1-Trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) Urea, a Selective and Potent Dual Inhibitor of Soluble Epoxide Hydrolase and p38 Kinase Intervenes in Alzheimer's Signaling in Human Nerve Cells

Abstract

Alzheimer's disease (AD) is the most common neurodegenerative disorder. Neuroinflammation is a prevalent pathogenic stress leading to neuronal death in AD. Targeting neuroinflammation to keep neurons alive is an attractive strategy for AD therapy. 1-Trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) is a potent inhibitor of soluble epoxide hydrolase (sEH) and can enter into the brain. It has good efficacy on a wide range of chronic inflammatory diseases in preclinical animal models. However, the anti-neuroinflammatory effects and molecular mechanisms of TPPU for potential AD interventions remain elusive. With an aim to develop multitarget therapeutics for neurodegenerative diseases, we screened TPPU against sEH from different mammalian species and a broad panel of human kinases in vitro for potential new targets relevant to neuroinflammation in AD. TPPU inhibits both human sEH and p38β kinase, two key regulators of inflammation, with nanomolar potencies and distinct selectivity. To further elucidate the molecular mechanisms, differentiated SH-SY5Y human neuroblastoma cells were used as an AD cell model, and we investigated the neuroprotection of TPPU against amyloid oligomers. We found that TPPU effectively prevents neuronal death by mitigating amyloid neurotoxicity, tau hyperphosphorylation, and mitochondrial dysfunction, promoting neurite outgrowth and suppressing activation and nuclear translocation of NF-κB for inflammatory responses in human nerve cells. The results indicate that TPPU is a potent and selective dual inhibitor of sEH and p38β kinase, showing a synergistic action in multiple AD signaling pathways. Our study sheds light upon TPPU and other sEH/p38β dual inhibitors for potential pharmacological interventions in AD.

Keywords: Alzheimer’s disease; dual inhibitor; mitochondrial dysfunction; neuroinflammation; neuroprotection; p38 mitogen-activated protein kinase; soluble epoxide hydrolase.

Figure 1.

Chemical structure and abbreviation of the sEH inhibitor, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU).

Figure 2.

(A) Inhibitory effects of TPPU on the activities of 40 kinases relevant to AD. Kinases were assayed in the presence of 1 μM TPPU or control (0.2% PEG400 vehicle). Data were the mean of duplicate of each of six independent experiments with ± SEM (n = 6). The data were analyzed by one-way ANOVA with Tukey’s multiple comparison test. **p < 0.01, ***p < 0.001, ****p < 0.0001 relative to the control. (B) TPPU inhibited p38β kinase activity with an IC₅₀ value of 0.27 μM. (C) TPPU inhibited p38γ kinase activity with an IC₅₀ value of 0.89 μM.

Figure 3.

Differentiated SH-SY5Y cells were a valid neuronal model. (A) Western blotting on a whole-cell lysate. Analysis was performed with antibodies against sEH (EPHX2), p38β kinase, and β-actin (loading control). Optical densities were normalized to β-actin. (B) Treatment with various concentrations of TPPU (10 to 1000 nM) for 24 h significantly decreased cellular sEH activities in SH-SY5Y cells. Analysis was performed with a sEH enzyme assay using a radiolabeled substrate t-DPPO. Data were the mean of three independent experiments with ± SEM (n = 3), *p < 0.05, ***p < 0.001.

Figure 4.

Morphological changes of SH-SY5Y cells upon treatments for 72 h. (A) 0.2% PEG 400 vehicle control. Differentiated cells with extended neurites. (B) 10 μM Aβ₄₂ treatment. Dying and nondifferentiated cells with retracted neurites. (C) Pretreatment of 100 nM TPPU followed by 10 μM Aβ₄₂ treatment. (D) Zoomed image showing protected well-differentiated neurons with extended neurites (arrow pointing). Micrographs represent the average morphologic characteristics of cell cultures under a given condition of 5–8 independent experimental replicates (n = 5–8). Scale bar = 100 μm.

Figure 5.

