Atorvastatin increases human serum levels of proprotein convertase subtilisin/kexin type 9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has gained attention as a key regulator of serum low density lipoprotein cholesterol (LDL-C) levels. This novel protease causes the degradation of hepatic low density lipoprotein receptors. In humans, gain-of-function mutations in PCSK9 cause a form of familial hypercholesterolemia, whereas loss-of-function mutations result in significantly decreased LDL-C levels and cardiovascular risk. Previous studies have demonstrated that statins upregulate PCSK9 mRNA expression in cultured cells and animal models. In light of these observations, we studied the effect of atorvastatin on circulating PCSK9 protein levels in humans using a sandwich ELISA to quantitate serum PCSK9 levels in patients treated with atorvastatin or placebo for 16 weeks. We observed that atorvastatin (40 mg/day) significantly increased circulating PCSK9 levels by 34% compared with baseline and placebo and decreased LDL-C levels by 42%. These results suggest that the addition of a PCSK9 inhibitor to statin therapy may result in even further LDL-C decreases.     

Fenofibrate treatment increases human serum proprotein convertase subtilisin kexin type 9 levels

Over the past several years, proprotein convertase subtilisin kexin type 9 (PCSK9) has gained significant attention as a key regulator of serum LDL-cholesterol (LDL-C) levels. In humans, gain-of-function mutations in PCSK9 cause a form of familial hypercholesterolemia, whereas loss-of-function mutations result in significantly decreased LDL-C and cardiovascular risk. Our laboratory was the first to demonstrate that atorvastatin increases PCSK9 serum levels, an observation that has since been confirmed by at least two other groups. In light of these observations, we studied the effect of another common lipid-lowering medication, fenofibrate, on circulating PCSK9 protein levels in patients treated with fenofibrate or placebo for 12 weeks. We observed that fenofibrate (200 mg per day) significantly increased circulating PCSK9 levels by 25% compared with baseline. Placebo treatment, in comparison, had no effect on PCSK9 levels. Interestingly, fenofibrate-induced increases in serum PCSK9 levels were highly correlated with fenofibrate-induced changes in HDL-C and triglyceride levels, as well as with fenofibrate-induced changes in LDL-C levels. These results suggest an explanation for why fibrates do not achieve as much LDL-C lowering as might otherwise be expected and indicate that the addition of a PCSK9 inhibitor to fibrate therapy may result in additional beneficial LDL-C lowering.     

A novel splicing variant of proprotein convertase subtilisin/kexin type 9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is the most recently identified member of the proprotein convertase family. Genetic and cell biology studies have suggested a critical role of PCSK9 in regulating low-density lipoprotein receptor (LDLR) protein levels and thus modulating plasma LDL cholesterol. Recent data on the molecular basis for PCSK9 action support the model in which PCSK9 is self-cleaved, secreted, and tightly bound to the EGF-A repeat of LDLR extracellular domain. PCSK9 binding to LDLR is essential for the ensuing receptor-mediated endocytosis and is speculated to lock LDLR in a specific conformation that favors degradation in lysosomal compartment instead of recycling back to plasma membrane. We report here a novel human PCSK9 splicing variant, which we named PCSK9sv. PCSK9sv had an in-frame deletion of the eighth exon of 58 amino acids and was expressed in multiple tissues, including liver, small intestine, prostate, uterus, brain, and adipose tissue. Unlike wild-type PCSK9, which is secreted, PCSK9sv expressed in human embryonic kidney HEK293 cells failed to process the prosegment intracellularly and thus was not secreted into the medium. Examination of potential functions revealed that PCSK9sv did not change the LDLR protein levels. Two mutations that have been reported in humans with the associated changes in plasma LDL cholesterol were within exon 8, and thus the expression and function of the two mutants were studied. Both N425S and A443T mutants were processed normally, secreted, and reduced LDLR levels. However, the physiological function of this novel splicing variant of PCSK9 has yet to be determined.     

Molecular characterization of proprotein convertase subtilisin/kexin type 9-mediated degradation of the LDLR

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that promotes degradation of cell surface LDL receptors (LDLRs) in selected cell types. Here we used genetic and pharmacological inhibitors to define the pathways involved in PCSK9-mediated LDLR degradation. Inactivating mutations in autosomal recessive hypercholesterolemia (ARH), an endocytic adaptor, blocked PCSK9-mediated LDLR degradation in lymphocytes but not in fibroblasts. Thus, ARH is not specifically required for PCSK9-mediated LDLR degradation. Knockdown of clathrin heavy chain with siRNAs prevented LDLR degradation. In contrast, prevention of ubiquitination of the LDLR cytoplasmic tail, inhibition of proteasomal activity, or disruption of proteins required for lysosomal targeting via macroautophagy (autophagy related 5 and 7) or the endosomal sorting complex required for trafficking (ESCRT) pathway (hepatocyte growth factor-regulated Tyr-kinase substrate and tumor suppressor gene 101) failed to block PCSK9-mediated LDLR degradation. These findings are consistent with a model in which the LDLR-PCSK9 complex is internalized via clathrin-mediated endocytosis and then routed to lysosomes via a mechanism that does not require ubiquitination and is distinct from the autophagy and proteosomal degradation pathways. Finally, the PCSK9-LDLR complex appears not to be transported by the canonical ESCRT pathway.     

