Development of the novel ACLY inhibitor 326E as a promising treatment for hypercholesterolemia

Abstract

Hepatic cholesterol accumulation is an important contributor to hypercholesterolemia, which results in atherosclerosis and cardiovascular disease (CVD). ATP-citrate lyase (ACLY) is a key lipogenic enzyme that converts cytosolic citrate derived from tricarboxylic acid cycle (TCA cycle) to acetyl-CoA in the cytoplasm. Therefore, ACLY represents a link between mitochondria oxidative phosphorylation and cytosolic de novo lipogenesis. In this study, we developed the small molecule 326E with an enedioic acid structural moiety as a novel ACLY inhibitor, and its CoA-conjugated form 326E-CoA inhibited ACLY activity with an IC₅₀ = 5.31 ± 1.2 μmol/L in vitro. 326E treatment reduced de novo lipogenesis, and increased cholesterol efflux in vitro and in vivo. 326E was rapidly absorbed after oral administration, exhibited a higher blood exposure than that of the approved ACLY inhibitor bempedoic acid (BA) used for hypercholesterolemia. Chronic 326E treatment in hamsters and rhesus monkeys resulted in remarkable improvement of hyperlipidemia. Once daily oral administration of 326E for 24 weeks prevented the occurrence of atherosclerosis in ApoE^-/- mice to a greater extent than that of BA treatment. Taken together, our data suggest that inhibition of ACLY by 326E represents a promising strategy for the treatment of hypercholesterolemia.

Keywords: ACLY inhibitor; ATP-Citrate lyase (ACLY); Atherosclerosis; Cholesterol efflux; Hypercholesterolemia; Lipogenesis; Liver.

Graphical abstract

Scheme 1

Synthesis of 326F.

Figure 1

Identification of 326E inhibits lipogenesis and gluconeogenesis in primary hepatocytes with more effective than BA. (A)–(B) Primary mouse hepatocytes were treated with the indicated concentration of 326F or BA for 24 h. 0.1 μCi/well [1,2-¹⁴C]-acetate was added into the culture medium at last 4 h of incubation. The contents of [1,2-¹⁴C]-acetate incorporated into fatty acids (A) and cholesterol (B) were measured. The radioactive content of lipids was normalized to DMSO group (n = 4); (C) Primary mouse hepatocytes were treated with the indicated concentration of 326F or BA for 6 h. The rate of gluconeogenesis in the medium induced by pyruvate (2 mmol/L) and lactate (20 mmol/L) was measured. The content of glucose output was normalized to DMSO group (n = 4); (D) Primary mouse hepatocytes were treated with the indicated concentration of 326F or BA for 6 h in the gluconeogenesis medium as (C), with or without suppled with 4.0 mmol/L l-glutamine in the indicated group. The content of glucose output was normalized to DMSO group (n = 4); (E) The structure of 326F, 326Z and 326E; (F)–(G) Primary mouse hepatocytes contain 0.1 μCi/well [1,2-¹⁴C]-acetate were treated for 4 h with the indicated concentration of 326F, 326E, 326Z and BA. The contents of [1,2-¹⁴C]-acetate incorporated into fatty acids (F) and cholesterol (G) were measured. The radioactive content of lipids was normalized to DMSO group (n = 3–4); (H) The Schematic diagram of the experiment. Male golden hamsters fed an HFHC diet for 2 weeks, then oral administration of 326E (10 mg/kg), 326Z (10 mg/kg) and BA (30 mg/kg) for another 2 weeks (n = 8). HFHC diet, high fat high cholesterol diet (normal chow supplemented with 0.5% cholesterol, 23% fat and 5% Fructose, w/w); (I)–(L) Serum levels of TG (I), TC (J), LDL-C (K) and HDL-C (L) in the groups were determined and plotted at indicated time. The hamsters were fasted overnight before the experiment. Data are mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 compared to DMSO or Veh. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared to indicated group. ns, not significant.

Scheme 2

Synthesis of 326E and 326Z. Condition: a) PPTS, DHP, DCM. b) n-BuLi, HMPA,THF. c) TsOH, MeOH. d) CBr₄, PPh₃, DCM. e) ethyl isobutyrate, LDA, THF. f) H₂, Ni(OAc)₂, NaBH₄, ethylene diamine, EtOH. g) KOH, EtOH, H₂O. h) LiAlH₄, Ethylene glycol diethyl ether.

