Proprotein Convertase Subtilisin-like Kexin Type Research Articles

Identification of 4-amino-2-Pyridones as new potent PCSK9 inhibitors: From phenotypic hit discovery to in vivo tolerability

Among the strategies to overcome the underperformance of statins in cardiovascular diseases (CVDs), the development of drugs targeting the Proprotein Convertase Subtilisin-like Kexin type 9 (PCSK9) is considered one of the most promising. However, only anti-PCSK9 biological drugs have been approved to date, and orally available small-molecules for the treatment of hypercholesterolemic conditions are still missing on the market. In the present work, we describe the application of a phenotypic approach to the identification and optimization of 4-amino-2-pyridone derivatives as a new chemotype with anti-PCSK9 activity. Starting from an in-house collection of compounds, functional assays on HepG2 cells followed by a chemistry-driven hit optimization campaign, led to the potent anti-PCSK9 candidate 5c. This compound, at 5 μM, totally blocked PCSK9 secretion from HepG2 cells, significantly increased LDL receptor (LDLR) expression, and acted cooperatively with simvastatin by reducing its induction of PCSK9 expression. Finally, compound 5c also proved to be well tolerated in C57BL/6J mice at the tested concentration (40 mg/kg) with no sign of toxicity or behavior modifications.

Improving Stability Enhances In Vivo Efficacy of a PCSK9 Inhibitory Peptide.

Optimization of peptide stability is essential for the development of peptides as bona fide alternatives to approved monoclonal antibodies. This is clearly the case for the many peptides reported to antagonize proprotein convertase subtilisin-like/kexin type 9 (PCSK9), a clinically validated target for lowering cholesterol. However, the effects of optimization of stability on in vivo activity and particularly the effects of binding to albumin, an emerging drug design paradigm, have not been studied for such peptide leads. In this study, we optimized a PCSK9 inhibitory peptide by mutagenesis and then by conjugation to a short lipidated tag to design P9-alb fusion peptides that have strong affinity to human serum albumin. Although attachment of the tag reduced activity against PCSK9, which was more evident in surface plasmon resonance binding and enzyme-linked immunosorbent competition assays than in cellular assays of activity, activity remained in the nanomolar range (∼40 nM). P9-alb peptides were exceptionally stable in human serum and had half-lives exceeding 48 h, correlating with longer half-lives in mice (40.8 min) compared to the unconjugated peptide. Furthermore, the decrease in in vitro binding was not deleterious to in vivo function, showing that engendering albumin binding improved low-density lipoprotein receptor recovery and cholesterol-lowering activity. Indeed, the peptide P9-albN2 achieved similar functional endpoints as the approved anti-PCSK9 antibody evolocumab, albeit at higher doses. Our study illustrates that optimization of stability instead of binding affinity is an effective way to improve in vivo function.

Ocular disorders associated with PCSK9 inhibitors: A pharmacovigilance disproportionality analysis.

To identify and characterize ocular adverse events (oAEs) that are significantly associated with proprotein convertase subtilisin-like/kexin type 9 (PCSK9) inhibitors using the US Food and Drug Administration Adverse Event Reporting System (FAERS). We conducted a disproportionality analysis of PCSK9 inhibitors in the FAERS (01/2004-12/2021). The association between PCSK9 inhibitors and oAEs was evaluated using the information component (IC) and the reporting odds ratio (ROR), and the difference in oAEs between evolocumab and alirocumab was compared using the ROR. Different sensitivity analyses were conducted to evaluate the robustness of results. A total of 103 531 reports involving at least 1 PCSK9 inhibitor were found in the FAERS. PCSK9 inhibitors were associated with higher reporting of increased lacrimation (IC 0.27 [95% confidence interval {CI} 0.02-0.45]; ROR 1.21 [95% CI 1.04-1.40]), seasonal allergy (IC 0.39 [95% CI 0.04-0.64]; ROR 1.32 [95% CI 1.07-1.62]) and eye operation (IC 0.66 [95% CI 0.04-1.10]; ROR 1.60 [95% CI 1.11-2.30]) compared with the full database, and there was no difference between evolocumab and alirocumab. Sensitivity analyses showed that the disproportionate signals of increased lacrimation disappeared after excluding cases with other lipid-lowering agents in the combined drugs. Except for eye operations, most of these adverse events occurred within 30 days of the first dose, and all 3 oAEs were mostly reported in women and individuals >65 years. This pharmacovigilance study identified a possible signal of ocular disorders associated with PCSK9 inhibitors and encourages paying attention to at-risk populations in PCSK9 inhibitors medication.

