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. 2020 Feb;52:102650.
doi: 10.1016/j.ebiom.2020.102650. Epub 2020 Feb 12.

A Small-Molecule Inhibitor of PCSK9 Transcription Ameliorates Atherosclerosis Through the Modulation of FoxO1/3 and HNF1α

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Free PMC article

A Small-Molecule Inhibitor of PCSK9 Transcription Ameliorates Atherosclerosis Through the Modulation of FoxO1/3 and HNF1α

Xuelei Wang et al. EBioMedicine. .
Free PMC article

Abstract

Background: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that down-regulates hepatic low-density lipoprotein receptor (LDLR) by binding and shuttling LDLR to lysosomes for degradation. The development of therapy that inhibits PCSK9 has attracted considerable attention for the management of cardiovascular disease risk. However, only monoclonal antibodies of PCSK9 have reached the clinic use. Oral administration of small-molecule transcriptional inhibitors has the potential to become a therapeutic option.

Methods: Here, we developed a cell-based small molecule screening platform to identify transcriptional inhibitors of PCSK9. Through high-throughput screening and a series of evaluation, we found several active compounds. After detailed investigation on the pharmacological effect and molecular mechanistic characterization, 7030B-C5 was identified as a potential small-molecule PCSK9 inhibitor.

Findings: Our data showed that 7030B-C5 down-regulated PCSK9 expression and increased the total cellular LDLR protein and its mediated LDL-C uptake by HepG2 cells. In both C57BL/6 J and ApoE KO mice, oral administration of 7030B-C5 reduced hepatic and plasma PCSK9 level and increased hepatic LDLR expression. Most importantly, 7030B-C5 inhibited lesions in en face aortas and aortic root in ApoE KO mice with a slight amelioration of lipid profiles. We further provide evidences suggesting that transcriptional regulation of PCSK9 by 7030B-C5 mostly depend on the transcriptional factor HNF1α and FoxO3. Furthermore, FoxO1 was found to play an important role in 7030B-C5 mediated integration of hepatic glucose and lipid metabolism.

Interpretation: 7030B-C5 with potential suppressive effect of PCSK9 expression may serve as a promising lead compound for drug development of cholesterol/glucose homeostasis and cardiovascular disease therapy. FUND: This work was supported by grants from the National Natural Science Foundation of China (81473214, 81402929, and 81621064), the Drug Innovation Major Project of China (2018ZX09711001-003-006, 2018ZX09711001-007 and 2018ZX09735001-002), CAMS Innovation Fund for Medical Sciences (2016-I2M-2-002, 2016-I2M-1-011 and 2017-I2M-1-008), Beijing Natural Science Foundation (7162129).

Keywords: Atherosclerosis; FoxO1/3; HNF1α; Lipid and glucose metabolism; PCSK9; Small-molecule inhibitor.

Conflict of interest statement

Declaration of Competing Interest The authors declare that there is no conflict of interest regarding the publication of this article.