TPPU inhibited Aβ₄₂ neurotoxicity in SH-SY5Y cells. Cell viability was determined with the MTS assay. Data were the mean of duplicate of six independent experiments with ± SEM (n = 6). Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test. #p < 0.05, ####p < 0.0001 relative to the vehicle control; ****p < 0.0001 relative to the 10 μM Aβ₄₂ treatment. (A) Cytotoxicity assessment of TPPU in SH-SY5Y cells. Cells were treated with varying concentrations of TPPU or 0.2% PEG 400 vehicle and incubated for 72 h. (B) Cells were pretreated with varying concentrations of TPPU or 0.2% PEG 400 vehicle for 1 h followed by 10 μM Aβ₄₂ treatment for 72 h. (C) TPPU inhibited neurotoxicity induced by 10 μM Aβ₄₂ with an EC₅₀ value of 48.6 nM. The results were normalized as the percentage of the neuroprotective activity relative to the control (100%) and the 10 μM Aβ₄₂ treatment (0%). Neuroprotection curve was analyzed by four-parameter regression. (D) Cotreatment of EETs with TPPU enhanced neuroprotection.

Figure 6.

A drug combination data analysis. Pairwise treatments of a selective sEH inhibitor (t-AUCB) and a selective p38α/β kinase inhibitor (SB202190) in differentiated SH-SY5Y cells. Each compound was neuroprotective against 10 μM Aβ₄₂ in a dose-dependent manner. Combinations of both compounds in the dose-response matrices showed a synergistic effect. Colors in 3D mesh showed different levels of neuroprotection that were presented in percentage relative to the Aβ₄₂ free control (100%) and the Aβ₄₂ treatment (0%). Data were the mean of three independent experiments (n = 3).

Figure 7.

TPPU attenuated Aβ₄₂ induced tau phosphorylation at the site S396 in a dose dependent manner. Differentiated SH-SY5Y cells were pretreated with various concentrations of TPPU or 0.2% PEG 400 vehicle for 1 h followed by treatment of 10 μM Aβ₄₂ for 72 h. The known selective p38 inhibitor, SB202190, at 0.05 μM was used as a reference control. ELISA analysis was performed with specific antibody against Tau pS396 to quantify cellular tau phosphorylation levels. Fold changes were calculated relative to the control with ± SEM (n = 6). Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test. ####p < 0.0001 relative to vehicle control; ****p < 0.0001 relative to the 10 μM Aβ₄₂ treatment.

Figure 8.

TPPU and EETs prevented Aβ₄₂-induced mitochondrial dysfunction in differentiated SH-SY5Y cells. Cellular mitochondrial membrane potential (Δψ_m) was evaluated with JC-10 assay. FCCP at 50 μM or Aβ₄₂ at 10 μM depolarized Δψ_m as indicated by increasing JC-10 monomer/aggregate fluorescence ratio (525/590 nm). Pretreatment with TPPU, EETs, or co-dose for both significantly prevented the depolarized condition of mitochondria in the presence of 10 μM Aβ₄₂ in 72 h. Data were the mean of duplicate of six independent experiments with ± SEM (n = 6). Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test. ####p < 0.0001 relative to the vehicle control; ***p < 0.001, ****p < 0.0001 relative to the 10 μM Aβ₄₂ treatment. FCCP at 50 μM was used as a reference control.

Figure 9.

TPPU suppressed activation and nuclear translocation of the transcription factor NF-κB in differentiated SH-SY5Y cells. NF-κB family members (p50, p52, p65, RelB, and c-Rel) in the nuclear extracts were monitored with TransAM NF-κB assay. 5 μM Aβ₄₂ treatment for 8 h significantly induced translocation of NF-κB to the nucleus. Pretreatment with various concentrations of TPPU (0.05 to 0.1 μM) or 0.1 μM DEX for 1 h followed by treatment of 5 μM Aβ₄₂ for 8 h significantly reduced levels of all five NF-κB subunits in neuronal nuclei. Data were the mean of duplicate of six independent experiments with ± SEM (n = 6). Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test. ####p < 0.0001 relative to vehicle control; ••••p < 0.0001 relative to the 5 μM Aβ₄₂ treatment. Dexamethasone (DEX) known as the NF-κB/p38 MAPK inhibitor for anti-inflammatory activities was used as a reference control.

Figure 10.

Proposed neuroprotective mechanisms of TPPU against Alzheimer’s disease.