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

Abstract

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.

A novel diacylglycerol kinase α-selective inhibitor, CU-3, induces cancer cell apoptosis and enhances immune response

“…Therefore, if a DGK ␣ -selective inhibitor is identifi ed and developed, it would reversely attenuate cancer cell proliferation and simultaneously activate T-cell function. There are two commercially available DGK inhibitors, 6-(2-{4-[(4-fl uorophenyl)phenylmethylene]1-piperidinyl}ethyl)-7-methyl-5 H -thiazolo-(3,2-a )pyrimidin-5-one (R59022 ) ( 14,15 ) and 3-(2-{4-[bis-(4-fl uorophenyl) methylene]1-piperidinyl}ethyl)-2,3-dihydro-2-thioxo-4(1 H ) quinazolinone (R59949 ) ( 16,17 ); however, we found that R59022 and R59949 only semiselectively inhibited type I, III, and V DGKs ␣ , , and , and type I and II DGKs ␣ , ␥ , ␦ , and , respectively ( 18 ). Moreover, the IC 50 values of R59022 and R59949 were ‫ف‬ 25 M and 18 M, respectively ( 18 ).…”

“…The expression plasmids, p3xFLAG-CMV-pig DGK ␣ ( 19 ), -rat DGK ␤ ( 19 ), -human DGK ␥ ( 19 ), -human DGK ␦ 1-⌬ SAM ( 20,21 ), -human DGK 1 ( 22 ), -human DGK ( 23 ), -human DGK ( 24 ), -human DGK ( 25 ), -human DGK ( 26 ) and -human DGK ( 27 ) were generated as described ( 18 ). cDNAs of DGK ␣ -⌬ 1-196 and DGK ␣ -⌬ 1-332 ( 28 ) were amplifi ed by PCR and inserted into p3x-FLAG-CMV vector.…”

“…Mobile phase A consisted of chloroform-methanol-ammonia (89:10:1), and mobile phase B S -transferase-fused pig DGK ␣ was expressed in Sf9 insect cells and purifi ed with a glutathione-Sepharose column (GE Healthcare). The DGK assay was performed using the ADP-Glo™ Kinase Assay Kit (Promega, Tokyo, Japan) at 30°C for 2 h as described previously ( 18 ). The chemiluminescence generated in this assay correlates to the amount of ADP generated, which is equivalent to the phosphatidic acid (PA) produced, in the kinase assay, indicating kinase activity.…”

“…For example, the conventional assay requires the use of a radioisotope ([ ␥ -32 P]ATP) and the manipulation of thin-layer chromatography with multiple extraction steps. We recently established a simple DGK assay ( 18 ) that is useful for constructing an HTS system for detecting DGK inhibitors from chemical compound libraries.…”

“…In this study, we screened a library containing 9,600 chemical compounds to fi nd DGK ␣ -specifi c inhibitors using the newly established DGK assay for HTS ( 18 ). As a result, we have obtained a promising compound, 5-[(2E)-3-(2-furyl)prop-2-enylidene]-3-[(phenylsulfonyl)amino]2-thioxo-1,3-thiazolidin-4-one (CU-3), which selectively inhibited DGK ␣ with an IC 50 value of 0.6 M. Moreover, CU-3 simultaneously enhanced the apoptosis of cancer cells and the activation of T cells.…”