compound class  (TargetMol)

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of the sEH Inhibitor Epoxykynin as a Potent Kynurenine Pathway Modulator

doi: 10.1021/acs.jmedchem.3c02245

Figure Lengend Snippet: Reduction of cellular Kyn levels by epoxykynin ( 1l ) and influence on IDO1 expression and Trp uptake. (A) Kyn assay in IFN-γ-treated HeLa cells. Cells were treated with IFN-γ, Trp, and epoxykynin for 48 h prior to measuring Kyn levels using para -dimethylaminobenzaldehyde ( p -DMAB, mean values ± SD, n = 3). (B) In vitro IDO1 enzymatic activity. Purified IDO1 was treated with epoxykynin or DMSO for 40 min at 37 °C prior to addition of Trp and incubation for 60 min at 37 °C. Kyn levels were detected using p -DMAB (mean values ± SD, n = 2). (C) IDO1 promoter-dependent reporter gene assay in HEK293T cells expressing firefly luciferase (Fluc) under the control of the IDO1 promoter and constitutive Renilla luciferase expression (Rluc). Cells were treated with IFN-γ to induce Fluc expression and simultaneously with epoxykynin for 48 h. Fluc values were normalized to the Rluc signal (mean values ± SD, n = 3). (D) Kyn assay in HEK293T cells transiently expressing human IDO1. Cells were treated with Trp and epoxykynin for 24 h prior to measuring Kyn levels with p -DMAB (mean values ± SD, n = 3). (E) IDO1 mRNA expression in HeLa cells that were treated with IFN-γ and epoxykynin or DMSO for 24 h prior to quantification of mRNA levels via qPCR (mean values ± SD, n = 3). (F) IDO1 protein levels in HeLa cells that were treated with IFN-γ and epoxykynin or DMSO for 24 h prior to quantification of protein levels via immunoblotting (mean values ± SD, n = 4). See also Figure S2 for complete blots. (G) Trp uptake in BxPC-3 cells for epoxykynin. BxPC-3 cells were starved for Trp for 72 h and treated with IFN-γ for 24 h prior to addition 5 mM l -leucine ( l -Leu), 1 mM 1-methyl- l -tryptophan (1-MT), or 5 μM epoxykynin for 30 min. Afterward, 50 μM Trp was added and the Trp uptake after 30 min was quantified with HPLC-MS/MS (mean values ± SD, n = 3). The dotted lines indicate signals of the respective DMSO controls that were set to 100%.

Article Snippet: The epoxykynin compound class was purchased from ChemDiv, US.

Techniques: Expressing, In Vitro, Activity Assay, Purification, Incubation, Reporter Gene Assay, Luciferase, Control, Western Blot, Tandem Mass Spectroscopy

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of the sEH Inhibitor Epoxykynin as a Potent Kynurenine Pathway Modulator

doi: 10.1021/acs.jmedchem.3c02245

Figure Lengend Snippet: Target identification for epoxykynin. (A) Structures of affinity probes 2a and 2b and control affinity probes 3a and 3b . (B) Influence of the pulldown probes 2a and 3a on Kyn levels. HeLa cells were treated with IFN-γ, Trp, and compounds for 48 h prior to measuring Kyn levels using p -DMAB (mean values ± SD, n = 3). The dotted line indicates the signal of the DMSO+IFN-γ control that was set to 100%. (C) Volcano plot for proteins enriched using affinity-based chemical proteomics (pulldown) with probe 2b (red) or control probe 3b (blue) created with VolcaNoseR. The affinity probes 2b and 3b were immobilized on NHS-activated beads and incubated for 2 h at 4 °C with lysate of HeLa cells that were treated with IFN-γ. Enriched proteins were analyzed using HRMS ( n = 2, N = 4, FDR 0.01), representative replicate is shown, see also Figure S3 . (D) Proteins from panel (C) that were significantly enriched with the affinity probe 2b . For a complete list of enriched proteins, see Tables S2 and S3 .

Article Snippet: The epoxykynin compound class was purchased from ChemDiv, US.

Techniques: Drug discovery, Control, Incubation

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of the sEH Inhibitor Epoxykynin as a Potent Kynurenine Pathway Modulator

