Paraoxonase-2 agonist vutiglabridin promotes autophagy activation and mitochondrial function to alleviate non-alcoholic steatohepatitis.

BACKGROUND AND PURPOSE: Only limited therapeutic agents have been developed for non-alcoholic steatohepatitis (NASH). Glabridin, a promising anti-obesity candidate, has only limited druggability due to its low in vivo chemical stability and bioavailability. Therefore, we developed vutiglabridin (VUTI), which is based on a glabridin backbone, and investigated its mechanism of action in treating NASH in animal models. EXPERIMENTAL APPROACH: Anti-NASH effects of VUTI were determined in in vitro fatty liver models, spheroids of primary human hepatocytes and L02 normal liver cell lines. To identify VUTI possible cellular target/s, biotin-labelled VUTI was synthesized and underwent chemical proteomic analysis. Further, the evaluation of VUTI therapeutic efficacy was carried out using an amylin-NASH and high-fat (HF) diet-induced obese (DIO) mouse models. This was carried out using transcriptomic, lipidomic and proteomic analyses of the livers from the amylin-NASH mouse model. KEY RESULTS: VUTI treatment markedly reduces hepatic steatosis, fibrosis and inflammation by promoting lipid catabolism, activating autophagy and improving mitochondrial dysfunction, all of which are hallmarks of effective NASH treatment. The cellular target of VUTI was identified as paraoxonase 2 (PON2), a newly proposed protein target for the treatment of NASH, VUTI enhanced PON2 activity. The results using PON2 knockdown cells demonstrated that PON2 is important for VUTI- activation of autophagy, promoting mitochondrial function, decreasing oxidative stress and alleviating lipid accumulation under lipotoxic condition. CONCLUSION AND IMPLICATIONS: Our data demonstrated that VUTI is a promising therapeutic for NASH. Targeting PON2 may be important for improving liver function in various immune-metabolic diseases including NASH.

Vutiglabridin Alleviates Cellular Senescence with Metabolic Regulation and Circadian Clock in Human Dermal Fibroblasts.

The process of cellular senescence, which is characterized by stable cell cycle arrest, is strongly associated with dysfunctional cellular metabolism and circadian rhythmicity, both of which are reported to result from and also be causal to cellular senescence. As a result, modifying any of them-senescence, metabolism, or the circadian clock-may affect all three simultaneously. Obesity accelerates aging by disrupting the homeostasis of reactive oxygen species (ROS) via an increased mitochondrial burden of fatty acid oxidation. As a result, if senescence, metabolism, and circadian rhythm are all linked, anti-obesity treatments may improve metabolic regulation while also alleviating senescence and circadian rhythm. Vutiglabridin is a small molecule in clinical trials that improves obesity by enhancing mitochondrial function. We found that chronic treatment of senescent primary human dermal fibroblasts (HDFs) with vutiglabridin alleviates all investigated markers of cellular senescence (SA-ß-gal, CDKN1A, CDKN2A) and dysfunctional cellular circadian rhythm (BMAL1) while remarkably preventing the alterations of mitochondrial function and structure that occur during the process of cellular senescence. Our results demonstrate the significant senescence-alleviating effects of vutiglabridin, specifically with the restoration of cellular circadian rhythmicity and metabolic regulation. These data support the potential development of vutiglabridin against aging-associated diseases and corroborate the intricate link between cellular senescence, metabolism, and the circadian clock.

Vutiglabridin Modulates Paraoxonase 1 and Ameliorates Diet-Induced Obesity in Hyperlipidemic Mice.

Vutiglabridin is a clinical-stage synthetic small molecule that is being developed for the treatment of obesity and its target proteins have not been fully identified. Paraoxonase-1 (PON1) is an HDL-associated plasma enzyme that hydrolyzes diverse substrates including oxidized low-density lipoprotein (LDL). Furthermore, PON1 harbors anti-inflammatory and antioxidant capacities and has been implicated as a potential therapeutic target for treating various metabolic diseases. In this study, we performed a non-biased target deconvolution of vutiglabridin using Nematic Protein Organisation Technique (NPOT) and identified PON1 as an interacting protein. We examined this interaction in detail and demonstrate that vutiglabridin binds to PON1 with high affinity and protects PON1 against oxidative damage. Vutiglabridin treatment significantly increased plasma PON1 levels and enzyme activity but not PON1 mRNA in wild-type C57BL/6J mice, suggesting that vutiglabridin modulates PON1 post-transcriptionally. We further investigated the effects of vutiglabridin in obese and hyperlipidemic LDLR-/- mice and found that it significantly increases plasma PON1 levels, while decreasing body weight, total fat mass, and plasma cholesterol levels. Overall, our results demonstrate that PON1 is a direct, interacting target of vutiglabridin, and that the modulation of PON1 by vutiglabridin may provide benefits for the treatment of hyperlipidemia and obesity.

Synthetic Glabridin Derivatives Inhibit LPS-Induced Inflammation via MAPKs and NF-κB Pathways in RAW264.7 Macrophages.

