Disease-related phenotypic assays enable unbiased discovery of novel bioactive small molecules and may provide novel insights into physiological systems and unprecedented molecular modes of action ...(MMOA). Herein, we report the identification and characterization of epoxykynin, a potent inhibitor of the soluble epoxide hydrolase (sEH). Epoxykynin was discovered by means of a cellular assay monitoring modulation of kynurenine (Kyn) levels in BxPC-3 cells upon stimulation with the cytokine interferon-γ (IFN-γ) and subsequent target identification employing affinity-based chemical proteomics. Increased Kyn levels are associated with immune suppression in the tumor microenvironment and, thus, the Kyn pathway and its key player indoleamine 2,3-dioxygenase 1 (IDO1) are appealing targets in immuno-oncology. However, targeting IDO1 directly has led to limited success in clinical investigations, demonstrating that alternative approaches to reduce Kyn levels are in high demand. We uncover a cross-talk between sEH and the Kyn pathway that may provide new opportunities to revert cancer-induced immune tolerance.
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Focused compound libraries are well-established tools for hit identification in drug discovery and chemical probe development. We present the compilation and application of a focused ...screening library of fatty acid mimetics (FAMs), which are compounds designed to bind the orthosteric site of proteins that endogenously accommodate natural fatty acids and lipid metabolites. This set complies with chemical properties of FAM and was found suitable for use also in cellular setting. Several hits were retrieved in screening the focused library against diverse fatty acid binding targets including the enzymes soluble epoxide hydrolase (sEH) and leukotriene A4 hydrolase (LTA4H), the nuclear receptors peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor α (RXRα), the carrier proteins fatty acid binding protein 4 and 5 (FABP4 and FABP5), as well as the G-protein coupled receptors leukotriene B4 receptor 1 (BLT1) and free-fatty acid receptor 1 (FFAR1). Thus, the focused FAM library is suitable to obtain chemical starting matter for fatty acid binding proteins and provides a valuable extension to available screening collections.
Soluble epoxide hydrolase (sEH) is a promising target for a number of inflammation-related diseases. In addition, inhibition of sEH has been shown to reduce neuroinflammation, which plays a critical ...role in the development of central nervous system (CNS) diseases such as Alzheimer’s disease. In this study, we present the rational design of a small fluorescent sEH inhibitor. Starting from the clinical candidate GSK2256294A, we replaced the triazine moiety with the 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) fluorophore. The resulting fluorescent sEH inhibitor displayed excellent potency in an in vitro enzyme activity assay (IC50 < 2 nM). The developed inhibitor is applicable in a NanoBRET-based assay system suitable for studying sEH target engagement in living cells. Furthermore, the inhibitor can be used to visualize sEH in sEH-transfected HEK293 cells and in primary mouse astrocytes by fluorescence microscopy.
