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

Abstract

Proprotein 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.