Protective Mechanism of Proprotein Convertase Subtilisin-Like Kexin Type 9 Inhibitor on Rats with Middle Cerebral Artery Occlusion-Induced Cerebral Ischemic Infarction.

The aim of this study was to research the mechanism of proprotein convertase subtilisin-like kexin type 9 (PCSK9) inhibitor in neural protective effect on rat cerebral ischemic reperfusion injury (I/RI). The transient middle cerebral artery occlusion (tMCAO) model of rats was prepared by the suture method, and PCSK9 inhibitor was injected intraperitoneally immediately after I/R. The rats were scored for neurological deficits and the cerebral infarction volume was measured. The brain tissues were collected and western blot (WB) was used to detect the expression of PCSK9. The rat cortical neural stem cells were treated with oxygen glucose deprivation (OGD) to establish a cell model of ischemia/reperfusion. WB was used to detect the expression of PCSK9 and the apoptosis-related pathway proteins. After interfering with the expression of PCSK9 siRNA, the cell viability (cell counting kit-8 assay) and apoptosis (TUNEL staining, Annexin V/PI method) were detected, and the cell proliferation was detected by EdU staining and flow cytometry. The expression of PCSK9 in the brain tissue of the MCAO group was dramatically increased. PCSK9 inhibitor can improve neurobehavioral scores and reduce apoptosis and infarct volume. An OGD model of neural stem cells in vitro was constructed. Inhibiting PCSK9 with si-PCSK9 can increase cell viability, promote cell proliferation, and also reduce cell apoptosis. Inhibition of PCSK9 can decrease the cerebral infarct volume in rats with cerebral I/RI and improve the neural function. Mechanically, inhibition of PCSK9 can lead to the decrease of nerve cell apoptosis and promotion of cell proliferation.

Associations of pro-protein convertase subtilisin-like kexin type 9, soluble low-density lipoprotein receptor and coronary artery disease: A case-control study

BackgroundLow-density lipoprotein receptor (LDLR) is the primary pathway for removal of cholesterol from the circulation, pro-protein convertase subtilisin-like kexin type 9 (PCSK9) is a secreted protease that binds to and promotes degradation of the LDLR protein. The goal of this case-control study was to investigate the role of soluble LDLR (sLDLR) and PCSK9 in coronary artery disease (CAD) and investigate the relationship between these two indices and CAD. MethodsIn a sample of 144 Chinese patients recruited between January 2018 and August 2018, 81 cases with mild and severe stenosis characterized by coronary angiograph (CAG) and 63 healthy controls were selected using the propensity score matching (PSM) based on demographics and medical history. sLDLR and PCSK9 concentrations were determined using enzyme-linked immunosorbent assay (ELISA), Immuno-precipitation (IP) and western blotting. Multivariable logistic models were used to assess the associations between the degree of coronary artery stenosis and the biomarkers of interest. ResultsHigher PCSK9 was found to be a significant predictor of coronary artery stenosis when comparing cases who had severe stenosis vs. controls (OR=1.016, 95%CI: 1.009, 1.024), and cases who had mild stenosis vs. controls (OR=1.009, 95%CI: 1.003, 1.015). sLDLR was positively corrected with PCSK9, which confounded the association between CAD and PCSK9. Compared to patients with mild-stenosis, patients with severe-stenosis also showed a higher level of PCSK9 (OR=1.007, 95%CI: 1.001, 1.013). ConclusionsThese findings suggest that elevated PSCK9 may contribute to the odds of developing CAD, with a higher degree of coronary artery stenosis.

A Series of Novel, Highly Potent, and Orally Bioavailable Next-Generation Tricyclic Peptide PCSK9 Inhibitors.

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) is a key regulator of plasma LDL-cholesterol (LDL-C) and a clinically validated target for the treatment of hypercholesterolemia and coronary artery disease. Starting from second-generation lead structures such as 2, we were able to refine these structures to obtain extremely potent bi- and tricyclic PCSK9 inhibitor peptides. Optimized molecules such as 44 demonstrated sufficient oral bioavailability to maintain therapeutic levels in rats and cynomolgus monkeys after dosing with an enabled formulation. We demonstrated target engagement and LDL lowering in cynomolgus monkeys essentially identical to those observed with the clinically approved, parenterally dosed antibodies. These molecules represent the first report of highly potent and orally bioavailable macrocyclic peptide PCSK9 inhibitors with overall profiles favorable for potential development as once-daily oral lipid-lowering agents. In this manuscript, we detail the design criteria and multiparameter optimization of this novel series of PCSK9 inhibitors.

