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In silico Screening of Chemical Libraries to Develop Inhibitors That Hamper the Interaction of PCSK9 with the LDL Receptor.

Min DK, Lee HS, Lee N, Lee CJ, Song HJ, Yang GE, Yoon D, Park SW - Yonsei Med. J. (2015)

Bottom Line: Inhibition of PCSK9 either by reducing its expression or by blocking its activity results in the upregulation of the LDLR and subsequently lowers the plasma concentration of LDL-cholesterol.Additionally, one particular molecule lowered the plasma concentration of total cholesterol and LDL-cholesterol significantly in wild-type mice, while such an effect was not observed in Pcsk9 knockout mice.Our findings strongly suggest that in silico screening of small molecules that inhibit the protein-protein interaction between PCSK9 and the LDLR is a potential modality for developing hypercholesterolemia therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Institute of Genetic Science, Integrated Genomic Research Center for Metabolic Regulation, Yonsei University College of Medicine, Seoul, Korea.

ABSTRACT

Purpose: Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low density lipoprotein receptor (LDLR) and promotes degradation of the LDLR. Inhibition of PCSK9 either by reducing its expression or by blocking its activity results in the upregulation of the LDLR and subsequently lowers the plasma concentration of LDL-cholesterol. As a modality to inhibit PCSK9 action, we searched the chemical library for small molecules that block the binding of PCSK9 to the LDLR.

Materials and methods: We selected 100 chemicals that bind to PCSK9 where the EGF-AB fragment of the LDLR binds via in silico screening of the ChemBridge chemical library, using the computational GOLD algorithm analysis. Effects of chemicals were evaluated using the PCSK9-LDLR binding assay, immunoblot analysis, and the LDL-cholesterol uptake assay in vitro, as well as the fast performance liquid chromatography assay for plasma lipoproteins in vivo.

Results: A set of chemicals were found that decreased the binding of PCSK9 to the EGF-AB fragment of the LDLR in a dose-dependent manner. They also increased the amount of the LDLR significantly and subsequently increased the uptake of fluorescence-labeled LDL in HepG2 cells. Additionally, one particular molecule lowered the plasma concentration of total cholesterol and LDL-cholesterol significantly in wild-type mice, while such an effect was not observed in Pcsk9 knockout mice.

Conclusion: Our findings strongly suggest that in silico screening of small molecules that inhibit the protein-protein interaction between PCSK9 and the LDLR is a potential modality for developing hypercholesterolemia therapeutics.

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Related in: MedlinePlus

In vivo effects of CB_36 in wild-type and Pcsk9 knockout mice. (A) FPLC profiles of plasma cholesterol from wild-type (WT) and Pcsk9-/- mice after injection with CB_36. The pooled plasma from mice described in Table 2 was fractionated by FPLC, and the concentration of cholesterol in each fraction was measured as described under "Materials and Methods." (B) Aliquots of liver lysates were subjected to SDS-polyacrylamide gel electrophoresis (livers from two mice were pooled for lanes 1-6 in WT and for lanes 7, 8, 10, and 11 in Pcsk9-/-), and amounts of Ldlr and Pcsk9 were determined by immunoblot analysis. Gapdh was used as an invariant control. FPLC, fast performance liquid chromatography.
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Figure 3: In vivo effects of CB_36 in wild-type and Pcsk9 knockout mice. (A) FPLC profiles of plasma cholesterol from wild-type (WT) and Pcsk9-/- mice after injection with CB_36. The pooled plasma from mice described in Table 2 was fractionated by FPLC, and the concentration of cholesterol in each fraction was measured as described under "Materials and Methods." (B) Aliquots of liver lysates were subjected to SDS-polyacrylamide gel electrophoresis (livers from two mice were pooled for lanes 1-6 in WT and for lanes 7, 8, 10, and 11 in Pcsk9-/-), and amounts of Ldlr and Pcsk9 were determined by immunoblot analysis. Gapdh was used as an invariant control. FPLC, fast performance liquid chromatography.

