WZ66, a novel acetyl-CoA carboxylase inhibitor, alleviates nonalcoholic steatohepatitis (NASH) in mice

Abstract

The global prevalence of nonalcoholic steatohepatitis (NASH) increases incredibly. NASH ends up to advanced liver disease, which is highly threatening to human health. Currently, treatment of NASH is very limited. Acetyl-CoA carboxylases (ACC1/ACC2) are proved as effective drug targets for NASH. We aimed to develop novel ACC inhibitors and evaluate their therapeutic value for NASH prevention. ACC inhibitors were obtained through structure-based drug design, synthesized, screened from ACC enzymatic measurement platform and elucidated in cell culture-based assays and animal models. The lipidome and microbiome analysis were integrated to assess the effects of WZ66 on lipids profiles in liver and plasma as well as gut microbiota in the intestine. WZ66 was identified as a novel ACC1/2 inhibitor. It entered systemic circulation rapidly and could accumulate in liver. WZ66 alleviated NASH-related liver features including steatosis, Kupffer cells and hepatic stellate cells activation in diet-induced obese mice. The triglycerides (TGs) and other lipids including diglycerides (DGs), phosphatidylcholine (PC) and sphingomyelin (SM) were decreased in WZ66-treated mice as evidenced by lipidome analysis in livers. The lipids profiles in plasma were also altered with WZ66 treatment. Plasma TG were moderately increased, while the activation of SREBP1c was not detected. WZ66 also downregulated the abundance of Allobaculum, Mucispirillum and Prevotella genera as well as Mucispirillum schaedleri species in gut microbiota. WZ66 is an ideal lead compound and a potential drug candidate deserving further investigation in the therapeutics of NASH.

Keywords: Acetyl-CoA carboxylase; WZ66; gut microbiome; nonalcoholic steatohepatitis; pharmacokinetics lipidome.

Scheme 1

Synthesis route of WZ66

Fig. 1

Identification of WZ66 as a novel ACC1/2 inhibitor. a Chemical structure of WZ66. b Model of WZ66 bound to the CT domain of humanized yeast ACC. Images depict a docked model of WZ66 and CT domain of ACC (PDB ID: 5CTB). c Inhibition of the ACC1 and ACC2 enzymatic activities by WZ66. Assays were performed in three independent experiments. d Malonyl-CoA concentration in AML12 cells treated with vehicle (Ctrl) or WZ66 (1 µM) for 24 h. e Representative images of Oil red O staining of AML12 cells. Cells were induced with palmitic acid (250 µM) for 24 h and treated with vehicle or WZ66 for another 24 h (scale bar = 50 µm, magnification = 100×). f (left) Acaca, Acacb, Fasn, Acly, Elovl5, Gpam, Pnpla3, Srebf1, Cers2, Cers4, Cers5, Cers6 mRNA expression in AML12 cells treated with vehicle or WZ66; (right) Acaca, Acacb, Fasn, Acly, Elovl5, Gpam, Pnpla3, Srebf1, Cers2, Cers4, Cers5, Cers6 mRNA expression in primary hepatocytes treated with vehicle or WZ66. ^*P < 0.05. g (left) Representative images of Oil red O staining of AML12 cells. After induction with palmitic acid (250 µM) for 24 h, AML12 cells were transfected with siRNA (siNC: negative control siRNA; siAcaca: Acaca siRNA) and treated with WZ66 (1 µM) for another 24 h (scale bar = 50 µm, magnification = 40×); (right) quantification of Oil red O staining. ^*P < 0.05. Experiments were repeated in three independent experiments. Data are represented as the mean ± SEM

Fig. 2

Pharmacokinetics and tissue biodistribution of WZ66 in mice. a Pharmacokinetics of WZ66 in plasma (n = 4 per group). Blood was collected from each mouse at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h. b Tissue biodistribution of WZ66 in the liver, kidney, lung, spleen, muscle, heart, fat, and brain (n = 8 including four time points). Data are represented as the mean ± SEM

Fig. 3

WZ66 attenuated hepatic steatosis. a Schematic of WZ66 and PF-05175157 treatment in HFD-induced obese mice. b Food intake and body weights (n = 7 per group). c Liver weights and liver/body weight ratios (n = 14 for vehicle; n = 13 for WZ66). d Representative images of H&E and Oil red O staining of liver sections from HFD-induced obese mice with vehicle, WZ66 (25 mg/kg), WZ66 (50 mg/kg), or PF-05175157 (50 mg/kg) treatment (scale bar = 50 µm, magnification = 20×). Data are represented as the mean ± SEM. ^*P < 0.05

