal.pone.0174742
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发表时间：
2017-03-30
期刊：
影响因子：
3.7
通讯作者：
Nakagawa, Terunaga
中科院分区：
文献类型：
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作者：
Azumaya, Caleigh M.;Days, Emily L.;Nakagawa, Terunaga
AMPA 受体 (AMPAR) 是配体门控离子通道，对突触传递和可塑性至关重要。它们的功能障碍与多种精神和神经疾病有关，从重度抑郁症到肌萎缩侧索硬化症。试图增强或抑制 AMPAR 活性本质上是一种难以平衡有效治疗和使人衰弱的副作用之间的行为。一种新探索的针对中枢神经系统 AMPAR 子集的策略是识别影响特定 AMPAR 辅助亚基复合物的化合物。这利用了已知 AMPAR 辅助亚基的不同时空表达模式，为设计大脑区域选择性化合物提供了手段。在这里，我们报告了一种基于高通量筛选的管道，可以识别对 GluA2-CNIH3 和 GluA2-stargazin 复合物具有选择性的化合物。这些化合物将帮助我们建立不断增长的 AMPAR 辅助亚基特异性抑制剂库，迄今为止，这些抑制剂都针对 TARP gamma-8。我们使用基于细胞的检测结合电压敏感染料 (VSD) 来识别谷氨酸门控阳离子流过共表达 GluA2 和辅助亚基的 HEK 细胞膜的变化。然后，我们使用钙流测定来进一步验证从 VSD 测定中挑选的命中。 VU0612951 和 VU0627849 是初始筛选的候选化合物，分别被鉴定为负变构调节剂和正变构调节剂（NAM 和 PAM）。它们对含有 stargazin 和 CNIH3 的复合物的 IC50/EC(50) 均低于单独的 GSG1L 或 AMPAR。我们还鉴定了一种候选化合物 VU0539491，它在 GIuA2(R)-CNIH3 和 GIuA2(Q) 复合物中具有 NAM 活性，在 GluA2(Q)GSG1L 复合物中具有 PAM 活性。
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 GIuA2-CNIH3 and GIuA2-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 gamma-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 GIuA2 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/EC(50)s 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 GIuA2(R)-CNIH3 and GIuA2(Q) complexes and PAM activity in GluA2(Q)GSG1L complexes.