Mentions: Due to the unavailability of the compound #7632817, which showed the most effective LDL-cholesterol uptake in HepG2 cells, the effect of only CB_36 was elucidated on the plasma cholesterol level in wild-type and Pcsk9-/- mice. The chemical #7338220 was not evaluated due to its weak effect on the uptake of Dil-LDL in HepG2 cells. C57BL/6J male mice (six per group) and Pcsk9-/- mice (five per group) were injected with CB_36 via tail vein at a concentration of 1 mg/kg for 2 consecutive days, and metabolic parameters were evaluated (Table 2). CB_36 significantly lowered the concentration of TC in wild-type mice by 18% compared to that in vehicle-treated mice (p<0.05), while the other phenotypic parameters in wild-type mice remained unchanged. More importantly, CB_36 had no effect on any parameters in Pcsk9-/- mice, suggesting that the action of CB_36 may involve a PCSK9-dependent pathway. The decrease in the plasma concentration of TC by CB_36 in wild-type mice was re-defined as a consequence of the decrease in LDL fractions in the lipoprotein profile determined by FPLC in wild-type mice (Fig. 3A; fraction numbers 15-22), while no change was observed in Pcsk9-/- mice. In wild-type mice, CB_36 also lowered the cholesterol level in fractions containing high-density lipoprotein (Fig. 3A; fraction numbers 23-30), of which the ApoE was also the ligand to the LDLR.22 However, in contrast to the results from HepG2 cells, CB_36 showed no differences in the amounts of LDLR and PCSK9 in the livers of wild-type or Pcsk9-/- mice (Fig. 3B). These results strongly suggest that CB_36 has the effect of lowering the TC level in plasma, particularly by lowering the LDL fraction of lipoproteins in a PCSK9-dependent manner, although amounts of LDLR and PCSK9 in the liver remained unchanged.


In silico Screening of Chemical Libraries to Develop Inhibitors That Hamper the Interaction of PCSK9 with the LDL Receptor.

Min DK, Lee HS, Lee N, Lee CJ, Song HJ, Yang GE, Yoon D, Park SW - Yonsei Med. J. (2015)

In vivo effects of CB_36 in wild-type and Pcsk9 knockout mice. (A) FPLC profiles of plasma cholesterol from wild-type (WT) and Pcsk9-/- mice after injection with CB_36. The pooled plasma from mice described in Table 2 was fractionated by FPLC, and the concentration of cholesterol in each fraction was measured as described under "Materials and Methods." (B) Aliquots of liver lysates were subjected to SDS-polyacrylamide gel electrophoresis (livers from two mice were pooled for lanes 1-6 in WT and for lanes 7, 8, 10, and 11 in Pcsk9-/-), and amounts of Ldlr and Pcsk9 were determined by immunoblot analysis. Gapdh was used as an invariant control. FPLC, fast performance liquid chromatography.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4541654&req=5

