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.

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to generate phosphatidic acid ( 1-6 ). To date, 10 mammalian DGK isozymes ( ␣ , ␤ , ␥ , ␦ , , , , , , and ) have been identifi ed. These DGK isozymes are divided into fi ve groups (type I-V) according to their structural features ( 1-6 ). Type I DGK isozymes (DGKs ␣ , ␤ , and ␥ ) commonly contain tandem repeats of two EFhand motif domains and are classifi ed as members of the EF-hand family of Ca 2+ binding proteins. In addition to the Ca 2+ binding EF-hand motifs, all type I DGK isozymes contain an N-terminal recoverin homology domain, two cysteine-rich C1 domains, and the C-terminal catalytic region ( 1-6 ).DGK ␣ ( 7, 8 ) is highly expressed in hepatocellular carcinoma and melanoma cells ( 9, 10 ). DGK ␣ expression is involved in hepatocellular carcinoma progression and is a Abstract Diacylglycerol kinase (DGK) consists of 10 isozymes. The ␣ -isozyme enhances the proliferation of cancer cells. However, DGK ␣ facilitates the nonresponsive state of immunity known as T-cell anergy; therefore, DGK ␣ enhances malignant traits and suppresses immune surveillance. The aim of this study was to identify a novel small molecule that selectively and potently inhibits DGK ␣ activity. We screened a library containing 9,600 chemical compounds using a newly established high-throughput DGK assay. Abbreviations: Con A, concanavalin A; CU-3, 5-[(2E)-3-(2-furyl) prop-2-enylidene]-3-[(phenylsulfonyl)amino]2-thioxo-1,3-thiazolidin-4-one; DG, diacylglycerol; DGK, diacylglycerol kinase; HTS, high-through put screening; IL-2, interleukin-2; PA, phosphatidic acid; PS, phosphatidylserine ; R59022, 6-(2-{4-[(4-fluorophenyl)phenylmethylene]1-piperidinyl}ethyl)-7-methyl-5 H -thiazolo-(3,2-a )pyrimidin-5-one; R59949, 3-(2-{4-[bis-(4-fl uorophenyl)methylene]1-piperidinyl}ethyl)-2,3-dihydro-2-thioxo-4(1 H )quinazolinone. This work was supported by Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. This work was also supported by MEXT/JSPS KAKENHI Grant Numbers 22370047 [Grant-in-Aid for Scientifi c Research (B)], 23116505 (Grant-in-Aid for Scientifi c Research on Innovative Areas), 25116704 (Grant-in-Aid for Scientifi c Research on Innovative Areas), 26291017 [Grant-in-Aid for Scientifi c Research (B)], and 15K14470 (Grant