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Background: Recently, it has become clear that drug-target residence time, in addition to affinity, often drives pharmacodynamic activity and disease efficacy in vivo. In the aim of determining the dissociation properties of test compounds, the kinase field has traditionally utilized purified proteins in various competitive probe displacement formats. Using such detection formats, kinetic parameters of drug-target binding can be quantified using analytical approaches described by Motulsky and Mahan. While quantitative, such approaches may fail to translate with cellular efficacy due to reliance on pure proteins or protein fragments. Furthermore, these methods are generally not amenable to high-throughput analysis for exploring intracellular drug-target residence time. Thus, a simple and quantitative method to evaluate the residence time of compounds inside living cells is highly desirable. Method: The NanoBRET™ Target Engagement Intracellular Kinase Assay was employed to investigate the kinetics of target engagement for various test compounds in living cells. This method utilizes BRET (Bioluminescence Resonance Energy Transfer) in cells by molecular proximity of the NanoBRET™ Tracer to the NanoLuc® luciferase-fused kinase. Quantitative determination of both of apparent affinity as well as binding kinetics for unodified test compounds can be achieved by competitive displacement of the Tracer. Full-length BTK fused with the NanoLuc® luciferase was transiently transfected in HEK293 cells and the cells were incubated at 37°C, 5% CO2 overnight. Transfected cells were harvested and suspended in 1 ml of Assay Medium in a conical tube. After incubation with test compound for 2 hours, the cells were washed with Assay Medium to remove unbound compound. Dosing of test compounds was an approximately IC80-90 concentration unless otherwise specified. The cell suspension was dispensed into a 96-well plate and the NanoBRET™ Tracer K-4 or K-5 was added to the wells following the addition of the Nano-Glo® Substrate Solution. BRET was measured repeatedly with the Glomax® Discover Multimode Microplate Reader. Data were fitted to the one-phase association equation or the kinetic equation developed by Malany to obtain the dissociation rate constatns of test compounds. Results: The kinetic constants using tracer K-4 or K-5 were successfully quantitated in live cells using NanoBRET™. Though the kinetic constants as determined by simple kobs fitting using tracer K-4 or K-5 were different, the constants as determined using the Malany approach for each tracer were in gratifying agreement. The results determined using the Malany equation reveal suitable differentiation of residence time for the irreversible and reversible compounds. As expected, longer residence time was observed for the irreversible/covalent inhibitors. Some reversible inhibitors also showed protracted residence time, offering opportunities for durable target inhibition in cells. Conclusion: We propose the method by which the data in the compound wash-out experiments are fitted to the kinetic equation of Malany as a simple and quantitative method to determine intracellular residence time of kinase inhibitors. Citation Format: Takeomi Inoue, Aki Emi, James D Vasta, Matthew B Robers, Yusuke Kawase. A new method to determine drug-target residence time of kinase inhibitors in living cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C085. doi:10.1158/1535-7163.TARG-19-C085

Phospholipase Cϵ (PLCϵ), an effector of Ras and Rap small GTPases, plays a crucial role in inflammation by augmenting proinflammatory cytokine expression. This proinflammatory function of PLCϵ is implicated in its facilitative role in tumor promotion and progression during skin and colorectal carcinogenesis, although their direct link remains to be established. Moreover, the molecular mechanism underlying these functions of PLCϵ remains unknown except that PKD works downstream of PLCϵ. Here we show by employing the colitis-induced colorectal carcinogenesis model, where Apc(Min) (/+) mice are administered with dextran sulfate sodium, that PLCϵ knock-out alleviates the colitis and suppresses the following tumorigenesis concomitant with marked attenuation of proinflammatory cytokine expression. In human colon epithelial Caco2 cells, TNF-α induces sustained expression of proinflammatory molecules and sustained activation of nuclear factor-κB (NF-κB) and PKD, the late phases of which are suppressed by not only siRNA-mediated PLCϵ knockdown but also treatment with a lysophosphatidic acid (LPA) receptor antagonist. Also, LPA stimulation induces these events in an early time course, suggesting that LPA mediates TNF-α signaling in an autocrine manner. Moreover, PLCϵ knockdown results in inhibition of phosphorylation of IκB by ribosomal S6 kinase (RSK) but not by IκB kinases. Subcellular fractionation suggests that enhanced phosphorylation of a scaffolding protein, PEA15 (phosphoprotein enriched in astrocytes 15), downstream of the PLCϵ-PKD axis causes sustained cytoplasmic localization of phosphorylated RSK, thereby facilitating IκB phosphorylation in the cytoplasm. These results suggest the crucial role of the TNF-α-LPA-LPA receptor-PLCϵ-PKD-PEA15-RSK-IκB-NF-κB pathway in facilitating inflammation and inflammation-associated carcinogenesis in the colon.

