Mammalian diacylglycerol kinase (DGK) represents a large enzyme family (Goto et al., 2006; Sakane et al., 2007; Mérida et al., 2008; Topham and Epand, 2009). To date, ten mammalian DGK isozymes, α (Sakane et al., 1990; Schaap et al., 1990), β (Goto and Kondo, 1993), γ (Goto et al., 1994; Kai et al., 1994), δ (Sakane et al., 1996), ε (Tang et al., 1996), ζ (Bunting et al., 1996; Goto and Kondo, 1996), η (Klauck et al., 1996), θ (Houssa et al., 1997), ι (Ding et al., 1998), and κ (Imai et al., 2005), have been identified. Moreover, several alternative splicing products—such as δ1 and δ2 (Sakane et al., 2002); η1–η3 (Murakami et al., 2003; Shionoya et al., 2015); ζ1 and ζ2 (Ding et al., 1997), and ι1–ι3 (Ito et al., 2004)—have also been found. These isozymes contain two or three characteristic protein kinase C (PKC)-like C1 domains (cysteine-rich, zinc finger structures) and the catalytic region in common and are subdivided into five groups, type I (α, β and γ), II (δ, η and κ), III (ε), IV (ζ and ι), and V (θ), according to their structural features (Goto et al., 2006; Sakane et al., 2007; Mérida et al., 2008; Topham and Epand, 2009). Each group is characterized by the subtype-specific functional domains, such as EF-hand motifs (type I), pleckstrin homology and sterile α motif domains (type II), ankyrin repeats (type IV), and ras-associating and pleckstrin homology domains (type V).

Our previous review (Sakane et al., 2008) showed that many interesting studies on DGK have brought DGK to the center stage of diverse biological events such as growth factor/cytokine-dependent cell proliferation and motility, seizure activity, immune responses, cardiovascular responses, and glucose metabolism. Therefore, from a medical point of view, DGK isoforms are implicated in the pathogenesis of a wide variety of diseases, for example, cancer, epilepsy, autoimmunity, cardiac hypertrophy, hypertension, and type II diabetes. Thus, DGKs have emerged as potential and attractive drug targets for curing these diseases.

Recent advances in genotyping technology have allowed for rapid genome-wide screening of common variants in large populations, launching a new era in the investigation of the genetic basis of complex diseases. DGK is no exception. Since our review was published (Sakane et al., 2008), additional interesting reports using genome-wide association studies (GWASs) have successively implied several new possible relationships between DGK isozymes and diseases. For example, DGKη (Baum et al., 2008; Ollila et al., 2009; Squassina et al., 2009; Weber et al., 2011; Zeng et al., 2011), DGKκ (van der Zanden et al., 2011; Carmichael et al., 2013), and DGKθ (Pankratz et al., 2009; Simón-Sánchez et al., 2011) have been suggested to be associated with susceptibility to bipolar disorder (BPD), hypospadias, and Parkinson's disease, respectively.

Among these isozymes, based on the results obtained for the GWASs of DGKH (DGKη gene), we recently investigated the relationship between DGKη and BPD. For this purpose, we generated DGKη-knockout (KO) mice and used these mice to perform behavioral and pharmacological tests. Intriguingly, we found that DGKη-knockout mice showed lithium (BPD remedy)-sensitive mania-like behaviors, suggesting that DGKη is one of key enzymes of the pathogenesis of BPD (Isozaki et al., 2016).

As mentioned in our previous review (Sakane et al., 2008), the development of DGK isozyme-specific inhibitors/activators is important both for fundamental research and for developing therapeutic strategies to treat a wide variety of pathological disorders. However, there was no available DGK isozyme-specific inhibitor/activator until recently. We have recently identified DGKα-selective inhibitors using a newly established high-throughput screening method (Sato et al., 2013). Because DGKα has both pro-tumoral and anti-immunogenic properties, the DGKα-selective inhibitors would simultaneously have anti-tumoral and pro-immunogenic (anti-tumor immunogenic) effects.

This mini review will focus primarily on the two abovementioned topics, recent GWASs and the development of DGK isozyme-specific inhibitors.

DGKα (Sakane et al., 1990; Schaap et al., 1990) is highly expressed in hepatocellular carcinoma and melanoma cells (Yanagisawa et al., 2007; Takeishi et al., 2012). DGKα expression is involved in hepatocellular carcinoma progression and is a positive regulator of the proliferative activity of hepatocellular carcinoma through the Ras/Raf/MEK/ERK pathway (Takeishi et al., 2012). In melanoma cells, DGKα positively regulates tumor necrosis factor-α-dependent nuclear factor-κB (p65) activation via the PKC ζ-mediated Ser311 phosphorylation of p65 (Kai et al., 2009). The growth of colon and breast cancer cell lines was significantly inhibited by DGKα-siRNA and R59949 (Torres-Ayuso et al., 2014). The DGKα/atypical PKC/β1 integrin signaling pathway is essential for matrix invasion of breast carcinoma cells (Rainero et al., 2014). Therefore, the suppression of DGKα activity is expected to inhibit the progression of these cancers. On the other hand, DGKα is abundantly expressed in T lymphocytes, where it facilitates the non-responsive state known as anergy (Olenchock et al., 2006; Zha et al., 2006). Anergy induction in T cells represents the main mechanism by which advanced tumors avoid immune action. Therefore, if a DGKα-selective inhibitor is identified and developed, it would reversely attenuate cancer cell proliferation and simultaneously activate T cell function and can be a dual effective compound.

