Chemical Proteomics Reveals Off-Targets of the Anandamide Reuptake Inhibitor WOBE437

Abstract

Anandamide or N-arachidonoylethanolamine (AEA) is a signaling lipid that modulates neurotransmitter release via activation of the type 1 cannabinoid receptor (CB₁R) in the brain. Termination of anandamide signaling is thought to be mediated via a facilitated cellular reuptake process that utilizes a purported transporter protein. Recently, WOBE437 has been reported as a novel, natural product-based inhibitor of AEA reuptake that is active in cellular and in vivo models. To profile its target interaction landscape, we synthesized pac-WOBE, a photoactivatable probe derivative of WOBE437, and performed chemical proteomics in mouse neuroblastoma Neuro-2a cells. Surprisingly WOBE437, unlike the widely used selective inhibitor of AEA uptake OMDM-1, was found to increase AEA uptake in Neuro-2a cells. In line with this, WOBE437 reduced the cellular levels of AEA and related N-acylethanolamines (NAEs). Using pac-WOBE, we identified saccharopine dehydrogenase-like oxidoreductase (SCCPDH), vesicle amine transport 1 (VAT1), and ferrochelatase (FECH) as WOBE437-interacting proteins in Neuro-2a cells. Further genetic studies indicated that SCCPDH and VAT1 were not responsible for the WOBE437-induced reduction in NAE levels. Regardless of the precise mechanism of action of WOB437 in AEA transport, we have identified SSCPHD, VAT1, and FECH as unprecedented off-targets of this molecule which should be taken into account when interpreting its cellular and in vivo effects.

Figure 1

Structures of WOBE437 and its probe derivatives.

Figure 2

Synthesis and characterization of WOBE437. (A) Reagents and conditions: (a) ethyl 2-(diethoxyphosphoryl)acetate, NaH, 0 °C, then (E)-dec-2-enal, −78 °C to rt, 63%; (b) NaOH, 60 °C, quant.; (c) 2-(3,4-dimethoxyphenyl)ethan-1-amine, HOAt, EDC, rt, 78%. (B) Endocannabinoid uptake was assayed in Neuro-2a cells, which were preincubated with OMDM-1 (40 μM) as a positive control or different concentrations of WOBE437 for 10 min. [³H]-AEA was added, and cells were incubated for an additional 15 min, washed, and harvested to measure radioactivity. Control experiments were also carried out under the same conditions at 4 °C in order to subtract passive diffusion from active uptake. Data are expressed as means ± SEM of three independent experiments, each performed in triplicate. *p < 0.05; ***p < 0.001 in comparison to vehicle-treated control (dotted line) using one-way ANOVA with Dunnett’s multiple comparisons correction.

Figure 3

WOBE437 disrupts cellular NAE levels within 10 min of treatment. Neuro-2a cells were treated with 10 μM WOBE437 or vehicle and harvested at the indicated time points to be analyzed by MS-based lipidomics. (A–C) Lipidomic data are presented as a volcano plot, and lipids with a fold-change threshold of ≥1.50 or ≤0.67 and a Benjamini-Hochberg false-discovery rate (FDR) ≤10% following a Student’s t-test are represented by colored circles indicating lipid class. (D) Fold-change of altered NAEs is represented as a function of time. The complete list of ratios at 30 min are depicted in Figure S1.

Figure 4

Synthesis and characterization of pac-WOBE (3). (A) Reagents and conditions: (a) 5-(2-aminoethyl)-2-methoxyphenol, HOBt, EDC, 0 °C to rt, 43%; (b) 3-(but-3-yn-1-yl)-3-(2-iodoethyl)-3H-diazirine, K₂CO₃, 60 °C, 29%. (B) Neuro-2a cells were treated with 10 μM WOBE437 or vehicle and subsequently with 0.1 μM pac-WOBE (3) or vehicle, irradiated, and lysed, and proteomes were conjugated to Cy5-N₃ using CuAAC chemistry and analyzed by SDS-PAGE and in-gel fluorescence scanning. Coomassie served as a protein loading control. Arrows indicate WOBE437-competed targets.

Figure 5

Identification and characterization of WOBE437 targets using pac-WOBE (3). Volcano plot depicting (A) UV enrichment and (B) WOBE437 competition of proteins labeled by in situ AfBPP in Neuro-2a cells using 1 μM pac-WOBE (3). UV enrichment is capped at 20-fold, p-value at 0.00001. A complete list of targets is available in Table S4. (C) Gel-based AfBPP profiling of overexpressing HEK-293-T cells using 0.1 μM pac-WOBE (3). (D,E) Representative gels of competition of 0.1 μM pac-WOBE (3) labeling of overexpressing HEK-293-T cells by 10 μM of the indicated compound. (F,G) Quantified residual labeling of indicated protein by 0.1 μM pac-WOBE (3) after preincubation with the indicated compound. Fluorescent signal was normalized to quantified Coomassie signal. Data represent means ± SD of three biological replicates. *p < 0.05; ***p < 0.001 in comparison to vehicle-treated control (dotted line) using one-way ANOVA with Dunnett’s multiple comparisons correction.

Figure 6

Partial SCCPDH and VAT1 knockouts were generated by CRISPR-Cas9. (A) SCCPDH and (C) VAT1 KO Neuro-2a lines were generated and checked by gel-based AfBPP using 0.1 μM pac-WOBE (3) for residual expression. VAT1 protein was tested by VAT1 Western blot. Coomassie served as a protein loading control. Lipid levels were tested by lipidomics on (B) SCCPDH KO and (D) VAT1 KO cells and compared to WT cells. Further characterization and the complete list of ratios are depicted in Figures S6 and S7. (E) Neuro-2a cells were treated with 10 μM WOBE437 or vehicle for 30 min and harvested to be analyzed by MS-based lipidomics. Lipid levels are displayed as ratio against the same type of cells treated with vehicle. Data represent means ± SEM (n = 4). One-way ANOVA with Dunnett’s multiple comparisons correction: not significant when compared to WT.