doi: 10.1021/acs.jmedchem.3c02245

Figure Lengend Snippet: Epoxykynin binds to sEH in vitro and in cellulo and inhibits the C-terminal lipid epoxide hydrolase activity (sEH-H). (A) Dose-dependent binding of epoxykynin to sEH as detected using nanoDSF. Purified sEH was treated with epoxykynin or DMSO for 10 min at room temperature prior to detection of the intrinsic tryptophan/tyrosine fluorescence upon thermal denaturation. Representative first derivatives of melting curves are shown ( n = 4, see also Figure S5 ). (B) Dose-dependent inhibition of sEH-H by epoxykynin. The epoxide hydrolase activity of purified sEH (sEH-H) was measured by means of the conversion of the fluorogenic sEH-H substrate PHOME upon treatment with epoxykynin (mean values ± SD, n = 3). See also Figure S6 . (C) Epoxykynin does not inhibit sEH-P. The phosphatase activity of purified sEH (sEH-P) was measured by means of an AttoPhos-based assay upon treatment with epoxykynin or AR9281 and ebselen as controls. Representative curves are shown ( n = 3, see also Figure S7). (D) Cellular thermal shift assay (CETSA) for sEH in Jurkat cells. Cells were treated with 10 μM epoxykynin or DMSO for 15 min prior to heat treatment and cell lysis. Soluble proteins were analyzed using immunoblotting. Representative immunoblots are shown ( n = 3, see also Figure S8 ). (E) Thermal stability of sEH upon compound treatment. Quantification of sEH band intensities from D (mean values ± SD, n = 3). (F) Dose-dependent displacement of a fluorescent tracer 4 by epoxykynin in HEK293T cells expressing NanoLuc-sEH. HEK293T cells that transiently express NanoLuc-sEH were treated with 60 nM of tracer and epoxykynin for 5 h prior to determination of the bioluminescence resonance energy transfer (BRET) ratio (mean values ± SD, n = 5). See Figure S9 for structure of fluorescent tracer 4 . (G, H) Knockdown (KD) of sEH decreases Kyn levels. HeLa cells were transfected with 50 nM nontargeting (NT) or EPHX2 -targeting siRNA for 48–96 h and treated with Trp and IFN-γ for 48 h prior to detection of EPHX2 mRNA (G) and Kyn levels with p -DMAB (H) (mean values ± SD, n = 3). (I) Overexpression of sEH in IFN-γ-HAP1 cells. HAP1 cells were transiently transfected with different amounts of sEH expression plasmid (1 μg (sEH-HAP1) or 3 μg (3XsEH-HAP1) plasmid DNA per 96-well plate) prior to treatment with epoxykynin, Trp and IFN-γ for 48 h. Kyn levels were quantified using p -DMAB (mean values ± SD, n = 3). The dotted lines indicate signals of the respective controls that were set to 100%. (J) Crystal structure of epoxykynin bound to human sEH-H (aa 228–547, PDB 8QZD ). Epoxykynin (wheat sticks) binds to the sEH-H active site (gray cartoon and sticks) and is stabilized by polar interactions with the two stabilizing residues Tyr383 and Tyr466 and with Asp335 of the catalytic triad Asp335-Asp496-His524 (indicated by the dotted black lines). The amino acids in the active site are labeled with the three-letter code. Heteroatoms of the ligand and amino acid side chains are depicted in red (oxygen), blue (nitrogen), dark red (bromine), and cyan (fluorine). Amino acids 497–500 are omitted for clarity.

Article Snippet: The epoxykynin compound class was purchased from ChemDiv, US.

Techniques: In Vitro, Activity Assay, Binding Assay, Nano Differential Scanning Fluorimetry, Purification, Fluorescence, Inhibition, Thermal Shift Assay, Lysis, Western Blot, Expressing, Bioluminescence Resonance Energy Transfer, Knockdown, Transfection, Over Expression, Plasmid Preparation, Labeling

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of the sEH Inhibitor Epoxykynin as a Potent Kynurenine Pathway Modulator

doi: 10.1021/acs.jmedchem.3c02245

Figure Lengend Snippet: sEH cross-talks with the Kyn pathway, thereby modulating cellular IDO1 and Kyn levels. (A) Knockdown (KD) of sEH. HeLa cells were transfected with nontargeting (NT) or EPHX2 -targeting siRNA for 48–72 h and treated with IFN-γ for 48 h prior to quantification of IDO1 protein levels via immunoblotting (mean values ± SD, n = 3). See also Figure S13 for complete blots. (B) Overexpression of sEH. HeLa cells were transfected with empty vector or pCMV3-EPHX2 for 24–48 h and treated with IFN-γ for 48 h prior to quantification of IDO1 protein levels via immunoblotting (mean values ± SD, n = 2). See also Figure S14 for complete blots. (C–D) Kyn assay in cell lysate. (C) BxPC-3 cells were treated with IFN-γ for 24 h prior to cell lysis. The lysate was treated with 10 μM epacadostat, 10 μM epoxykynin or DMSO for 45 min prior to detection of Kyn levels (mean values ± SD, n = 3). (D) BxPC-3 cells were treated with 5 μM epoxykynin, 50 nM EPHX2 -targeting siRNA or DMSO and IFN-γ for 48 h prior to cell lysis and detection of Kyn levels (mean values ± SD, n = 3).

Article Snippet: The epoxykynin compound class was purchased from ChemDiv, US.

Techniques: Knockdown, Transfection, Western Blot, Over Expression, Plasmid Preparation, Lysis