Fig. 4

WZ66 inhibited Kupffer cell activation and infiltration as well as decreased hepatic stellate cell activation. a Hepatic mRNA of chemokines (n = 7 for vehicle; n = 6 for WZ66). b (left) Adgre1 (F4/80) mRNA level in the liver (n = 14 for vehicle; n = 13 for WZ66; n = 10 for PF-05175157); (right) representative images of F4/80 immunofluorescent staining of liver sections from HFD-fed obese mice with vehicle or WZ66 treatment (scale bar = 50 µm, magnification = 20×). c (left) Col1a1 mRNA level in the liver (n = 14 for Vehicle; n = 13 for WZ66; n = 10 for PF-05175157); (right) representative images of sirius red staining of liver sections from obese mice with vehicle or WZ66 treatment (scale bar = 50 µm, magnification = 20×). d (left) Acta2 mRNA level in the liver (n = 14 for vehicle; n = 13 for WZ66; n = 10 for PF-05175157); (right) representative images of α-SMA immunohistochemical staining of liver sections from obese mice with vehicle or WZ66 treatment (scale bar = 50 μm, magnification = 20×). e Western blot for α-SMA and quantitative analysis of the bands (n = 4 per group). Data are represented as the mean ± SEM. ^*P < 0.05

Fig. 5

Treatment with WZ66 reduced lipid accumulation in the liver. a Heatmap of the relative abundance of lipids in the livers from the vehicle group and WZ66-treated group (n = 7 per group). b The amount of total lipids in the livers (n = 7 per group). c The relative abundance of each class of lipids in the livers (n = 7 per group). d The lipids that changed significantly after treatment with WZ66 (n = 7 per group). The scale of the y-axis is presented as log10. e Acaca, Acacb, Acly, Fasn, Elovl1, Elovl3, Elovl4, Elovl5, Elovl6, Elovl7, Lpin1, Lpin2, Lpin3, Srebf1, Gpam, Scap, Pnpla3, Scd1, Cers2, Cers4, Cers5, and Cers6 mRNA levels in the livers (n = 7 per group). f Western blot for mature SREBP1c and quantitative analysis of the bands (n = 4 per group). Data are represented as the mean ± SEM. ^*P < 0.05

Fig. 6

WZ66 modulated the lipid profiles in plasma. a Heatmap of the relative abundance of the lipids in plasma (n = 7 per group). b The relative abundance of each class of lipids in plasma (n = 7 per group). (c) The lipids that changed significantly following treatment with WZ66 (n = 7 per group). The scale of the y-axis is presented as log10. d Total cholesterol, HDL-cholesterol, LDL-cholesterol, and vLDL-cholesterol in plasma (n = 7 per group). Data are represented as the mean ± SEM. ^*P < 0.05

Fig. 7

WZ66 regulated the gut microbiota composition. a Phylogenetic diversity in diet-induced obese mice with vehicle and WZ66 treatment (n = 7 per group). b Principal component analysis (PCA) of the gut microbiota in vehicle and WZ66-treated obese mice (n = 7 per group). c (left) The relative abundance of gut microbiota at the phylum level; (right) the abundance from the phylum Deferribacteres decreased in WZ66-treated obese mice. d The relative abundance of gut microbiota at the genus level. e (left) The relative abundance of 17 genera with abundance >1%; the abundance of Allobaculum, Mucispirillum, and Prevotella were downregulated significantly between vehicle and WZ66-treated obese mice. (right) The relative abundance of Mucispirillum schaedleri species decreased in the WZ66-treated group. Statistical analysis was performed using the negative binomial test in the “edgeR” R package after TMM normalization, and the Benjiamini & Hochberg corrected ^*P < 0.05 was considered significant

Fig. 8

A proposed working model for WZ66 in the prevention of nonalcoholic steatohepatitis (NASH). ACC inhibition by WZ66 led to a decrease in de novo lipogenesis (DNL), which was evidenced by the lowered levels of triglycerides (TGs), diglycerides (DGs), phosphatidylcholine (PC), and sphingomyelin (SM) in the liver. The reduced lipid accumulation might promote the transition of Kupffer cells and hepatic stellate cells from the activated state to the inactivate state, leading to decreased Kupffer cell activation and collagen expression. The changes in lipid profiles in the liver caused an altered lipidome in the plasma, in which TG(18:1/20:4/18:3), TG(56:7), TG(58:8), and LysoPC(19:0) increased while PC(P-36:4) or (O-36:4), PC(18:0/20:3), and PE(O-18:0/22:6) decreased. All of the above changes due to WZ66 administration directly or indirectly induced alterations in the gut microbiota, as evidenced by the decreased abundance of the genera Allobaculum, Mucispirillum, and Prevotella in the intestine