Fig 2: PON1 is identified as a potential target of vutiglabridin through NPOT assay. (A) Schematic representation of the Nematic Protein Organization Technique (NPOT) analysis of vutiglabridin on protein homogenates from the liver of healthy wild-type (WT) and diet-induced obesity (DIO) mice with validation by biophysics methods. In total, 568 proteins were identified as potentially interacting proteins, and 296 proteins (52%) were present in both conditions. (B) Ranked list of Gene Ontology (GO) cellular components and (C) molecular functions of the identified proteins in both conditions. (D) Ranked list of Gene Ontology biological processes of the identified proteins in both conditions with hydrolase activity as the molecular function. X-axis shows gene count. Yellow star marks the inclusion of PON1 protein. (E) The interactome network of 37 proteins that are involved with phospholipid metabolic process, steroid metabolic process, and lipid transport, which are downstream of the molecular function of lipid metabolic process. PON1 is identified as the central protein involved in all three processes.

Fig 3: Vutiglabridin protects against diet-induced obesity in hyperlipidemic mice. (A) LDLR−/− mice were utilized to investigate the role of vutiglabridin against diet-induced obesity and hyperlipidemia. Mice were fed four different diets for three weeks: Chow ± vutiglabridin or Western diet (WD) ± vutiglabridin. Dosing of vutiglabridin was at 100 mg/kg. Both male and female mice, between 8–12 weeks of age, with five mice per group were used. The mice were weighed prior to the start of the experiment. At the end of the third week of feeding, the experiment was terminated. (B) Changes in body weights were determined between the weight of the mice before the start of the experiment and final weights prior to termination. (C) Fat mass changes were assessed and quantified as percentage of final fat mass over total body weight × 100. (D) Muscle (lean) mass changes were assessed and quantified as percentage of final muscle mass over total body weight × 100. Plasma total cholesterol levels (E) were also determined. PON1 expression was evaluated at the end of the study in all groups via Western blot analysis, as reflected in the representative image (F). PON1 expression was normalized to transferrin and quantified as a ratio of PON1 to transferrin (G). The plasma paraoxonase activity as PON1 specific enzyme activity was measured in WD-fed groups (H). The data are represented as mean ± SEM. One-way ANOVA with Tukey’s multiple comparison test was performed. Student’s t-test was performed for (H). Statistically significant data are noted as * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 4: Vutiglabridin binds to rePON1 and increases it enzyme activity under oxidative stress conditions. (A) Chemical structures of glabridin, (R)-vutiglabridin, and (S)-vutiglabridin in stick model are shown in green, cyan, and purple, respectively. The oxygen and hydrogen atoms are in red and white, respectively. In the 3D structure of PON1 protein, the key residues of the binding site are represented by light red stick models. In the docked conformation of the three compounds, hydrogen bond, hydrophobic interaction, and unfavorable bump are shown as green, pink, and red dotted lines, respectively. The specific hydrogen bond and hydrophobic interaction sites, and the cluster analysis of compounds out of 50 trials and the GOLD fitness scores are written below each compound. (B) Glabridin (Gla), (R)-vutiglabridin, (S)-vutiglabridin, and vutiglabridin were incubated with rePON1 under oxidative stress conditions induced by oxidized linoleic acid at 7 μM. Lactonase activity was measured by spectrometry. Results are presented as the percentage of absorbance compared to the non-treated control (Con). The data are shown as mean ± SEM (n = 3). One-way ANOVA with Sidak’s multiple comparison test was performed. Statistically significant data are noted as * p < 0.05, ** p < 0.01, *** p < 0.001. (C) SPR analysis showing sensorgram of the interaction between vutiglabridin and rePON1 captured by anti-DDK/FLAG Tag antibody on the chip. The compound was diluted at the indicated concentrations. The sensorgram corresponds to normalized vutiglabridin signal (subtracted of reference surface and the buffer signal).