Scheme 3

Synthesis of the CoA thioester of 326E and BA.

Figure 2

326E inhibits ACLY activity and suppresses lipid synthesis in primary hepatocytes isolated from mice. (A)–(B) Dose-dependent inhibition of purified, recombinant ACLY after incubated with BA-CoA (A) and 326E-CoA (B), the activity at control was defined as 100%; (C)–(D) Recombinant human ACLY was incubated with indicated concentrations of 326E-CoA coenzyme A (C) or citrate (D). K_i was calculated by Michaelis-Menten kinetic analysis; (E)–(F) Hepatocytes from mouse were exposed to DMEM with control or multiple doses of 326E for 4 h in the presence of [1,2-¹⁴C]-acetate (E) or [¹⁴C]-citrate (F) as noted, and IC₅₀ for de novo lipogenesis in the hepatocytes were generated; (G)–(J) Hepatocytes from mouse were exposed to DMEM with control or 326E for 4 h in the presence of [³C]-H₂O or [¹⁴C]-glucose as noted, fatty acids (G, I) and cholesterol (H, J) biogenesis in the hepatocytes were showed; (K)–(L) ACSLs inhibitor triacsin C attenuates 326E induced lipogenesis in fatty acids (K) and cholesterol synthesis (L). Triacsin C (5 μmol/L) was pre-treated for 30 min before 326E treatment; (M)–(N) Hepatocytes isolated from mice or HepG2 cell lines were exposed to DMEM with control or 326E for 4 h in the presence of [1,2-¹⁴C]-acetate as noted, and the radioactive contents of ¹⁴C-fatty acids (M) and ¹⁴C-cholesterol (N) incorporated by [1,2-¹⁴C]-acetate were counted by liquid scintillation counter; (O)–(P) Hepatocytes isolated from Acly f/f mice or Acly f/f:cre mice were exposed to DMEM with control, 326E or BA for 4 h in the presence of 0.1 μCi/well [¹⁴C]-citrate as noted, and the radioactive contents of ¹⁴C-fatty acids (O) and ¹⁴C-cholesterol (P) incorporated by [¹⁴C]-citrate were counted by liquid scintillation counter. Data are mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 compared toControl (Con). ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared to indicated group. ns, not significant.

Figure 3

326E treatment reduces hepatic de novo lipogenesis and VLDL-TGs secretion. (A) Schematic diagram of 326E treatment for hepatic de novo lipogenesis. Briefly, male C57BL/6J mice were fasted for 48 h followed by refeeding for another 48h. Animals then received 326E treatment at multi-dose followed by an intraperitoneally injection with 2 μCi [1,2-¹⁴C] acetate (in B, C) and 5 μCi [1,2-¹⁴C] acetate (in D, E) 1 h later dosing. One hour after [1,2-¹⁴C] acetate injection, the mice were sacrificed after anesthetization, and the hepatic lipogenesis was calculated; (B)–(C) The rate of ¹⁴C-labeled hepatic de novo lipogenesis after 326E (10, 30, 100 mg/kg) and BA (30 mg/kg) treatment (n = 5–6); (D)–(E) The rate of ¹⁴C-labeled de novo lipogenesis after 326E (30 mg/kg) in the liver, leg muscle, ileum, kidney, heart and abdominal fat (a-Fat) (n = 7–8); (F)–(I) Plasma TG (F) and TC (H) concentration under tyloxapol (600 mg/kg) injection were measured after 326E and BA treatment at the indicated dose (n = 10). The area under the curve of TG level (G) and TC (I) in 4 h of VLDL-TGs secretion were shown. Data are mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 compared to Veh. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared to indicated group. ns, not significant.