Figures

Fig. 1
Fig. 1
Identification of novel PCSK9 inhibitors using cell-based high-throughput screening (HTS) assays. (a) The construction of recombinant plasmid pGL4-PCSK9-P. Human PCSK9 promoter region spanning −2112 to −1 bp, relative to the ATG start codon, was amplified by PCR, verified by DNA sequencing and cloned into pGL4-Basic vector between the Xho I and Hind III sites to produce pGL4-PCSK9-P. (b) The samples in compound library were screened by the established cell-based HTS assay for their capability of inhibiting PCSK9 transcription. In total, 6328 compounds at 25 μg/mL were screened. The red dot represented PCSK9 inhibitors 7030B-C5, 7031B-H9, 7045B-E7 and 7045B-F7 we identified. The blue dot represented positive control berberine (BBR). (c) HTS hit triage workflow resulting in the identification of 7030B-C5, 7031B-H9, 7045B-E7 and 7045B-F7, with the number of compounds selected at each step. (d) The inhibitory activity and chemical structure of a novel PCSK9 inhibitor 7030B-C5, 7031B-H9, 7045B-E7 and 7045B-F7. The stable pGL4-PCSK9-P transfected HepG2 cells were treated with 7030B-C5, 7031B-H9, 7045B-E7 or 7045B-F7 for 24 h and the luciferase activities were measured. The data represent the mean ± SEM of at least three independent experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2
Fig. 2
Effects of 7030B-C5 on PCSK9 and LDLR expression in hepatic cells. (a) HepG2 cells were treated with 7030B-C5 in a series of concentration for 24 h. The mRNA level of PCSK9 was measured by RT-qPCR analysis. (b) HepG2 cells were treated with 7030B-C5 in a series of concentration for 24 h. Expression of PCSK9 and LDLR protein was measured by Western blot. (c) HepG2 cells were treated with 7030B-C5 in 12.5 μM with different times. After treatment, cellular proteins were extracted and used to determine PCSK9 protein by Western blot. (d) HepG2 cells were treated with 7030B-C5 in a series of concentrations for 24 h. Secreted form of PCSK9 protein and cellular PCSK9 proteins were determined. (e) Huh7 cells were treated with different concentrations of 7030B-C5 for 24 h. Expression of PCSK9 and LDLR protein was measured by western blot. (f) Human primary hepatocytes were treated with 7030B-C5 for 24 h. Expression of PCSK9 and LDLR protein was determined. (g) HepG2 cells were treated with vehicle or 7030B-C5 for 24 h. The cells were incubated with DiI-LDL (5 μg/mL) at 37 °C for 4 h, and then the LDL uptake activity was measured by flow cytometric analysis. Values are presented as means ± SEM (n = 3). *p < 0.05 vs. control in the corresponding group.
Fig. 3
Fig. 3
Administration of 7030B-C5 to ApoE KO mice reduces atherosclerosis progression. Male ApoE KO mice were intragastrically injected with vehicle and 7030B-C5 (10 mg/kg per day, 30 mg/kg per day), respectively, for 12 weeks. At the end of experiment, aorta, serum and liver samples were individually collected and used for the following assays. (a) The body weight course of ApoE KO mice fed an HFD without (control) or with 7030B-C5. *p < 0.05 HFD vs. 10 mg/kg group, #p < 0.05 HFD vs. 30 mg/kg group, (b) Plasma total cholesterol, LDL-C, HDL-C collected from the ApoE KO mice at the 12th week after 7030B-C5 treatment. And serum PCSK9 levels were determined by an ELISA kit. (c) Plasma was pooled per group and the distribution of cholesterol over the individual lipoproteins was determined after separation by fast protein liquid chromatography (FPLC). (d and e) Atherosclerotic plaques in the en face aorta and aortic root were determined by Oil-Red-O staining, and the plaques area were quantified using ImageJ. a, chow; b, HFD; c, 7030B-C5 (10 mg/kg); d, 7030B-C5 (30 mg/kg). Scale bar = 500 μm. (f) Expression of PCSK9 and LDLR protein in the liver was determined by Western blot. Representative images are shown. Values are presented as means ± SEM (n = 10 per group). *p < 0.05 vs. control in the corresponding group.
Fig. 4
Fig. 4
7030B-C5 reduces PCSK9 expression transcriptionally in a HNF1α and FoxO3-responsive element-dependent manner. (a) The constructs of human PCSK9 promoter-luciferase reporters. Position +1 was designated as the nucleotide preceding the ATG start codon. Position −1 is the 3′ end of PCSK9 promoter inserts in common to all promoter-reporter constructs. The 5′ ends of the promoters in each promoter-reporter construct are marked by numbers at left, and the name of each construct is shown at right. (b) HepG2 cells were transfected with the PCSK9 promoter-luciferase reporter constructs PCSK9-P D1-D7 for 24 h and then treated with vehicle (0.1% DMSO) or 7030B-C5 for 24 h. (c and d) Deletion analysis of PCSK9 promoter suggested that HNF1/IRE and HINFP-bs might be the key cis-regulatory elements responsible for the regulation of PCSK9 by 7030B-C5. Mut-1 represents core nucleotide sequence of the HNF1/IRE site mutation, mut-2 represents core nucleotide sequence of the HINFP-bs site mutation. (e) HepG2 cells were treated with vehicle or 7030B-C5 in a series of concentrations for 24 h. Expression of HNF1α HINFP and FoxO3 protein were measured by Western blot. (f) HepG2 cells was transfected with siRNA negative control (siNC) or siRNA for the knockdown of FoxO3 (siFoxO3), HNF1α (siHNF1α), HINFP (siHINFP) and the level of FoxO3, HNF1α and HINFP protein was determined by Western blot analysis. (g) HepG2 cells were treated with 7030B-C5 (12.5 μM) for 24 h. Chromatin was isolated followed by immunoprecipitation with normal IgG, anti-HNF1α or anti-FoxO3 antibody. The PCR was conducted with primers for HNF1 or IRE in the PCSK9 promoter. (h) Regulation of PI3K/Akt pathway by 7030B-C5 in HepG2. Representative images are shown. Values are presented as means ± SEM (n = 3). *p < 0.05, #p < 0.05 vs. control in the corresponding group.
Fig. 5
Fig. 5
7030B-C5 regulates glucose metabolism through FoxO1. (a) Male ApoE KO mice were intragastrically injected with vehicle and 7030B-C5 (10 mg/kg per day, 30 mg/kg per day), respectively, for 12 weeks. At the end of experiment, liver samples were collected. The liver lipid accumulation was determined by Oil-Red-O staining and quantified by using ImageJ. a, chow; b, HFD; c, 7030B-C5 (10 mg/kg); d, 7030B-C5 (30 mg/kg). Scale bar = 500 μm. Representative images are shown. (b) Volcano plots for all differentially expressed genes in comparison. Horizontal coordinate is the value of fold change between 7030B-C5 group/HFD difference genes and the vertical coordinates is the p value. The left side represents the differentially down-regulated gene, the right side represents the differentially up-regulated gene, and the green and red dots represent the differential gene p < 0.05, blank dots represent the non-significantly differential gene. (c) GO pathway enrichment analysis of 321 differentially expressed genes. (d) KEGG pathway enrichment analysis of 42 differentially expressed genes involved in glucose and lipid metabolism. (e) Male ApoE KO mice were intragastrically injected with vehicle and 7030B-C5 (10 mg/kg per day, 30 mg/kg per day), respectively, for 12 weeks. At the end of experiment, triglycerides, glucose, glycated serum protein (GSP) and glycated albumin (GA) in the serum were collected from the ApoE KO mice at the 12th week after 7030B-C5 treatment. (f) Effects of 7030B-C5 on FoxO1 expression in HepG2 cells. HepG2 cells were treated with 7030B-C5 in a series of concentrations for 24 h. Expression of FoxO1 protein was measured by Western blot. (g) The interaction of FoxO1 with IRE element was determined by luciferase reporter system. (h) HepG2 cells were treated with 7030B-C5 in a series of concentrations for 24 h. The mRNA level of PEPCK, G6Pase, MTP and ApoC-III was measured by RT-qPCR analysis. Values are presented as means ± SEM (n = 10 per group). *p < 0.05 vs. control in the corresponding group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 6
Fig. 6
Schematic diagram of the regulatory mechanism of 7030B-C5 on lipid and glucose metabolism.

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