Glabridin is a polyphenolic compound with reported anti-inflammatory and anti-oxidative effects. In the previous study, we synthesized glabridin derivatives-HSG4112, (S)-HSG4112, and HGR4113-based on the structure-activity relationship study of glabridin to improve its biological efficacy and chemical stability. In the present study, we investigated the anti-inflammatory effects of the glabridin derivatives in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. We found that the synthetic glabridin derivatives significantly and dose-dependently suppressed the production of nitric oxide (NO) and prostaglandin E2 (PGE2), and decreased the level of inducible nitric oxygen synthase (iNOS) and cyclooxygenase-2 (COX-2) and the expression of pro-inflammatory cytokines interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor alpha (TNF-α). The synthetic glabridin derivatives inhibited the nuclear translocation of the NF-κB by inhibiting phosphorylation of the inhibitor of κB alpha (IκB-α), and distinctively inhibited the phosphorylation of ERK, JNK, and p38 MAPKs. In addition, the compounds increased the expression of antioxidant protein heme oxygenase (HO-1) by inducing nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) through ERK and p38 MAPKs. Taken together, these results indicate that the synthetic glabridin derivatives exert strong anti-inflammatory effects in LPS-stimulated macrophages through MAPKs and NF-κB pathways, and support their development as potential therapeutics against inflammatory diseases.

Paraoxonase-2 contributes to promoting lipid metabolism and mitochondrial function via autophagy activation.

Non-alcoholic fatty liver disease (NAFLD) is an increasingly prevalent immuno-metabolic disease that can progress to hepatic cirrhosis and cancer. NAFLD pathogenesis is extremely complex and is characterized by oxidative stress, impaired mitochondrial function and lipid metabolism, and cellular inflammation. Thus, in-depth research on its underlying mechanisms and subsequent investigation into a potential drug target that has overarching effects on these features will help in the discovery of effective treatments for NAFLD. Our study examines the role of endogenous paraoxonase-2 (PON2), a membrane protein with reported antioxidant activity, in an in vitro cell model of NAFLD. We found that the hepatic loss of PON2 activity aggravated steatosis and oxidative stress under lipotoxic conditions, and our transcriptome analysis revealed that the loss of PON2 disrupts the activation of numerous functional pathways closely related to NAFLD pathogenesis, including mitochondrial respiratory capacity, lipid metabolism, and hepatic fibrosis and inflammation. We found that PON2 promoted the activation of the autophagy pathway, specifically the mitophagy cargo sequestration, which could potentially aid PON2 in alleviating oxidative stress, mitochondrial dysfunction, lipid accumulation, and inflammation. These results provide a mechanistic foundation for the prospect of PON2 as a drug target, leading to the development of novel therapeutics for NAFLD.

Accelerated aging phenotypes in the retinal pigment epithelium of Zmpste24-deficient mice.

Age-related macular degeneration (AMD) is a chronic and progressive disease characterized by degeneration of the retinal pigment epithelium (RPE) and retina that ultimately leads to loss of vision. The pathological mechanisms of AMD are not fully known. Cellular senescence, which is a state of cell cycle arrest induced by DNA-damage or aging, is hypothesized to critically affect the pathogenesis of AMD. In this study, we examined the relationship between cellular senescence and RPE/retinal degeneration in mouse models of natural aging and accelerated aging. We performed a bulk RNA sequencing of the RPE cells from adult (8 months old) and naturally-aged old (24 months old) mice and found that common signatures of senescence and AMD pathology - inflammation, apoptosis, and blood vessel formation - are upregulated in the RPE of old mice. Next, we investigated markers of senescence and the degree of RPE/retinal degeneration in Zmpste24-deficient (Zmpste24-/-) mice, which is a model for progeria and accelerated aging. We found that Zmpste24-/- mice display markedly greater level of senescence-related markers in RPE and significant RPE/retinal degeneration compared to wild-type mice, in a manner consistent with natural aging. Overall, these results provide support for the association between cellular senescence of RPE and the pathogenesis of AMD, and suggest the use of Zmpste24-/- mice as a novel senescent RPE model of AMD.

Discovery and preclinical efficacy of HSG4112, a synthetic structural analog of glabridin, for the treatment of obesity.

BACKGROUND: HSG4112 is a clinical-stage drug candidate for the treatment of obesity. Here, we report its discovery and preclinical efficacy. METHODS: In high-fat diet (HFD)-induced obese male C57BL/6J mice, we tested the weight loss effect of synthetic compounds derived from a structure-activity relationship (SAR) study of glabridin, a natural compound known to reduce body weight and influence energy homeostasis. After selecting HSG4112 as our optimized compound from this discovery method, we characterized its pharmacological effects on parameters related to obesity through in vivo metabolic and biochemical measurements, histology and gene expression analysis, and indirect calorimetry. RESULTS: Through the SAR study, we identified four novel components of glabridin pertinent for its anti-obesity activity, and found that HSG4112, an optimized structural analog of glabridin, markedly supersedes glabridin in weight reduction efficacy and chemical stability. Six-week administration of HSG4112 to HFD-induced obese mice led to dose-dependent normalization of obesity-related parameters, including body weight, muscle and adipose tissue weight, adipocyte size, and serum leptin/insulin/glucose levels. The weight reduction induced by HSG4112 was partially mediated by decreased food intake and mainly mediated by increased energy expenditure, with no change in physical activity. Accordingly, the pattern of transcriptional changes was aligned with increased energy expenditure in the liver and muscles. Following significant body weight reduction, robust amelioration of histopathology and blood markers of fatty liver were also observed. CONCLUSIONS: Our study demonstrates the key chemical components of glabridin pertinent to its weight loss effects and suggests HSG4112 as a promising novel drug candidate for the pharmacological treatment of obesity.