New CETP inhibitor K-312 reduces PCSK9 expression: a potential effect on LDL cholesterol metabolism

Abstract

Despite significant reduction of cardiovascular events by statin treatment, substantial residual risk persists, driving emerging needs for the development of new therapies. We identified a novel cholesteryl ester transfer protein (CETP) inhibitor, K-312, that raises HDL and lowers LDL cholesterol levels in animals. K-312 also suppresses hepatocyte expression of proprotein convertase subtilisin/kexin 9 (PCSK9), a molecule that increases LDL cholesterol. We explored the underlying mechanism for the reduction of PCSK9 expression by K-312. K-312 inhibited in vitro human plasma CETP activity (IC50; 0.06 M). Administration of K-312 to cholesterol-fed New Zealand White rabbits for 18 wk raised HDL cholesterol, decreased LDL cholesterol, and attenuated aortic atherosclerosis. Our search for additional beneficial characteristics of this compound revealed that K-312 decreases PCSK9 expression in human primary hepatocytes and in the human hepatoma cell line HepG2. siRNA silencing of CETP in HepG2 did not compromise the suppression of PCSK9 by K-312, suggesting a mechanism independent of CETP. In HepG2 cells, K-312 treatment decreased the active forms of sterol regulatory element-binding proteins (SREBP-1 and -2) that regulate promoter activity of PCSK9. Chromatin immunoprecipitation assays demonstrated that K-312 decreased the occupancy of SREBP-1 and SREBP-2 on the sterol regulatory element of the PCSK9 promoter. PCSK9 protein levels decreased by K-312 treatment in the circulating blood of cholesterol-fed rabbits, as determined by two independent mass spectrometry approaches, including the recently developed, highly sensitive parallel reaction monitoring method. New CETP inhibitor K-312 decreases LDL cholesterol and PCSK9 levels, serving as a new therapy for dyslipidemia and cardiovascular disease. cholesteryl ester transfer protein; proprotein convertase subtilisin/ kexin 9; atherosclerosis; sterol regulatory element-binding protein; lipoproteins; mass spectrometry; parallel reaction monitoring

Multiple apolipoprotein kinetics measured in human HDL by high-resolution/accurate mass parallel reaction monitoring

“…5). HR/AM-PRM performed on a quadrupole Orbitrap fi rst selects the peptide of interest and then fragments and scans all fragment ions simultaneously ( 7,8,45,47 ). Because PRM itself is a relatively recent method, MRM [sometimes referred to as selected reaction monitoring (SRM)] is the for the fi rst time.…”

CETP Deficiency and Concerns in CETP Inhibitor Development

“…One of the potential causes of lowering LDL is reduction of PCSK9 levels as shown in the studies of anacetrapib and a new small CETP inhibitor (K-312) (35,36).…”

Current Trends and Future Perspectives of State-of-the-Art Proteomics Technologies Applied to Cardiovascular Disease Research

“…We generated an in vitro synthesized form of rabbit PCSK9 and used PRM to develop a rabbit PCSK9 spectral library and confirmed that K-312 did in fact lower circulating PCSK9 by >50%. 10, 25 Targeted proteomics has also been applied to in vivo metabolism studies of mostly LDL and HDL apolipoproteins during lipid-lowering clinical trials. Subjects are injected with amino acid tracer (D3-Leu), which is taken up by primarily the liver Most of these clinical metabolism studies have depended on SRM/MRM to detect very low levels of D3-Leu tracer incorporation into apolipoproteins.…”

“…21-23 Also, the less abundant proteins can be enriched by cellular fractionation methods such as those that separate cellular compartments or those that use immunopurification strategies ( Figure 1C). 24, 25 The dynamic range capabilities of MS continually improve, but the identification and quantification of low-abundance proteins still depend on sample preparation procedures that shift these low signals into the detection range of the instrument either by depletion and/or enrichment approaches.any developments in the field of proteomics have come from the yeast model system 1-3 and from cancer research ( Figure 1A). In addition, immortalized cells (eg, HeLa, HEK293T, RAW264.7, THP-1) have provided mass spectrometrists ample protein with limited variability across biological replicates, factors that are important to establish technical reproducibility when novel workflows are being developed.…”

“…21-23 Also, the less abundant proteins can be enriched by cellular fractionation methods such as those that separate cellular compartments or those that use immunopurification strategies ( Figure 1C). 24, 25 The dynamic range capabilities of MS continually improve, but the identification and quantification of low-abundance proteins still depend on sample preparation procedures that shift these low signals into the detection range of the instrument either by depletion and/or enrichment approaches.…”