Secreted proprotein convertase subtilisin/kexin type 9 reduces both hepatic and extrahepatic low-density lipoprotein receptors in vivo

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that is known to reduce hepatic low-density lipoprotein receptor (LDLR) levels and increase plasma LDL cholesterol. It is not clear, however, whether secreted PCSK9 degrades extrahepatic LDLRs. We present evidence that recombinant PCSK9, either injected intravenously into or expressed in the liver of C57BL/6 mice, significantly reduced LDLR levels in multiple extrahepatic tissues. During the initial characterization, we found that injected human recombinant PCSK9 at 30 μg/mouse had a half-life of 15 min in serum in mice. Hepatic LDLR levels were reduced within 30 min and the degradation of hepatic LDLR reached the maximum 2 h after the initial protein injection. Endocytosis of PCSK9 in liver occurred within 5 min of protein injection and internalized PCSK9 was only barely detectable within 1 h. When extrahepatic LDLRs were examined by Western blotting analysis, we found significant reductions of LDLRs in multiple extrahepatic tissues including lung, adipose and kidney along with the more dramatic reduction of LDLRs in liver. These studies were further extended using adenoviral expression of human PCSK9 in C57BL/6 mice to demonstrate that PCSK9 produced in liver impacted extrahepatic tissue LDLR levels as well. Taken together, our studies indicate that secreted PCSK9 can potentially impact extrahepatic tissue cholesterol homeostasis by regulating extrahepatic tissue LDLR levels.     

Dual mechanisms for the fibrate-mediated repression of proprotein convertase subtilisin/kexin type 9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is associated with familial autosomal dominant hypercholesterolemia and is a natural inhibitor of the LDL receptor (LDLr). PCSK9 is degraded by other proprotein convertases: PC5/6A and furin. Both PCSK9 and the LDLr are up-regulated by the hypocholesterolemic statins. Thus, inhibitors or repressors of PCSK9 should amplify their beneficial effects. In the present study, we showed that PPARalpha activation counteracts PCSK9 induction by statins by repressing PCSK9 promoter activity and by increasing PC5/6A and furin expression. Quantification of mRNA and protein levels showed that various fibrates decreased PCSK9 and increased PC5/6A and furin expression. Fenofibric acid (FA) reduced PCSK9 protein content in immortalized human hepatocytes (IHH) as well as its cellular secretion. FA suppressed PCSK9 induction by statins or by the liver X receptor agonist TO901317. PCSK9 repression is occurring at the promoter level. We showed that PC5/6A and furin fibrate-mediated up-regulation is PPARalpha-dependent. As a functional test, we observed that FA increased by 30% the effect of pravastatin on the LDLr activity in vitro. In conclusion, fibrates simultaneously decreased PCSK9 expression while increasing PC5/6A and furin expression, indicating a broad action of PPARalpha activation in proprotein convertase-mediated lipid homeostasis. Moreover, this study validates the functional relevance of a combined therapy associating PCSK9 repressors and statins.     

Affinity and kinetics of proprotein convertase subtilisin/kexin type 9 binding to low-density lipoprotein receptors on HepG2 cells

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that regulates the number of cell surface low-density lipoprotein receptors (LDLRs) and the levels of low-density lipoprotein cholesterol in plasma. Intact cells have not previously been used to determine the characteristics of binding of PCSK9 to LDLR. Using PCSK9 iodinated by the tyramine cellobiose (TC) method ([(125)I]TC-PCSK9), we measured the affinity and kinetics of binding of PCSK9 to LDLR on HepG2 cells at 4 °C. The extent of [(125)I]TC-PCSK9 binding increased as cell surface LDLR density increased. Unlabeled wild-type and two gain-of-function mutants of PCSK9 reduced binding of [(125)I]TC-PCSK9. The Scatchard plot of the binding-inhibition curve was curvilinear, indicative of high-affinity and low-affinity sites for PCSK9 binding on HepG2 cells. Nonlinear regression analysis of the binding data also indicated that a two-site model better fitted the data. The time course of [(125)I]TC-PCSK9 binding showed two phases in the association kinetics. Dissociation of [(125)I]TC-PCSK9 also occurred in two phases. Unlabeled PCSK9 accelerated the dissociation of [(125)I]TC-PCSK9. At low pH, only one phase of dissociation was apparent. Furthermore, the dissociation of [(125)I]TC-PCSK9 under pre-equilibrium conditions was faster than under equilibrium conditions. Overall, the data suggest that PCSK9 binding to cell surface LDLR cannot be described by a simple bimolecular reaction. Possible interpretations that can account for these observations are discussed.     

