Supplementary Material for: Evaluations of the Selectivities of the Diacylglycerol Kinase Inhibitors R59022 and R59949 Among Diacylglycerol Kinase Isozymes Using a New Non-Radioactive Assay Method

Ten mammalian diacylglycerol kinase (DGK) isozymes (a-γ) have been identified. Recent studies have revealed that DGK isozymes play pivotal roles in a wide variety of pathophysiological functions. Thus, it is important to be able to easily check DGK activity in each pathophysiological event. Moreover, the conventional DGK assay is quite laborious because it requires the use of a radioisotope and thin-layer chromatography including multiple extraction steps. In order to minimize the laborious procedures, we established a non-radioactive, single well, two-step DGK assay system. We demonstrated that, compared to the conventional method, the new assay system has comparable sensitivity and much higher efficiency, and is effective in detecting potential agents with high reliability (Z'-factor = 0.69 ± 0.12; n = 3). Using the newly developed assay, we comprehensively evaluated the DGK isozyme selectivities of commercially available DGK inhibitors, R59022 and R59949, in vitro. We found that among 10 isozymes, R59022 strongly inhibited type I DGKa and moderately attenuated type III DGKe and type V DGKθ, and that R59949 strongly inhibited type I DGK a and γ, and moderately attenuated type II DGK d and γ.

目前已鉴定出10种哺乳动物二酰甘油激酶（diacylglycerol kinase, DGK）同工酶。近年研究表明，DGK同工酶在诸多病理生理过程中发挥关键调控作用。因此，便捷检测各病理生理事件中的DGK活性具有重要意义。此外，传统DGK检测方法操作繁琐：其需使用放射性同位素，并结合包含多步萃取步骤的薄层色谱技术。为简化繁琐的实验流程，本研究建立了一种非放射性、单孔双步DGK检测体系。研究表明，相较于传统检测方法，该新型检测体系灵敏度相当，且效率显著提升，同时可高可靠性地筛选潜在活性物质（Z因子=0.69±0.12；n=3）。利用该新型检测体系，本研究体外全面评估了市售DGK抑制剂R59022与R59949的DGK同工酶选择性。研究发现，在10种同工酶中，R59022可强力抑制I型DGKα，并中度抑制III型DGKε与V型DGKθ；而R59949可强力抑制I型DGKα与DGKγ，并中度抑制II型DGKδ与DGKγ。