Table 1.
Inhibitor potency in hABHD12-HEK293 lysates for commercially available triterpenes (1–11) and triterpenoids (12–15) as well as their calculated lipophilicity values (logD).
Figure 1.
Inhibitor potency in hABHD12-HEK293 lysates for compounds 16–23 as well as their calculated lipophilicity values (logD).
Data are mean ± SEM from three independent experiments.
Figure 2.
Inhibitor potency in hABHD12-HEK293 lysates for compounds 24–30 as well as their calculated lipophilicity values (logD).
Data are mean ± SEM from three independent experiments.
Figure 3.
Inhibitor potency in hABHD12-HEK293 lysates for compounds 31–39 as well as their calculated lipophilicity values (logD).
Data are mean ± SEM from three independent experiments.
Figure 4.
Inhibitor potency in hABHD12-HEK293 lysates for compounds 40–43 as well as their calculated lipophilicity values (logD).
Data are mean ± SEM from three independent experiments.
Figure 5.
Collection of the features used to define the pharmacophoric model for the binding of the triterpenes to the ABHD12.
Color coding of pharmacophoric features: green, hydrophobic site; magenta, acceptor site; cyan, donor site; gray cage, ligand shape constraint. B. The best solution for the alignment of the most active compound (33) to pharmacophore model (the same color coding as above except the ligand surface omitted for the clarity).
Figure 6.
Competitive ABPP to unveil triterpenoid targets among the serine hydrolases in proteomes of HEK293 cell lysates (A–B) and mouse brain membranes (C).
Lysates (25 µg) or membranes (100 µg) were treated for 1 h with DMSO or the indicated concentrations of the inhibitors, after which TAMRA-FP labelling was conducted for 5 min (lysates) or for 1 hour (membranes), as described in Methods. The proteins were resolved in 10% SDS-PAGE together with protein standards. TAMRA-FP labeling was visualized after in-gel fluorescence imaging as described in experimental procedures. Molecular weight markers are shown in both gel images. A. Transient expression of hABHD12 in HEK293 cells results in the appearance of a ∼46 kDa protein band (black arrow), comigrating together with an endogenous serine hydrolase with similar size. The endogenous band is weakly visible in Mock lysates and does not correspond to ABHD12 [4]. Note that probe labeling to hABHD12 is fully prevented by THL (10 µM), it is also dose-dependently inhibited by compound 23 whereas compound 22 is ineffective. B. Quantitative data (mean ± SEM) on the effects of selected triterpenoids on probe labeling of the hABHD12 band (black arrow in A) combined from three independent experiments with hABHD12-HEK lysates. Statistical differences in band intensities were tested using one-way ANOVA, followed by Tukey's multiple comparison test (**P<0.01 and ***P<0.001). C. ABHD12 is the sole triterpenoid target among the serine hydrolases in mouse brain membrane proteome. THL (10 µM) was used as a positive control and consistent with previous data [1], [34], [35], THL prevented TAMRA-FP labeling of serine hydrolases migrating at ∼63 kDa (BAT5), ∼46 kDa (ABHD12) and ∼33 kDa (ABHD6). Note that compound 23 and maslinic acid (compound 8) dose-dependently inhibit TAMRA-FP labeling of ABHD12 already at 20 µM whereas compound 22 is ineffective, even at 200 µM concentration. Note also that with the exception of heightened TAMRA-FP labeling of an unidentified serine hydrolase migrating at ∼90 kDa (indicated by the asterix), no additional targets are evident for maslinic acid or compound 23 among the metabolic serine hydrolases. The gel is representative from three independent ABPP runs with similar outcome.