MS Binding Assays for Glycine Transporter 2 (GlyT2) Employing Org25543 as Reporter Ligand.,ChemMedChem

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MS Binding Assays for Glycine Transporter 2 (GlyT2) Employing Org25543 as Reporter Ligand.
ChemMedChem ( IF 3.6 ) Pub Date : 2020-07-30 , DOI: 10.1002/cmdc.202000342
Thomas M Ackermann ₁ , Lars Allmendinger ₁ , Georg Höfner ₁ , Klaus T Wanner ₁

Affiliation

This study describes the first binding assay for glycine transporter 2 (GlyT2) following the concept of MS Binding Assays. The selective GlyT2 inhibitor Org25543 was employed as a reporter ligand and it was quantified with a highly sensitive and rapid LC‐ESI‐MS/MS method. Binding of Org25543 at GlyT2 was characterized in kinetic and saturation experiments with an off‐rate of 7.07×10⁻³ s⁻¹, an on‐rate of 1.01×10⁶ M⁻¹ s⁻¹, and an equilibrium dissociation constant of 7.45 nM. Furthermore, the inhibitory constants of 19 GlyT ligands were determined in competition experiments. The validity of the GlyT2 affinities determined with the binding assay was examined by a comparison with published inhibitory potencies from various functional assays. With the capability for affinity determination towards GlyT2 the developed MS Binding Assays provide the first tool for affinity profiling of potential ligands and it represents a valuable new alternative to functional assays addressing GlyT2.

中文翻译：

使用 Org25543 作为报告配体对甘氨酸转运蛋白 2 (GlyT2) 进行 MS 结合测定。

本研究描述了遵循 MS 结合测定概念的第一个甘氨酸转运蛋白 2 (GlyT2) 结合测定。采用选择性 GlyT2 抑制剂 Org25543 作为报告配体，并通过高灵敏度和快速的 LC-ESI-MS/MS 方法对其进行定量。Org25543 在 GlyT2 上的结合在动力学和饱和实验中进行了表征，解离速率为 7.07×10 ^-3 s ^-1，结合速率为 1.01×10 ⁶ M ^-1 s ^-1，平衡解离常数为 7.45纳米。此外，在竞争实验中测定了 19 个 GlyT 配体的抑制常数。通过与已发表的各种功能测定的抑制效力进行比较，检查了结合测定确定的 GlyT2 亲和力的有效性。凭借对 GlyT2 的亲和力测定能力，开发的 MS 结合测定为潜在配体的亲和力分析提供了第一个工具，它代表了针对 GlyT2 的功能测定的一种有价值的新替代方案。

更新日期：2020-07-30

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