Series of Novel and Highly Potent Cyclic Peptide PCSK9 Inhibitors Derived from an mRNA Display Screen and Optimized via Structure-Based Design.

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) is a key regulator of plasma LDL-cholesterol (LDL-C) and a clinically validated target for the treatment of hypercholesterolemia and coronary artery disease. In this paper, we describe a series of novel cyclic peptides derived from an mRNA display screen which inhibit the protein-protein interaction between PCSK9 and LDLR. Using a structure-based drug design approach, we were able to modify our original screening lead 2 to optimize the potency and metabolic stability and minimize the molecular weight to provide novel bicyclic next-generation PCSK9 inhibitor peptides such as 78. These next-generation peptides serve as a critical foundation for continued exploration of potential oral, once-a-day PCSK9 therapeutics for the treatment of cardiovascular disease.

Statins plus ezetimibe in the era of proprotein convertase subtilisin/ kexin type 9 inhibitors.

Statins are first‑line agents used in patients with dyslipidemia, which show established benefits in reducing low‑density lipoprotein cholesterol (LDL‑C) levels and decreasing the rate of cardiovascular events. However, a considerable number of patients on statins do not achieve target LDL‑C levels, even at maximally tolerated statin doses, or are intolerant to intensive statin therapy. These patients can benefit from the addition of a nonstatin lipid‑lowering agent, and recent cholesterol guidelines have put greater focus on combination lipid‑lowering therapy. In patients who cannot achieve target treatment goals with statin therapy alone, the addition of a cholesterol absorption inhibitor, ezetimibe, leads to further LDL‑C reduction with good tolerability and decreases cardiovascular morbidity and mortality. The more recent proprotein convertase subtilisin‑like / kexin type 9 (PCSK‑9) inhibitors can lower LDL‑C by additional 45% to 65% and are also well tolerated. These complementary approaches for LDL‑C lowering in patients treated with statins decrease LDL‑C levels more effectively than statin monotherapy. As no threshold level has been established below which LDL‑C lowering benefits disappear, the early application of a combination treatment strategy may lead to improved cardiovascular outcomes, particularly in high‑risk patients. This review examines the rationale, advantages, and potential barriers to combination lipid‑lowering therapy with reference to the current guideline recommendations.

Accumulation of Pericardial Fat Is Associated With Alterations in Heart Rate Variability Patterns in Hypercholesterolemic Pigs.

Heart rate variability (HRV) and pulse rate variability are indices of autonomic cardiac modulation. Increased pericardial fat is associated with worse cardiovascular outcomes. We hypothesized that progressive increases in pericardial fat volume and inflammation prospectively dampen HRV in hypercholesterolemic pigs. WT (wild type) or PCSK9 (proprotein convertase subtilisin-like/kexin type-9) gain-of-function Ossabaw mini-pigs were studied in vivo before and after 3 and 6 months of a normal diet (WT-normal diet, n=4; PCSK9-normal diet, n=6) or high-fat diet (HFD; WT-HFD, n=3; PCSK9-HFD, n=6). The arterial pulse waveform was obtained from an arterial telemetry transmitter to analyze HRV indices, including SD (SD of all pulse-to-pulse intervals over a single 5-minute period), root mean square of successive differences, proportion >50 ms of normal-to-normal R-R intervals, and the calculated ratio of low-to-high frequency distributions (low-frequency power/high-frequency power). Pericardial fat volumes were evaluated using multidetector computed tomography and its inflammation by gene expression of TNF (tumor necrosis factor)-α. Plasma lipid panel and norepinephrine level were also measured. At diet completion, hypercholesterolemic PCSK9-HFD had significantly (P<0.05 versus baseline) depressed HRV (SD of all pulse-to-pulse intervals over a single 5-minute period, root mean square of successive differences, proportion >50 ms, high-frequency power, low-frequency power), and both HFD groups had higher sympathovagal balance (SD of all pulse-to-pulse intervals over a single 5-minute period/root mean square of successive differences, low-frequency power/high-frequency power) compared with normal diet. Pericardial fat volumes and LDL (low-density lipoprotein) cholesterol concentrations correlated inversely with HRV and directly with sympathovagal balance, while sympathovagal balance correlated directly with plasma norepinephrine. Pericardial fat TNF-α expression was upregulated in PCSK9-HFD, colocalized with nerve fibers, and correlated inversely with root mean square of successive differences and proportion >50 ms. Progressive pericardial fat expansion and inflammation are associated with a fall in HRV in Ossabaw mini-pigs, implying aggravated autonomic imbalance. Hence, pericardial fat accumulation is associated with alterations in HRV and the autonomic nervous system. Visual Overview: A visual overview is available for this article.