Figure 3: In vivo effects of CB_36 in wild-type and Pcsk9 knockout mice. (A) FPLC profiles of plasma cholesterol from wild-type (WT) and Pcsk9-/- mice after injection with CB_36. The pooled plasma from mice described in Table 2 was fractionated by FPLC, and the concentration of cholesterol in each fraction was measured as described under "Materials and Methods." (B) Aliquots of liver lysates were subjected to SDS-polyacrylamide gel electrophoresis (livers from two mice were pooled for lanes 1-6 in WT and for lanes 7, 8, 10, and 11 in Pcsk9-/-), and amounts of Ldlr and Pcsk9 were determined by immunoblot analysis. Gapdh was used as an invariant control. FPLC, fast performance liquid chromatography.
Mentions: Due to the unavailability of the compound #7632817, which showed the most effective LDL-cholesterol uptake in HepG2 cells, the effect of only CB_36 was elucidated on the plasma cholesterol level in wild-type and Pcsk9-/- mice. The chemical #7338220 was not evaluated due to its weak effect on the uptake of Dil-LDL in HepG2 cells. C57BL/6J male mice (six per group) and Pcsk9-/- mice (five per group) were injected with CB_36 via tail vein at a concentration of 1 mg/kg for 2 consecutive days, and metabolic parameters were evaluated (Table 2). CB_36 significantly lowered the concentration of TC in wild-type mice by 18% compared to that in vehicle-treated mice (p<0.05), while the other phenotypic parameters in wild-type mice remained unchanged. More importantly, CB_36 had no effect on any parameters in Pcsk9-/- mice, suggesting that the action of CB_36 may involve a PCSK9-dependent pathway. The decrease in the plasma concentration of TC by CB_36 in wild-type mice was re-defined as a consequence of the decrease in LDL fractions in the lipoprotein profile determined by FPLC in wild-type mice (Fig. 3A; fraction numbers 15-22), while no change was observed in Pcsk9-/- mice. In wild-type mice, CB_36 also lowered the cholesterol level in fractions containing high-density lipoprotein (Fig. 3A; fraction numbers 23-30), of which the ApoE was also the ligand to the LDLR.22 However, in contrast to the results from HepG2 cells, CB_36 showed no differences in the amounts of LDLR and PCSK9 in the livers of wild-type or Pcsk9-/- mice (Fig. 3B). These results strongly suggest that CB_36 has the effect of lowering the TC level in plasma, particularly by lowering the LDL fraction of lipoproteins in a PCSK9-dependent manner, although amounts of LDLR and PCSK9 in the liver remained unchanged.

Bottom Line: Inhibition of PCSK9 either by reducing its expression or by blocking its activity results in the upregulation of the LDLR and subsequently lowers the plasma concentration of LDL-cholesterol.Additionally, one particular molecule lowered the plasma concentration of total cholesterol and LDL-cholesterol significantly in wild-type mice, while such an effect was not observed in Pcsk9 knockout mice.Our findings strongly suggest that in silico screening of small molecules that inhibit the protein-protein interaction between PCSK9 and the LDLR is a potential modality for developing hypercholesterolemia therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Institute of Genetic Science, Integrated Genomic Research Center for Metabolic Regulation, Yonsei University College of Medicine, Seoul, Korea.

ABSTRACT

Purpose: Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low density lipoprotein receptor (LDLR) and promotes degradation of the LDLR. Inhibition of PCSK9 either by reducing its expression or by blocking its activity results in the upregulation of the LDLR and subsequently lowers the plasma concentration of LDL-cholesterol. As a modality to inhibit PCSK9 action, we searched the chemical library for small molecules that block the binding of PCSK9 to the LDLR.

Materials and methods: We selected 100 chemicals that bind to PCSK9 where the EGF-AB fragment of the LDLR binds via in silico screening of the ChemBridge chemical library, using the computational GOLD algorithm analysis. Effects of chemicals were evaluated using the PCSK9-LDLR binding assay, immunoblot analysis, and the LDL-cholesterol uptake assay in vitro, as well as the fast performance liquid chromatography assay for plasma lipoproteins in vivo.

Results: A set of chemicals were found that decreased the binding of PCSK9 to the EGF-AB fragment of the LDLR in a dose-dependent manner. They also increased the amount of the LDLR significantly and subsequently increased the uptake of fluorescence-labeled LDL in HepG2 cells. Additionally, one particular molecule lowered the plasma concentration of total cholesterol and LDL-cholesterol significantly in wild-type mice, while such an effect was not observed in Pcsk9 knockout mice.

Conclusion: Our findings strongly suggest that in silico screening of small molecules that inhibit the protein-protein interaction between PCSK9 and the LDLR is a potential modality for developing hypercholesterolemia therapeutics.

Show MeSH
Related in: MedlinePlus