Yonsei Med J.  2015 Sep;56(5):1251-1257. 10.3349/ymj.2015.56.5.1251.

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

• KMID: 2163616
• DOI: http://doi.org/10.3349/ymj.2015.56.5.1251

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.

Keyword

PCSK9; in silico; protein-protein interaction; LDL receptor; hypercholesterolemia; inhibitor

MeSH Terms

Animals
Cholesterol/*blood
Cholesterol, LDL/blood
Hep G2 Cells
Humans
Mice
Mice, Knockout
Proprotein Convertases/*metabolism
Receptors, LDL/*metabolism
Serine Endopeptidases/*metabolism
*Small Molecule Libraries
Cholesterol
Cholesterol, LDL
Proprotein Convertases
Receptors, LDL
Serine Endopeptidases
Small Molecule Libraries

Figure

• 

  Fig. 1 Structures of CB_36 and its analogs. Numbers represent the ChemBridge ID.

• 

  Fig. 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.

• 

  Fig. 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.

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