We started the “Dual effective DGKα-selective inhibitor project” in 2009. To develop highly effective and DGKα-selective inhibitors, a system for high-throughput screening is required; however, the conventional DGK assay is quite laborious and requires technical skill. For example, the conventional assay requires the use of a radioisotope ([γ-³²P]ATP) and the manipulation of thin-layer chromatography with multiple extraction steps. We recently established a simple DGK assay (Sato et al., 2013) that is useful for constructing a high-throughput screening system for detecting DGK inhibitors from chemical compound libraries.

We screened a library containing core 9600 compounds (Drug Discovery Initiative, The University of Tokyo) using a high-throughput chemiluminescence-based assay. We obtained several compounds that inhibited the α-isozyme of DGK. Among the compounds, CU-3, 5-[(2E)-3-(2-furyl)prop-2-enylidene]-3-[(phenylsulfonyl)amino]-2-thioxo-1,3-thiazolidin-4-one was identified as a potent and selective inhibitor against the DGKα (Liu et al., 2016). Compared with commercially available DGK inhibitors, such as R59022 and R59949 (Sato et al., 2013), CU-3 exhibited higher efficiency and selectivity against DGKα. The IC₅₀ value of CU-3 (0.6 μM) was markedly lower than the values of R59022 and R59949 (~25 and 18 μM, respectively; Sato et al., 2013). R59022 and R59949 only semi-selectively inhibited type I, III and V DGKs α, ε, and θ, and type I and II DGKs α, γ, δ, and κ, respectively (Sato et al., 2013). However, the IC₅₀ value of CU-3 for DGKα was at least ~12 times lower than the values for other DGK isozymes. Therefore, this study is the first report of a highly α-isozyme selective inhibitor. The target of CU-3 is the catalytic domain of DGKα, and CU-3 competitively reduced the affinity of DGKα for ATP but not diacylglycerol or phosphatidylserine, strongly suggesting that CU-3 competes with ATP binding.

CU-3 induced apoptosis in HepG2 hepatocellular carcinoma and HeLa cervical cancer cells (Liu et al., 2016). Supporting our results, Torres-Ayuso et al. (Torres-Ayuso et al., 2014) also demonstrated that the growth of colon and breast cancer cell lines was significantly inhibited by DGKα-siRNA and R59949. In addition, Dominguez et al. reported that DGKα-siRNA and R59022 negatively affected the proliferation of glioblastoma, melanoma, breast cancer, and cervical cancer cells (Dominguez et al., 2013). The authors also observed that in marked contrast to cancer cells, R59022 did not weaken the growth of non-cancerous astrocytes and fibroblasts (Dominguez et al., 2013). CU-3 also failed to increase the caspase 3/7 activity of the non-cancer-derived COS-7 cells. These findings suggest that CU-3 selectively induces apoptosis.

In addition to the induction of cancer cell apoptosis, we found that CU-3 promoted IL-2 production, which is one of the indicators of T cell activation. Because inactivation (anergy induction) of T cells is the main mechanism by which advanced tumors to avoid immune action, it is expected that CU-3 is able to activate cancer immunity.

General anti-cancer drugs inhibit the proliferation and function of both cancer and bone marrow cells (Chabner and Roberts, 2005; Pérez-Herrero and Fernández-Medarde, 2015). Therefore, they induce not only the attenuation of cancer cell proliferation but also bone marrow suppression/myelosuppression, which is one of the most commonly observed side-effects of anti-cancer drugs. However, there is no drug that has both pro-tumoral and anti-immunogenic effects. The DGKα-selective inhibitor would simultaneously have anti-tumoral and pro-immunogenic effects (Figure 1). Therefore, in addition to the direct effects on apoptosis induction in cancer cells, CU-3 can indirectly induce the death of cancer cells through activation of the immune system. Moreover, CU-3 can be an effective tool for biological science concerning cancer and immunity.

CU-3 still does not have sufficient isozyme selectivity and efficiency as an excellent inhibitor. Moreover, comprehensive studies where other kinase groups are tested have not been performed. Further refinement of CU-3 and/or identification/development of new candidates using larger chemical compound libraries are required. Finally, our current results encourage us to identify and develop specific inhibitors/activators against every DGK isozyme that can be effective regulators and drugs against a wide variety of physiological events and diseases, although the ten DGK isozymes are highly similar to each other.

All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.