Structure and Function of Proprotein Convertase Subtilisin/kexin Type 9 (PCSK9) in Hyperlipidemia and Atherosclerosis.

One of the important factors in Low-Density Lipoprotein (LDL) metabolism is the LDL receptor (LDLR) by its capacity to bind and subsequently clear cholesterol derived from LDL (LDL-C) in the circulation. Proprotein Convertase Subtilisin-like Kexin type 9 (PCSK9) is a newly discovered serine protease that destroys LDLR in the liver and thereby controls the levels of LDL in plasma. Inhibition of PCSK9-mediated degradation of LDLR has, therefore, become a novel target for lipid-lowering therapy. We review the current understanding of the structure and function of PCSK9 as well as its implications for the treatment of hyperlipidemia and atherosclerosis. New treatments such as monoclonal antibodies against PCSK9 may be useful agents to lower plasma levels of LDL and hence prevent atherosclerosis. PCSK9's mechanism of action is not yet fully clarified. However, treatments that target PCSK9 have shown striking early efficacy and promise to improve the lives of countless patients with hyperlipidemia and atherosclerosis.

Heterozygous Null LDLR Mutation in a Familial Hypercholesterolemia Patient With an Atypical Presentation Because of Alcohol Abuse

Patients with familial hypercholesterolemia (FH; Online Mendelian Inheritance in Man #143890) have lifelong elevations in low-density lipoprotein cholesterol (LDL-C) that result in deposition of cholesterol in tendons— referred to as tendon xanthomas and occurring in ≈19% of heterozygous FH patients1—and an increased risk of premature cardiovascular disease. FH is an autosomal dominant disorder resulting from mutations in the LDL receptor ( LDLR ), APOB (apolipoprotein B), or PCSK9 (proprotein convertase subtilisin-like kexin type 9) genes.2 Heterozygous mutations in LDLR are the most common cause of FH. In addition to elevated LDL-C, patients harboring LDLR mutations may have lower levels of high-density lipoprotein cholesterol and only mild, if any, elevations in triglycerides.3–5 Severe hypertriglyceridemia is rarely seen in FH patients.5 We report an alcoholic patient who presented with severely elevated serum triglycerides and both tuberous and tendon xanthomas in whom the diagnosis of FH was confirmed by genetic testing. A 75-year-old Hispanic man was referred to us for genetic testing as part of a larger research protocol.6 His medical history included 3-vessel coronary artery bypass graft at age 47 and a myocardial infarction at age 60, hypothyroidism, osteoporosis, gastro-esophageal reflux, hypertension, recurrent major depressive disorder with a history of a suicide attempt, heavy alcohol and tobacco abuse, and Alzheimer dementia (initially diagnosed at age 72 with superimposed frontal lobe damage from prior alcohol use). Hyperlipidemia was first diagnosed at age 34, although his wife noted that he had Achilles tendon xanthomas and arcus senilis since age 22 (when they first met). He had been tried on multiple medications (he could not recall exact names) and ultimately underwent ileal bypass surgery at age 41 for treatment. The ileal bypass was reversed at age 60 because of multiple episodes of intestinal obstruction. At age 66, he was …

An association analysis between a missense polymorphism at the pig PCSK9 gene and serum lipid and meat quality traits in Duroc pigs

