Shalaly Nancy Dekki, Aneiros Eduardo, Blank Michael, Mueller Johan, Nyman Eva, Blind Michael, Dabrowski Michael A, Andersson Christin V, Sandberg Kristian
Department of Neuroscience, AstraZeneca R&D, Södertälje, Sweden and Division of Protein Technology, School of Biotechnology, Royal Institute of Technology, Stockholm, Sweden
Department of Neuroscience, AstraZeneca R&D, Södertälje, Sweden and Lead Discovery Technologies, Merck Serono SA, Geneva, Switzerland.
J Biomol Screen. 2015 Oct;20(9):1112-23. doi: 10.1177/1087057115590575. Epub 2015 Jun 12.
According to the gate control theory of pain, the glycine receptors (GlyRs) are putative targets for development of therapeutic analgesics. A possible approach for novel analgesics is to develop a positive modulator of the glycine-activated Cl(-) channels. Unfortunately, there has been limited success in developing drug-like small molecules to study the impact of agonists or positive modulators on GlyRs. Eight RNA aptamers with low nanomolar affinity to GlyRα1 were generated, and their pharmacological properties analyzed. Cytochemistry using fluorescein-labeled aptamers demonstrated GlyRα1-dependent binding to the plasma membrane but also intracellular binding. Using a fluorescent membrane potential assay, we could identify five aptamers to be positive modulators. The positive modulation of one of the aptamers was confirmed by patch-clamp electrophysiology on L(tk) cells expressing GlyRα1 and/or GlyRα1β. This aptamer potentiated whole-cell Cl(-) currents in the presence of low concentrations of glycine. To our knowledge, this is the first demonstration ever of RNA aptamers acting as positive modulators for an ion channel. We believe that these aptamers are unique and valuable tools for further studies of GlyR biology and possibly also as tools for assay development in identifying small-molecule agonists and positive modulators.
根据疼痛的闸门控制理论，甘氨酸受体（GlyRs）是开发治疗性镇痛药的潜在靶点。开发新型镇痛药的一种可能方法是开发甘氨酸激活的Cl(-)通道的正性调节剂。不幸的是，在开发类似药物的小分子以研究激动剂或正性调节剂对GlyRs的影响方面，取得的成功有限。我们生成了8种对GlyRα1具有低纳摩尔亲和力的RNA适配体，并分析了它们的药理学特性。使用荧光素标记的适配体进行细胞化学分析表明，GlyRα1不仅依赖于质膜结合，还存在细胞内结合。使用荧光膜电位测定法，我们鉴定出5种适配体为正性调节剂。在表达GlyRα1和/或GlyRα1β的L(tk)细胞上，通过膜片钳电生理学证实了其中一种适配体的正性调节作用。在低浓度甘氨酸存在下，这种适配体增强了全细胞Cl(-)电流。据我们所知，这是首次证明RNA适配体可作为离子通道的正性调节剂。我们相信，这些适配体是进一步研究GlyR生物学的独特且有价值的工具，也可能作为鉴定小分子激动剂和正性调节剂的检测方法开发工具。
