High-Throughput Implementation of the NanoBRET Target Engagement Intracellular Kinase Assay to Reveal Differential Compound Engagement by SIK2/3 Isoforms
High-Throughput Implementation of the NanoBRET Target Engagement Intracellular Kinase Assay to Reveal Differential Compound Engagement by SIK2/3 Isoforms
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DOI：
10.1177/2472555219893277
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发表时间：
2019-12-18
期刊：
影响因子：
3.1
通讯作者：
Symons, Antony
中科院分区：
文献类型：
--
作者：
Jin, Hyun Yong;Tudor, Yanyan;Symons, Antony
在活细胞中实时量化小分子配体的靶标结合(TE)在技术上仍然具有挑战性。在高通量环境中对这种相互作用进行系统的量化，有望在病理生理和生物相关的细胞环境中识别目标特定的、有效的小分子。盐诱导蛋白激酶(SIKs)属于AMP激活蛋白激酶(AMPK)家族的一个亚家族，在人类中由三种亚型(SIK1、SIK2和SIK3)组成。它们调节免疫细胞中促炎和抗炎细胞因子的产生。尽管PAN-SIK抑制剂足以逆转依赖SIK的炎症反应，但与抑制SIK3相关的明显毒性表明，需要异构体特异性抑制才能在可接受的安全范围内实现治疗效果。在这里，我们使用了一种灵敏的能量转移技术--NanoBRET TE细胞内激酶分析，来直接测量分子接近度，并对过度表达SIK2或SIK3的HEK293T细胞的TE进行定量。我们对530种化合物的384孔高通量筛选表明，NanoBRET TE细胞内激酶分析足够灵敏和可靠，可以揭示候选化合物与两种SIK异构体的不同结合，并进一步突显了高通量实施NanoBRET TE细胞内激酶分析用于靶向驱动小分子筛选的可行性。
The real-time quantification of target engagement (TE) by small-molecule ligands in living cells remains technically challenging. Systematic quantification of such interactions in a high-throughput setting holds promise for identification of target-specific, potent small molecules within a pathophysiological and biologically relevant cellular context. The salt-inducible kinases (SIKs) belong to a subfamily of the AMP-activated protein kinase (AMPK) family and are composed of three isoforms in humans (SIK1, SIK2, and SIK3). They modulate the production of pro- and anti-inflammatory cytokines in immune cells. Although pan-SIK inhibitors are sufficient to reverse SIK-dependent inflammatory responses, the apparent toxicity associated with SIK3 inhibition suggests that isoform-specific inhibition is required to realize therapeutic benefit with acceptable safety margins. Here, we used the NanoBRET TE intracellular kinase assay, a sensitive energy transfer technique, to directly measure molecular proximity and quantify TE in HEK293T cells overexpressing SIK2 or SIK3. Our 384-well high-throughput screening of 530 compounds demonstrates that the NanoBRET TE intracellular kinase assay was sensitive and robust enough to reveal differential engagement of candidate compounds with the two SIK isoforms and further highlights the feasibility of high-throughput implementation of NanoBRET TE intracellular kinase assays for target-driven small-molecule screening.