A genome-wide association analysis in a Duroc pig population allowed us detecting a genomic region on pig chromosome 6 (141–147Mb) that was associated with serum cholesterol (CHOL), triglyceride (TRIG) and low-density lipoprotein (LDL) concentrations. This region contains the proprotein convertase subtilisin-like kexin type 9 (PCSK9) gene (SSC6, 145Mb), which has a key role in the regulation of CD36, LDL receptor and very low density lipoprotein (VLDL) receptor levels. In the current work, we have genotyped by pyrosequencing a missense PCSK9 c.1222G>A mutation (E408K) in 273 Duroc pigs. The performance of an association analysis with the GEMMA software did not reveal any association between PCSK9 genotype and serum lipid concentrations, evidencing that this polymorphism is not the causal mutation of the CHOL, TRIG, and LDL SSC6 QTL. However, we detected an association, that was highly significant at the nominal level, between PCSK9 genotype and palmitelaidic content at the gluteus medius muscle (P-value = 0.008). There is evidence that PCSK9 induces the degradation of CD36, a key long-chain fatty acid transporter, and that it may decrease the uptake of palmitate. However, the E408K polymorphism analysed in the current work is not predicted to be deleterious, suggesting that the associations found are probably due to the linkage of this polymorphism with a causal mutation yet to be found.

Transcription Factor Hepatocyte Nuclear Factor-1β Regulates Renal Cholesterol Metabolism.

HNF-1β is a tissue-specific transcription factor that is expressed in the kidney and other epithelial organs. Humans with mutations in HNF-1β develop kidney cysts, and HNF-1β regulates the transcription of several cystic disease genes. However, the complete spectrum of HNF-1β-regulated genes and pathways is not known. Here, using chromatin immunoprecipitation/next generation sequencing and gene expression profiling, we identified 1545 protein-coding genes that are directly regulated by HNF-1β in murine kidney epithelial cells. Pathway analysis predicted that HNF-1β regulates cholesterol metabolism. Expression of dominant negative mutant HNF-1β or kidney-specific inactivation of HNF-1β decreased the expression of genes that are essential for cholesterol synthesis, including sterol regulatory element binding factor 2 (Srebf2) and 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr). HNF-1β mutant cells also expressed lower levels of cholesterol biosynthetic intermediates and had a lower rate of cholesterol synthesis than control cells. Additionally, depletion of cholesterol in the culture medium mitigated the inhibitory effects of mutant HNF-1β on the proteins encoded by Srebf2 and Hmgcr, and HNF-1β directly controlled the renal epithelial expression of proprotein convertase subtilisin-like kexin type 9, a key regulator of cholesterol uptake. These findings reveal a novel role of HNF-1β in a transcriptional network that regulates intrarenal cholesterol metabolism.

Abstract 10829: Biological and Pharmacological Properties of the Protease Proprotein Convertase Subtilisin-Like Kexin Type 9 Antagonists (Best of Basic Science Abstract)

Protease proprotein convertase subtilisin-like kexin type 9 (PCSK9) is a new important target for the treatment of hypercholesterolemia. Currently, several pharmaceutical companies are racing to develop new drugs against PCSK9, which could be the newest weapon against heart disease since the introduction of statins in the 80’s. However, those companies have focused primarily on the development of mAbs, not small molecules, as drugs. PCSK9 is a serine protease that is synthesized as a zymogen that undergoes autocatalytic processing and secretion. Secreted PCSK9 binds to the LDL-receptor (LDLR) and chaperones it to the degradation pathway. This study focused on the identification of antagonists of the PCSK9/LDLR interaction and explored their mechanism of action using a range of functional assays, as well as determined pharmacokinetic (PK) and pharmacodynamic (PD) properties of the lead compounds. Potential inhibitors of the PCSK9/LDLR interaction were identified through a virtual screen at the PCSK9 surface where the LDLR-EGF-A binds. The top hits exhibited inhibition of PCSK9/LDLR binding in the low micromolar (μM) range, a 5-fold upregulation of the LDLR at an inhibitor concentration of 1.6 μM in recombinant cell-based assays, and an increase in the fluorescently labeled DiI-LDL uptake in the nanomolar (nM) range in situ . Pharmacological studies revealed that subcutaneous injection of 4 mg/kg of one of these compounds resulted in a 32% reduction of the total cholesterol and 20% reduction in LDL-C levels in mice fed a high fat/high cholesterol diet. Moreover, administration of 40mg/kg of atorvastatin resulted in a 27% total cholesterol reduction and 20% reduction in LDL-C level. Additional studies were performed to evaluate the effect of the combination of our PCSK9 inhibitor with atorvastatin. Administration of the PCSK9 inhibitor (4 mg/ kg) in combination with atorvastatin (40 mg/kg) resulted in more than a 40% reduction in LDL-C in mice fed a high fat/high cholesterol diet. In vivo PK and PD evaluation of our lead PCSK9 inhibitor demonstrated that it was efficacious and exhibited 13% oral bioavailability. To our knowledge, no small molecule orally bioavailable antagonist against PCSK9 has been reported, only mAbs.