Antisense inhibition of proprotein convertase subtilisin/kexin type 9 reduces serum LDL in hyperlipidemic mice

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of a family of proteases that is thought to promote the degradation of the low density lipoprotein receptor (LDLR) through an as yet undefined mechanism. We developed second generation antisense oligonucleotide (ASO) inhibitors targeting murine PCSK9 to determine their potential as lipid-lowering agents. Administration of a PCSK9 ASO to high fat-fed mice for 6 weeks reduced total cholesterol and LDL by 53% and 38%, respectively. Moreover, inhibition of PCSK9 expression resulted in a 2-fold increase in hepatic LDLR protein levels. This phenotype closely resembles that reported previously in Pcsk9-deficient mice. The absence of cholesterol lowering in Ldlr-deficient mice effectively demonstrated a critical role for this receptor in mediating the lipid-lowering effects of PCSK9 inhibition. Antisense inhibition of PCSK9 is an attractive and novel therapeutic approach for treating hypercholesterolemia in human.     

Characterization of a quantitative method to measure free proprotein convertase subtilisin/kexin type 9 in human serum

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that plays an important role in the regulation of serum low-density lipoprotein (LDL) cholesterol by downregulation of LDL receptor, and as such is considered a novel target in cholesterol lowering therapy. In support of the drug development program for Evolocumab, a fully human IgG₂ antibody that targets PCSK9, a quantitative ELISA to measure free PCSK9 in human serum was developed. PCSK9 serves as a biomarker of pharmacological response during treatment, and measuring levels of the free ligand post-dosing was of interest as an aid to establishing the pharmacokinetic and pharmacodynamic properties of the therapeutic. Given the complexities associated with the measurement of free ligand in the presence of high concentrations of circulating drug, it was important to challenge the method with experiments designed to assess ex vivo conditions that have the potential to affect the binding equilibrium of drug and ligand within test samples during routine sampling handling and assay conditions. Herein, we report results of experiments that were conducted to characterize the assay in alignment with regulatory guidance and industry standards, and to establish evidence that the method is measuring the free ligand in circulation at the time serum was collected. A robust supporting data package was generated that demonstrates the method specifically and reproducibly measures the free ligand, and is suitable for its intended use.     

Novel domain interaction regulates secretion of proprotein convertase subtilisin/kexin type 9 (PCSK9) protein

PCSK9 (proprotein convertase subtilisin/kexin type 9) has emerged as a novel therapeutic target for hypercholesterolemia due to its LDL receptor (LDLR)-reducing activity. Although its structure has been solved, the lack of a detailed understanding of the structure-function relation hinders efforts to develop small molecule inhibitors. In this study, we used mutagenesis and transfection approaches to investigate the roles of the prodomain (PD) and the C-terminal domain (CD) and its modules (CM1-3) in the secretion and function of PCSK9. Deletion of PD residues 31-40, 41-50, or 51-60 did not affect the self-cleavage, secretion, or LDLR-degrading activity of PCSK9, whereas deletion of residues 61-70 abolished all of these functions. Deletion of the entire CD protein did not impair PCSK9 self-cleavage or secretion but completely abolished LDLR-degrading activity. Deletion of any one or two of the CD modules did not affect self-cleavage but influenced secretion and LDLR-reducing activity. Furthermore, in cotransfection experiments, a secretion-defective PD deletion mutant (ΔPD) was efficiently secreted in the presence of CD deletion mutants. This was due to the transfer of PD from the cotransfected CD mutants to the ΔPD mutant. Finally, we found that a discrete CD protein fragment competed with full-length PCSK9 for binding to LDLR in vitro and attenuated PCSK9-mediated hypercholesterolemia in mice. These results show a previously unrecognized domain interaction as a critical determinant in PCSK9 secretion and function. This knowledge should fuel efforts to develop novel approaches to PCSK9 inhibition.     