Figure 4

Treatment of ACLY inhibitors increase cholesterol efflux and reduce hepatic cholesterol in HCD mice. (A)–(B) The mRNA levels of Abcg5 and Abcg8 (A) and intracellular cholesterol (B) were detected in the primary mouse hepatocytes after 326E and BA (50 μmol/L) treatment for 24 h. During the incubation, 15 μg/mL cholesterol (containing 420 μg/mL M-β-CD) was added. The LXR agonist T0901317 (T0, 1 μmol/L) were used as positive control in (B); (C) The schematic diagram of cholesterol efflux in the primary hepatocytes was shown; (D) The cholesterol efflux rate was detected after 326E and BA (50 μmol/L) treatment for 12 h in primary hepatocytes. The LXR agonist T0901317 (1 μmol/L) were used as positive control; (E) Hepatic mRNA of Abcg5 and Abcg8 were measured after a single dose of 326E and BA 30 mg/kg for 24 h in HCD mice (n = 5–6); (F) The content of cholesterol in the gallbladder was detected after a single dose of 326E and BA 30 mg/kg for 24 h in HCD mice (n = 5–6); (G)–(K) ³H-cholesterol and ³H-bile acids content in the feces (H, I) and liver (J, K) were counted with scintillation liquid. Briefly, HCD mice were oral of 326E or BA for 7 days. On the 7th day of treatment, mice were received an intravenous tail vein injection of 2.5 μCi ³H-cholesterol, which was dissolved in 100 uL intralipid (20%). Fecal samples were collected at indicated time after ³H-cholesterol injection. Livers were harvested 24 h later at last dosing (G). The radioactive cholesterol (H, J) and ³H-bile acids (I, K) were detected (n = 5–6); (L)–(M) The HCD mice were oral given with 326E and BA for 7 days. The livers were harvested after 4 h fasting. The representative pictures H&E staining (L) were shown. The contents of hepatic cholesterol (TC) and triglycerides (TG) were measured (M) (n = 5–6). Data are mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 compared toVeh. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared to indicated group. ns, not significant.

Figure 5

Effects of 326E on lipid levels in blood of HFHC diet induced hyperlipidemia. (A) Schematic diagram of 326E for hyperlipidemia treatment. Briefly, male golden hamsters were induced by HFHC diet for 2 weeks. Animals then received 326E or BA treatment for another 2 weeks (n = 8). HFHC diet, high fat high cholesterol diet (normal chow supplemented with 0.5% cholesterol, 23% fat and 5% fructose, w/w); (B) Body weight gain were shown after 326E treatment for 2 weeks; (C)–(F) Serum levels of TG (C), TC (D), LDL-C (E), and HDL-C (F) before and during the treatment period were shown. The oral dose of BA was 30 mg/kg. Data are mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 compared to Veh.

Figure 6

Lipid lowering efficacy and safety evaluation of 326E in monkeys with hyperlipidemia. (A) Schematic diagram of 326E treatment in rhesus monkey. Briefly, 6–20 years old male rhesus monkey with mild or moderate hyperlipidemia for more than 2 years (LDL-C:1.94–2.45 mmol/L) were selected, and oral treated with Veh, atorvastatin (atorva, 2.4 mg/kg/day) or 326E (20 mg/kg/day). n = 3 monkey per group; (B)–(C) Body weight change after 2 weeks treatment; (D)–(K) Plasma levels of TG (D), TC (F), LDL-C (H), ApoB100 (J) in rhesus monkey before and after 2 weeks treatment; percentage lipid lowering effect were showed compared to baseline TG (E), TC (G), LDL-C (I), ApoB100 (K) were shown; (L)–(M) Pharmacokinetic curve in each monkey (M1, M2, M3) was shown after the first dose of 326E at indicated time (L), and percentage of LDL-C lowering after 326E treated for 2 weeks in each monkey (M); (N)–(O) Plasma levels of ALT (N) and AST (O) before and after 326E or atorvastatin treatment. Data are mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, Veh compared to 326E; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, Veh compared to atorva.

Figure 7

Treatment of 326E decreases atherosclerosis in ApoE^−/− mice. (A)–(B) Male 6–8 weeks ApoE^−/− mice were induced by Western diet for 23–24 weeks. During the inducement, mice were received 326E or BA treatment once daily (n = 10). Western diet (normal chow supplemented with 0.2% cholesterol, w/w). Cholesterol levels (A) and triglycerides levels (B) in liver after 24 weeks treatment (n = 10); (C)–(D) Representative photographs of aortas from different groups after 24 weeks treatment (C); the quantifications (D) of the lesion areas in C (n = 10); (E)–(F) Representative H&E staining of cross-sections of aortic roots in different groups after 24 weeks treatment (E); the quantifications (F) of the lesion areas in E (n = 8–10); (G) Plasma contents of hsCRP in the indicated groups after 24 weeks treatment (n = 10); (H)–(L) Quantitative mRNA expression of Cd68, F4/80, Il-1β, Abcg5, Abcg8 in the liver after 326E and BA treatment. Data are mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 compared to Veh. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared to indicated group. ns, not significant.