Abstract 10306: Identification and Characterization of Orally Bioavailable Small Molecule Protease Proprotein Convertase Subtilisin-like Kexin Type 9 Inhibitors (Best of Basic Science Abstract)

Intervention with drugs to reduce low density lipoprotein-cholesterol (LDL-C) has proven to decrease mortality and morbidity. Here, we report the development of small molecules that lower LDL-C by targeting the LDL-receptor (LDLR) degradation pathway, which is modulated by the protease proprotein convertase subtilisin-like kexin type 9 (PCSK9). PCSK9 is synthesized as a 72-kDa zymogen that undergoes autocatalytic processing between the prodomain and catalytic domain, an important step that is absolutely required for secretion of PCSK9 and its function. Secreted PCSK9 binds to the LDLR and enhances its degradation. We have identified inhibitors of the autocatalytic processing by virtual docking of millions of commercial compounds into the atomic structure of the PCSK9 active site. Top scoring compounds that showed the best fit in the active site were selected for screening. These compounds were tested for their ability to inhibit the autocatalytic processing/secretion of PCSK9. The most potent compounds exhibited a concentration-dependent inhibition of the PCSK9 processing/secretion with IC50’s in the nanomolar range. These compounds were tested for selectivity against other PCSK’s. The data demonstrated that these compounds were selective in that they inhibit PCSK9 processing/secretion without affecting the secretion of other related PCSK’s. Furthermore, these compounds exhibited a 5- to 10-fold LDLR upregulation at 1.6 μM in two recombinant cell-based assays as well as exhibited an increase in the fluorescently labeled DiI-LDL uptake in situ in the nanomolar range. In animal studies, intraperitoneal (IP) injection of these compounds administered alone resulted in more than 20% reduction in LDL-C in mice fed a high fat/high cholesterol diet and over 40% reduction in LDL-C if administered in combination with atorvastatin. In vivo pharmacokinetic (PK) and pharmacodynamic (PD) evaluation of these compounds demonstrated that they are orally efficacious, with one compound exhibiting 43% oral bioavailability. Thus, identifying small molecule, orally active PCSK9 modulators represents a significant advance and opportunityfor drug development.

Interactions of several genetic polymorphisms and alcohol consumption on blood pressure levels.

This study aimed to detect the interactions of several single nucleotide polymorphisms (SNPs) and alcohol consumption on blood pressure levels. Genotypes of 10 SNPs in the ATP-binding cassette transporter A1 (ABCA-1), acyl-CoA:cholesterol acyltransferase-1 (ACAT-1), low density lipoprotein receptor (LDLR), hepatic lipase gene (LIPC), endothelial lipase gene (LIPG), methylenetetrahydrofolate reductase (MTHFR), the E3 ubiquitin ligase myosin regulatory light chain-interacting protein (MYLIP), proprotein convertase subtilisin-like kexin type 9 (PCSK9), peroxisome proliferator-activated receptor delta (PPARD), and Scavenger receptor class B type 1 (SCARB1) genes were determined in 616 nondrinkers and 608 drinkers. The genotypic frequencies of LDLR rs5925, LIPC rs2070895, MTHFR rs1801133, and MYLIP rs3757354 SNPs were significantly different between nondrinkers and drinkers. The levels of systolic blood pressure (ABCA-1 rs2066715 and rs2070895), diastolic blood pressure (rs2070895), and pulse pressure (PP) (rs2066715, ACAT-1 rs1044925, and rs1801133) in nondrinkers, and systolic blood pressure (rs1044925 and SCARB1 rs5888), diastolic blood pressure (rs1044925 and LIPG rs2000813), and PP (PCSK9 rs505151 and rs5888) in drinkers were different among the genotypes (P < 0.005-0.001). The interactions of several SNPs and alcohol consumption on systolic blood pressure (rs2066715, rs1044925, rs5925, rs2070895, rs1801133, rs3757354, PPARD rs2016520, and rs5888), diastolic blood pressure (rs2066715, rs1044925, rs5925, rs2000813, rs3757354, and rs2016520), and PP (rs1044925, rs2070895, rs1801133, rs3757354, rs505151, and rs5888) were observed (P < 0.005-0.001). The differences in blood pressure levels between the nondrinkers and drinkers might be partially attributed to the interactions of these SNPs and alcohol consumption.