Regulation of epithelial sodium channel trafficking by proprotein convertase subtilisin/kexin type 9 (PCSK9)

The epithelial Na(+) channel (ENaC) is critical for Na(+) homeostasis and blood pressure control. Defects in its regulation cause inherited forms of hypertension and hypotension. Previous work found that ENaC gating is regulated by proteases through cleavage of the extracellular domains of the α and γ subunits. Here we tested the hypothesis that ENaC is regulated by proprotein convertase subtilisin/kexin type 9 (PCSK9), a protease that modulates the risk of cardiovascular disease. PCSK9 reduced ENaC current in Xenopus oocytes and in epithelia. This occurred through a decrease in ENaC protein at the cell surface and in the total cellular pool, an effect that did not require the catalytic activity of PCSK9. PCSK9 interacted with all three ENaC subunits and decreased their trafficking to the cell surface by increasing proteasomal degradation. In contrast to its previously reported effects on the LDL receptor, PCSK9 did not alter ENaC endocytosis or degradation of the pool of ENaC at the cell surface. These results support a role for PCSK9 in the regulation of ENaC trafficking in the biosynthetic pathway, likely by increasing endoplasmic reticulum-associated degradation. By reducing ENaC channel number, PCSK9 could modulate epithelial Na(+) absorption, a major contributor to blood pressure control.     

Characterization of a quantitative method to measure free proprotein convertase subtilisin/kexin type 9 in human serum

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that plays an important role in the regulation of serum low-density lipoprotein (LDL) cholesterol by downregulation of LDL receptor, and as such is considered a novel target in cholesterol lowering therapy. In support of the drug development program for Evolocumab, a fully human IgG₂ antibody that targets PCSK9, a quantitative ELISA to measure free PCSK9 in human serum was developed. PCSK9 serves as a biomarker of pharmacological response during treatment, and measuring levels of the free ligand post-dosing was of interest as an aid to establishing the pharmacokinetic and pharmacodynamic properties of the therapeutic. Given the complexities associated with the measurement of free ligand in the presence of high concentrations of circulating drug, it was important to challenge the method with experiments designed to assess ex vivo conditions that have the potential to affect the binding equilibrium of drug and ligand within test samples during routine sampling handling and assay conditions. Herein, we report results of experiments that were conducted to characterize the assay in alignment with regulatory guidance and industry standards, and to establish evidence that the method is measuring the free ligand in circulation at the time serum was collected. A robust supporting data package was generated that demonstrates the method specifically and reproducibly measures the free ligand, and is suitable for its intended use.     

New hypotheses about the structure–function of proprotein convertase subtilisin/kexin type 9: Analysis of the epidermal growth factor‐like repeat A docking site using WaterMap

LDL cholesterol (LDL‐C) is cleared from plasma via cellular uptake and internalization processes that are largely mediated by the low‐density lipoprotein cholesterol receptor (LDL‐R). LDL‐R is targeted for lysosomal degradation by association with proprotein convertase subtilisin‐kexin type 9 (PCSK9). Gain of function mutations in PCSK9 can result in excessive loss of receptors and dyslipidemia. On the other hand, receptor‐sparing phenomena, including loss‐of‐function mutations or inhibition of PCSK9, can lead to enhanced clearance of plasma lipids. We hypothesize that desolvation and resolvation processes, in many cases, constitute rate‐determining steps for protein–ligand association and dissociation, respectively. To test this hypothesis, we analyzed and compared the predicted desolvation properties of wild‐type versus gain‐of‐function mutant Asp374Tyr PCSK9 using WaterMap, a new in silico method for predicting the preferred locations and thermodynamic properties of water solvating proteins (“hydration sites”). We compared these results with binding kinetics data for PCSK9, full‐length LDL‐R ectodomain, and isolated EGF‐A repeat. We propose that the fast k_on and entropically driven thermodynamics observed for PCSK9‐EGF‐A binding stem from the functional replacement of water occupying stable PCSK9 hydration sites (i.e., exchange of PCSK9 H‐bonds from water to polar EGF‐A groups). We further propose that the relatively fast k_off observed for EGF‐A unbinding stems from the limited displacement of solvent occupying unstable hydration sites. Conversely, the slower k_off observed for EGF‐A and LDL‐R unbinding from Asp374Tyr PCSK9 stems from the destabilizing effects of this mutation on PCSK9 hydration sites, with a concomitant increase in the persistence of the bound complex. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Discovery and SAR analysis of phenylbenzo[d][1,3]dioxole-based proprotein convertase subtilisin/kexin type 9 inhibitors

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a novel therapeutic target for the development of cholesterol-lowering drugs. In the discovery of PCSK9/LDLR (low-density lipoprotein receptor) protein-protein interaction (PPI) impairing small molecules, a total of 47 phenylbenzo[d][1,3] dioxole-based compounds were designed and synthesised. The result revealed that the 4-chlorobenzyl substitution in the amino group is important for the PPI disrupting activity. In the hepatocyte-based functional tests, active compounds such as A12, B1, B3, B4 and B14, restored the LDLR levels on the surface of hepatic HepG2 cells and increased extracellular LDL uptake in the presence of PCSK9. It is notable that molecule B14 exhibited good performance in all the evaluations. Collectively, novel structures targeting PCSK9/LDLR PPI have been developed with hypolipidemic potential. Further structural modification of derived active compounds is promising in the discovery of lead compounds with improved activity for the treatment of hyperlipidaemia-related disorders.