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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

Effects of CB_36 and its analogs in HepG2 cells. (A) At 18 h after treatment of CB_36, amounts of LDLR and PCSK9 were determined by immunoblot analysis. (B) Fluorescence-labeled Dil-LDL was incubated for an additional 2 h, and the uptake of Dil-LDL was analyzed by fluorescence microscopy. (C) The intensity of fluorescence was quantitated by flow cytometry analysis. Each value represents the ratio of the mean fluorescence intensity relative to that in vehicle-treated cells (DM). Error bars represent the SD of triplicate reactions. Similar results were obtained from at least three independent experiments. *p<0.05, †p<0.01 Student's t-test when compared with values in DMSO-treated cells. PCSK9, proprotein convertase subtilisin/kexin type 9; LDL, low density lipoprotein; LDLR, LDL receptor; GAPDH, glyceraledhyde-3-phosphate dehydrogenase; DMSO, dimethyl sulfoxide.
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Figure 2: Effects of CB_36 and its analogs in HepG2 cells. (A) At 18 h after treatment of CB_36, amounts of LDLR and PCSK9 were determined by immunoblot analysis. (B) Fluorescence-labeled Dil-LDL was incubated for an additional 2 h, and the uptake of Dil-LDL was analyzed by fluorescence microscopy. (C) The intensity of fluorescence was quantitated by flow cytometry analysis. Each value represents the ratio of the mean fluorescence intensity relative to that in vehicle-treated cells (DM). Error bars represent the SD of triplicate reactions. Similar results were obtained from at least three independent experiments. *p<0.05, †p<0.01 Student's t-test when compared with values in DMSO-treated cells. PCSK9, proprotein convertase subtilisin/kexin type 9; LDL, low density lipoprotein; LDLR, LDL receptor; GAPDH, glyceraledhyde-3-phosphate dehydrogenase; DMSO, dimethyl sulfoxide.

Mentions: Effects of the chemical with ChemBridge ID #7926604 (lab ID, CB_36), which had the highest docking score (Supplementary Table 1, only online), and its three-dimensional analogs (#7632817 and #7338220) proposed by ChemBridge's online website (http://www.hit2lead.com/) were evaluated for in vitro parameters in HepG2 cells. The structures of these chemicals are depicted in Fig. 1. The concentrations of chemicals applied were determined experimentally and set as low as possible. All three chemicals increased the expression of the LDLR and PCSK9 in a dose-dependent manner (Fig. 2A). The decrease in the LDLR and PCSK9 by the compound #7632817 at a concentration of 20 µg/mL appeared to be due to cytotoxicity (Fig. 2A, lane 12). The uptake of Dil-LDL was increased accordingly with the increase in LDLR and PCSK9 expression (Fig. 2B). When the intensity of the fluorescence in cells was quantitated using flow cytometry analysis, CB_36 at 5 µg/mL increased the uptake of Dil-LDL by a factor of 1.69 compared to the vehicle (DMSO) (Fig. 2C). Interestingly, #7632817, the compound that had the most similar three-dimensional structure (94%) and was predicted not by the GOLD algorithm but by ChemBridge, increased the Dil-LDL uptake most strongly by a factor of 2.13. Chemical #7338220 (75% similarity) was relatively less effective in increasing the LDL uptake (by a factor of 1.37 at a concentration of 30 µg/mL). These results suggest that CB_36 and its analogs function to increase the uptake of LDL cholesterol in HepG2 cells despite the simultaneous increase in the amount of PCSK9.


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)

Effects of CB_36 and its analogs in HepG2 cells. (A) At 18 h after treatment of CB_36, amounts of LDLR and PCSK9 were determined by immunoblot analysis. (B) Fluorescence-labeled Dil-LDL was incubated for an additional 2 h, and the uptake of Dil-LDL was analyzed by fluorescence microscopy. (C) The intensity of fluorescence was quantitated by flow cytometry analysis. Each value represents the ratio of the mean fluorescence intensity relative to that in vehicle-treated cells (DM). Error bars represent the SD of triplicate reactions. Similar results were obtained from at least three independent experiments. *p<0.05, †p<0.01 Student's t-test when compared with values in DMSO-treated cells. PCSK9, proprotein convertase subtilisin/kexin type 9; LDL, low density lipoprotein; LDLR, LDL receptor; GAPDH, glyceraledhyde-3-phosphate dehydrogenase; DMSO, dimethyl sulfoxide.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Effects of CB_36 and its analogs in HepG2 cells. (A) At 18 h after treatment of CB_36, amounts of LDLR and PCSK9 were determined by immunoblot analysis. (B) Fluorescence-labeled Dil-LDL was incubated for an additional 2 h, and the uptake of Dil-LDL was analyzed by fluorescence microscopy. (C) The intensity of fluorescence was quantitated by flow cytometry analysis. Each value represents the ratio of the mean fluorescence intensity relative to that in vehicle-treated cells (DM). Error bars represent the SD of triplicate reactions. Similar results were obtained from at least three independent experiments. *p<0.05, †p<0.01 Student's t-test when compared with values in DMSO-treated cells. PCSK9, proprotein convertase subtilisin/kexin type 9; LDL, low density lipoprotein; LDLR, LDL receptor; GAPDH, glyceraledhyde-3-phosphate dehydrogenase; DMSO, dimethyl sulfoxide.
Mentions: Effects of the chemical with ChemBridge ID #7926604 (lab ID, CB_36), which had the highest docking score (Supplementary Table 1, only online), and its three-dimensional analogs (#7632817 and #7338220) proposed by ChemBridge's online website (http://www.hit2lead.com/) were evaluated for in vitro parameters in HepG2 cells. The structures of these chemicals are depicted in Fig. 1. The concentrations of chemicals applied were determined experimentally and set as low as possible. All three chemicals increased the expression of the LDLR and PCSK9 in a dose-dependent manner (Fig. 2A). The decrease in the LDLR and PCSK9 by the compound #7632817 at a concentration of 20 µg/mL appeared to be due to cytotoxicity (Fig. 2A, lane 12). The uptake of Dil-LDL was increased accordingly with the increase in LDLR and PCSK9 expression (Fig. 2B). When the intensity of the fluorescence in cells was quantitated using flow cytometry analysis, CB_36 at 5 µg/mL increased the uptake of Dil-LDL by a factor of 1.69 compared to the vehicle (DMSO) (Fig. 2C). Interestingly, #7632817, the compound that had the most similar three-dimensional structure (94%) and was predicted not by the GOLD algorithm but by ChemBridge, increased the Dil-LDL uptake most strongly by a factor of 2.13. Chemical #7338220 (75% similarity) was relatively less effective in increasing the LDL uptake (by a factor of 1.37 at a concentration of 30 µg/mL). These results suggest that CB_36 and its analogs function to increase the uptake of LDL cholesterol in HepG2 cells despite the simultaneous increase in the amount of PCSK9.

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