Development of chemicals which reduce the expression of PCSK9 as therapeutics for treatment of hypercholesterolemia

Dept. of Medical Science/박사Proprotein Convertase Subtilisin/kexin type 9 (PC1SK9), the ninth member of subtilisin serine protease, promotes the degradation of the low density lipoprotein receptor (LDLR), thereby increasing the plasma concentration of LDL-cholesterol. Several studies have strongly suggested that inhibition of PCSK9 action is a promising therapeutic modality to treat hypercholesterolemia. As a strategy for development of PCSK9 inhibitors, the chemical library that consists of 3,000 randomly selected compounds was primarily screened by western blot analyses for the chemicals that reduce the amount of protein levels of PCSK9 with reciprocal increase in the LDLR expression in HepG2 cells. A set of chemicals (C935 and related chemicals) with the the common scaffold structure of 1,4-naphthoquinone reduced the amounts of the protein and mRNA for PCSK9, and transcriptional activity of the PCSK9 promoter, while they increased the amount of the LDLR protein. Functional relevance of the increased amount of in the LDLR was confirmed by the increased uptake of fluorescence-labeled LDL as well as the increase in the LDLR protein level. These results suggest that these chemicals increase the uptake of LDL into the cells by the increased LDLR expression which may be driven by reduction of PCSK9 expression in HepG2 cells. To elucidate the mechanism by which selected chemicals to reduce the transcriptional activity of PCSK9, microarray analysis was performed in HepG2 cells after treatment of chemicals. Among the genes of which amounts of mRNA was changed by C935, Nuclear factor (erythroid-derived 2)-like 2 (NRF2) was deduced to play an important role on regulation of the PCSK9 expression, although the reduction of PCSK9 by C935 does not involve NRF2 directly. These findings suggest that decrease in the PCSK9 expression by NRF2 is a novel mechanism of cells to unburden to synthesize cholesterol de novo synthesis under oxidative and/or electrophilic stress conditions. In addition, it is suggested that modulation of NRF2 activation along with PCSK9 might serves as a new target of lowering the plasma concentration of LDL Cholesterol.ope

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

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.ope

Grasp55 -/- Mice Display Impaired Fat Absorption and Resistance to High-Fat Diet-Induced Obesity

The Golgi apparatus plays a central role in the intracellular transport of macromolecules. However, molecular mechanisms of Golgi-mediated lipid transport remain poorly understood. Here, we show that genetic inactivation of the Golgi-resident protein GRASP55 in mice reduces whole-body fat mass via impaired intestinal fat absorption and evokes resistance to high-fat diet induced body weight gain. Mechanistic analyses reveal that GRASP55 participates in the Golgi-mediated lipid droplet (LD) targeting of some LD-associated lipases, such as ATGL and MGL, which is required for sustained lipid supply for chylomicron assembly and secretion. Consequently, GRASP55 deficiency leads to reduced chylomicron secretion and abnormally large LD formation in intestinal epithelial cells upon exogenous lipid challenge. Notably, deletion of dGrasp in Drosophila causes similar defects of lipid accumulation in the midgut. These results highlight the importance of the Golgi complex in cellular lipid regulation, which is evolutionary conserved, and uncover potential therapeutic targets for obesity-associated diseases.ope

Welsh onion extract inhibits PCSK9 expression contributing to the maintenance of the LDLR level under lipid depletion conditions of HepG2 cells

Statins mediate the transactivation of PCSK9, which in turn limits their cholesterol-lowering effects via LDL receptor (LDLR) degradation. The objective of the present study was to investigate the mechanism of action by which Welsh onion (Allium fistulosum L. [family Amaryllidaceae]) extract (WOE) regulates LDLR and PCSK9. HepG2 cells were cultured under lipid depletion conditions using a medium supplemented with delipidated serum (DLPS). WOE (50, 100, 200, and 400 μg ml-1) significantly attenuated the DLPS-mediated increases in LDLR, PCSK9, and SREBP2 gene expression. While WOE treatment maintained the DLPS-mediated increases in LDLR protein expression, it dose-dependently and significantly attenuated the DLPS-mediated increases in the protein content of PCSK9. The suppression of PCSK9 was associated with the WOE-mediated reductions in SREBP2, but not HNF1α. WOE also dose-dependently reduced PCSK9 protein expression that was otherwise markedly induced by concomitant statin treatment. WOE-mediated PCSK9 inhibition contributed to LDLR lysosomal degradation suppression, and subsequent LDLR protein stabilization. HPLC analysis indicated that WOE contains kaempferol, quercetin, ferulic acid, and p-coumaric acid. Kaempferol and p-coumaric acid contributed to the maintenance of LDLR expression by inhibiting PCSK9 in lipid depleted HepG2 cells. Altogether, these findings suggest that WOE inhibits PCSK9 transcription and protein expression via the reduction of SREBP2, and decreased PCSK9 further contributes to LDLR degradation prevention and LDLR protein stabilization under conditions of lipoprotein deficiency. The PCSK9 inhibition-mediated mechanism of WOE was likely attributed to the action of kaempferol and p-coumaric acid present in WOE.restrictio