Proprotein convertase subtilisin kexin type 9 (PCSK9) lowers the abundance of surface low-density lipoprotein (LDL) receptor through an undefined mechanism. The structure of human PCSK9 shows the subtilisin-like catalytic site blocked by the prodomain in a noncovalent complex and inaccessible to exogenous ligands, and that the C-terminal domain has a novel fold. Biosensor studies show that PCSK9 binds the extracellular domain of LDL receptor with K(d) = 170 nM at the neutral pH of plasma, but with a K(d) as low as 1 nM at the acidic pH of endosomes. The D374Y gain-of-function mutant, associated with hypercholesterolemia and early-onset cardiovascular disease, binds the receptor 25 times more tightly than wild-type PCSK9 at neutral pH and remains exclusively in a high-affinity complex at the acidic pH. PCSK9 may diminish LDL receptors by a mechanism that requires direct binding but not necessarily receptor proteolysis.

Sterol regulatory element-binding proteins (SREBPs) are major transcriptional regulators of cholesterol, fatty acid, and glucose metabolism. Genetic disruption of SREBP activity reduces plasma and liver levels of cholesterol and triglycerides and insulin-stimulated lipogenesis, suggesting that SREBP is a viable target for pharmacological intervention. The proprotein convertase SREBP site 1 protease (S1P) is an important posttranscriptional regulator of SREBP activation. This report demonstrates that 10 M PF-429242 (Bioorg Med Chem Lett 17:4411-4414, 2007), a recently described reversible, competitive aminopyrrolidineamide inhibitor of S1P, inhibits endogenous SREBP processing in Chinese hamster ovary cells. The same compound also down-regulates the signal from an SREluciferase reporter gene in human embryonic kidney 293 cells and the expression of endogenous SREBP target genes in cultured HepG2 cells. In HepG2 cells, PF-429242 inhibited cholesterol synthesis, with an IC 50 of 0.5 M. In mice treated with PF-429242 for 24 h, the expression of hepatic SREBP target genes was suppressed, and the hepatic rates of cholesterol and fatty acid synthesis were reduced. Taken together, these data establish that small-molecule S1P inhibitors are capable of reducing cholesterol and fatty acid synthesis in vivo and, therefore, represent a potential new class of therapeutic agents for dyslipidemia and for a variety of cardiometabolic risk factors associated with diabetes, obesity, and the metabolic syndrome.

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tions in PCSK9 cause hypercholesterolemia in humans ( 1-4 ), whereas loss-of-function mutations in humans lead to a reduction in LDL cholesterol and a marked decrease in the risk of coronary heart disease ( 5-7 ). PCSK9 is a member of the subtilisin serine protease family and the proteinase K subfamily. The protein is synthesized as a proprotein that autocatalytically cleaves to generate mature protein ( 8 ). PCSK9 protein is secreted as a complex containing cleaved N-terminal prodomain, which remains associated with the catalytic domain ( 8 ). The catalytic activity is required for PCSK9 maturation and secretion ( 9 ) but appears not to be essential for the reduction of LDLR (LDL receptor) by secreted PCSK9 ( 10 ).PCSK9 is predominantly expressed in liver, intestine, and to a lesser extent, in kidney ( 8 ). PCSK9 gene expression is regulated by cholesterol via sterol regulatory element-binding protein (SREBP) pathways ( 11,12 ). In mice, PCSK9 gene expression is downregulated by dietary cholesterol and dramatically upregulated by hepatic overexpression of the transactivation domain of SREBP-1a and SREBP-2 ( 12 ). Deletion of PCSK9 in mice leads to elevated liver LDLR protein (but not mRNA) and to reduced plasma total cholesterol, mainly HDL cholesterol ( 13 ). Furthermore, PCSK9 defi ciency increases LDL clearance and enhances the response to statin ( 13 ). Knockdown of PCSK9 by antisense approach also decreases total cholesterol in high-fat-fed mice in an LDLR dependent manner ( 14 ). Hepatic overexpression of murine or human PCSK9 by adenovirus ( 9, 15, 16 ) or transgene ( 17 ) results in the reduction of liver LDLR protein and elevation of LDL cholesterol. Transgenic mice overexpressing PCSK9 in liver accumulated PCSK9 to a level of 100-400 g/ml in plasma and reduced liver LDLR protein in wild-type mice that are parabiotically Abstract Proprotein convertase subtilisin/kexin type 9 (PCSK9) is predominantly expressed in liver and regulates cholesterol metabolism by down regulating liver LDL receptor (LDLR) proteins. Here we report transgenic overexpression of human PCSK9 in kidney increased plasma levels of PCSK9 and subsequently led to a dramatic reduction in liver LDLR proteins. The regulation of LDLR by PCSK9 displayed tissue specifi city, with liver being the most responsive tissue. Even though the PCSK9 transgene was highly expressed in kidney, LDLR proteins were suppressed to a lower extent in this tissue than in liver. Adrenal LDLR proteins were not regulated by elevated plasma PCSK9. hPCSK9 transgene expression and subsequent reduction of liver LDLR led to increases in plasma total cholesterol, LDL cholesterol, and ApoB, which were further increased by a highfat, high-cholesterol diet. We also observed that the size distribution of hPCSK9 in transgenic mouse plasma was heterogeneous. In chow-fed mice, the majority of PCSK9 proteins were in free forms; however, feeding a high-fat, high-cholesterol diet resulted in a shift of hPCSK9 distribution toward larger complexes. PCSK9 distribution in human plasma...