Abstract 13436: A Novel CETP Inhibitor, K-312, Suppresses PCSK9 Expression Through the Modulation of Its Promoter Activity

Abstract

Background: Inhibition of proprotein convertase subtilisin/ kexin 9 (PCSK9) reduces LDL cholesterol levels. Although RNA and antibody therapeutics against PCSK9 are available, no small molecule inhibitors have been reported. A synthetic compound K-312, developed by us as a novel cholesteryl ester transfer protein (CETP) inhibitor, raises HDL- and lowers LDL-cholesterol levels in animals. We found that K-312 also suppresses PCSK9 expression in hepatocytes. We have explored the underlying mechanism for this effect.

Methods and Results: K-312 decreased PCSK9 mRNA expression by >90% in the human hepatocyte cell line HepG2. K-312 did not affect stability of PCSK9 mRNA as examined via actinomycin D treatment, suggesting its effects on transcriptional regulation. Luciferase assay on deletion mutants of the PCSK9 promoter revealed that K-312 (3 µM, 24 hours) suppressed its transcriptional activity up to 52% by targeting the region within 450 bp upstream of the translation start site, where sterol-regulatory element (SRE), and hepatocyte nuclear factor 1 (HNF1) and histone nuclear factor P (HINFP) binding sites exist. Each site binds to SREBP1/2, HNF1α/β and HINFP, respectively. Mutation of SRE, HNF1 or HINFP binding site diminished suppressive effects of PCSK9 promoter activity by K-312, suggesting the role of these sites. As the binding sites for SREBP1/2, HNF1α/β and HINFP on the PCSK9 promoter are located closely, K-312 may interfere with the interaction between these transcription factors. K-312 (3 µM) also decreased mRNA expression levels of SREBP1 and 2 by 57% and 27%, respectively, but not HNF1α,HNF1β or HINFP. In addition, K-312 treatment decreased the active form of SREBP1. To further examine the effect of K-312 on the binding of SREBPs to SRE, we performed transcription factor binding assay using immobilized human PCSK9 SRE oligo DNA. We found that the addition of K-312 in the binding reaction inhibited binding of SREBP1 or 2 to SRE.

Conclusions: Our findings suggest that the CETP inhibitor K-312 has a unique mechanism of action on suppression of PCSK9 transcription in part by inhibiting SREBP binding to SRE on the PCSK9 promoter and suppressing SREBP expression. K-312 may decrease LDL cholesterol levels through the inhibition of both CETP and PCSK9.