The transcription factor sterol regulatory element-binding protein 1c (SREBP1c) plays an important role in the control of fatty acid metabolism in the liver. Evidence suggests that mammalian target of rapamycin (mTOR) complex 1 (mTORC1) contributes to the regulation of SREBP1c expression, but signaling downstream of mTORC1 remains unclear. We have now shown that medium rich in branched-chain amino acids stimulates expression of the SREBP1c gene in cultured hepatocytes in a manner sensitive both to rapamycin, a pharmacological inhibitor of mTORC1, and to a short hairpin RNA (shRNA) specific for S6 kinase 1 (S6K1), a downstream effector of mTORC1. The phosphorylation of S6K1 was increased in the liver of obese db/db mice. Furthermore, depletion of hepatic S6K1 in db/db mice with the use of an adenovirus vector encoding S6K1 shRNA resulted in down-regulation of SREBP1c gene expression in the liver as well as a reduced hepatic triglyceride content and serum triglyceride concentration. These results thus suggest that S6K1 regulates SREBP1c expression both in cultured hepatocytes and in mouse liver, and that increased hepatic activity of S6K1 contributes at least in part to the pathogenesis of obesity-induced hepatic steatosis and hypertriglyceridemia.

The transcription factor sterol regulatory element-binding protein 1c (SREBP1c) plays an important role in the regulation of fatty acid metabolism in the liver. Although the importance of phosphoinositide 3-kinase in the regulation of SREBP1c expression is widely accepted, the role of mammalian target of rapamycin (mTOR) in such regulation has remained unclear. We have now shown that the insulin-induced increase in the abundance of SREBP1c mRNA in cultured AML12 mouse hepatocytes was largely abolished by LY294002, an inhibitor of phosphoinositide 3-kinase, but was reduced only slightly by rapamycin, an inhibitor of mTOR. Forced expression of a constitutively active form of Akt containing a myristoylation signal sequence (MyrAkt) in these cells with the use of an adenoviral vector resulted in the phosphorylation of p70 S6 kinase, a downstream target of mTOR signaling, and this effect was inhibited by rapamycin. MyrAkt also increased the abundance of SREBP1c mRNA and protein as well as the expression of the SREBP1c target genes for fatty acid synthase and stearoyl-CoA desaturase 1. These effects of MyrAkt were also markedly inhibited by LY294002 and by rapamycin. These results thus suggest that mTOR signaling plays a major role in Akt-mediated up-regulation of SREBP1c expression but that it plays only a minor role in insulin-induced expression of this transcription factor.

Gene related to anergy in lymphocytes (GRAIL) is an E3 ubiquitin ligase that regulates energy in T-lymphocytes. Whereas, the relevance of GRAIL to T lymphocyte function is well established, the role of this protein in other cell types remains unknown. Given that GRAIL is abundant in the liver, we investigated the potential function of GRAIL in nutrient metabolism by generating mice in which the expression of GRAIL is reduced specifically in the liver. Adenovirus-mediated transfer of a short hairpin RNA specific for GRAIL mRNA markedly reduced the amounts of GRAIL mRNA and protein in the liver. Blood glucose levels of the mice with hepatic GRAIL deficiency did not differ from those of control animals in the fasted or fed states. However, these mice manifested glucose intolerance in association with a normal increase in plasma insulin levels during glucose challenge. The mice also manifested an increase in the serum concentration of free fatty acids, whereas the serum levels of cholesterol and triglyceride were unchanged. The hepatic abundance of mRNAs for glucose-6-phosphatase, catalytic (a key enzyme in hepatic glucose production) and for sterol regulatory element-binding transcription factor 1 (an important transcriptional regulator of lipogenesis) was increased in the mice with hepatic GRAIL deficiency, possibly contributing to the metabolic abnormalities of these animals. Our results thus demonstrate that GRAIL in the liver is essential for maintenance of normal glucose and lipid metabolism in living animals.

Cell division in many mammalian tissues is associated with specific times of day, but just how the circadian clock controls this timing has not been clear. Here, we show in the regenerating liver (of mice) that the circadian clock controls the expression of cell cycle–related genes that in turn modulate the expression of active Cyclin B1-Cdc2 kinase, a key regulator of mitosis. Among these genes, expression of wee1 was directly regulated by the molecular components of the circadian clockwork. In contrast, the circadian clockwork oscillated independently of the cell cycle in single cells. Thus, the intracellular circadian clockwork can control the cell-division cycle directly and unidirectionally in proliferating cells.