Abstract 9761: Novel Modulators of Low Density Lipoprotein Receptor Metabolism

Intervention with drugs to reduce Low Density Lipoprotein-cholesterol (LDL-C) has proven to decrease the risk of subsequent cardiovascular events, including mortality. Our goal is to develop novel, small molecule, LDL-C lowering drugs by targeting the Low Density Lipoprotein Receptor (LDLR) degradation pathway, which is modulated by the protease proprotein convertase subtilisin-like kexin type 9 (PCSK9). PCSK9 has been shown to bind to the epidermal growth factor-like repeat A (EGF-A) domain of the LDLR; it enhances the degradation of the LDLR by an unknown. We successfully integrated virtual screening methods and cell based assays into a simple, efficient procedure and identified compounds that interfere with the PCSK9-LDLR interaction. Using the atomic coordinates of the crystal structure of the PCSK9/LDLR-EGF-A complex, about a million drug-like compounds were docked in silico into the region of PCSK9 where LDLR-EGF-A binds. We selected 179 top scoring compounds that showed the best fit in the binding site as well as the greatest number of interactions with PCSK9. These compounds were tested in (a) our PCSK9/LDLR dependent HEK293 cell-based recombinant assay and (b) the endogenously expressed LDLR in HepG2/PCSK9 dependent cell based assay for their ability to upregulate the LDLR. From these screening campaigns, eleven compounds consistently exhibited concentration dependent increase in the LDLR as comparable to control, with IC 50 s in the low micromolar range. Three of the top picks were shown to exhibit a 5 to 10 fold up-regulation at 1.6 μM in two cell lines. In addition, these compounds also exhibited an increase in the fluorescently labeled DiI-LDL uptake in the nanomolar range in situ . Furthermore, in a two week animal study, subcutaneous injection of 4 mg/kg of one of these compounds resulted in a 32% reduction of the total cholesterol level in mice fed a high fat/high cholesterol diet as compared to 27% cholesterol reduction obtained with 40 mg/kg Atorvastatin. To our knowledge, no small molecule antagonist against PCSK9 has been reported, only mAbs. Thus, identifying such an oral small molecule PCSK9 inhibitor represents a tremendous advance and opportunity for drug development.

Effects of Niacin, Statin, and Fenofibrate on Circulating Proprotein Convertase Subtilisin/Kexin Type 9 Levels in Patients With Dyslipidemia

Recent trials demonstrated substantial improvement in lipid parameters with inhibition of proprotein convertase subtilisin-like/kexin type 9 (PCSK9). Although statins and fibrates have been reported to increase plasma PCSK9 levels, the effect of niacin on PCSK9 is unknown. We investigated the impact of niacin, atorvastatin, and fenofibrate on PCSK9 levels in 3 distinct studies. A statin-only study randomized 74 hypercholesterolemic patients to placebo, atorvastatin 10 mg/day, or atorvastatin 80 mg/day for 16 weeks. A dose-related increase in PCSK9 was noted such that atorvastatin 80 mg increased PCSK9 by a mean +27% (95% confidence interval [CI] +12 to +42), confirming the effect of statin therapy on raising PCSK9. A second study randomized 70 patients with carotid atherosclerosis to simvastatin 20 mg/day, simvastatin 80 mg/day, or simvastatin 20 mg/extended-release (ER) niacin 2 g/day. PCSK9 levels were increased with statin therapy, but decreased with the simvastatin 20 mg/ER niacin combination (mean -13%, CI -3 to -23). A final study involved 19 dyslipidemic participants on atorvastatin 10 mg with serial addition of fenofibric acid 135 mg followed by ER niacin 2 g/day. Fenofibric acid led to a +23% (CI +10 to +36, p = 0.001) increase in PCSK9; the addition of niacin resulted in a subsequent -17% decrease (CI -19 to -5, p = 0.004). A positive association was noted between change in PCSK9 and low-density lipoprotein cholesterol levels (r = 0.62, p = 0.006) with the addition of niacin. In conclusion, niacin therapy offsets the increase in PCSK9 levels noted with statin and fibrate therapy. A portion of the low-density lipoprotein cholesterol reduction seen with niacin therapy may be due to reduction in PCSK9.

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