Proteolytic cleavage of antigen extends the durability of an anti-PCSK9 monoclonal antibody.

Lilly PCSK9 antibody LY3015014 (LY) is a monoclonal antibody (mAb) that neutralizes proprotein convertase subtilisin-kexin type 9 (PCSK9). LY decreases LDL cholesterol in monkeys and, unlike other PCSK9 mAbs, does not cause an accumulation of intact PCSK9 in serum. Comparing the epitope of LY with other clinically tested PCSK9 mAbs, it was noted that the LY epitope excludes the furin cleavage site in PCSK9, whereas other mAbs span this site. In vitro exposure of PCSK9 to furin resulted in degradation of PCSK9 bound to LY, whereas cleavage was blocked by other mAbs. These other mAbs caused a significant accumulation of serum PCSK9 and displayed a shorter duration of LDL-cholesterol lowering than LY when administered to mice expressing the WT human PCSK9. In mice expressing a noncleavable variant of human PCSK9, LY behaved like a cleavage-blocking mAb, in that it caused significant PCSK9 accumulation, its duration of LDL lowering was reduced, and its clearance (CL) from serum was accelerated. Thus, LY neutralizes PCSK9 and allows its proteolytic degradation to proceed, which limits PCSK9 accumulation, reduces the CL rate of LY, and extends its duration of action. PCSK9 mAbs with this property are likely to achieve longer durability and require lower doses than mAbs that cause antigen to accumulate.

Statin-associated muscle symptoms: impact on statin therapy-European Atherosclerosis Society Consensus Panel Statement on Assessment, Aetiology and Management.

Statin-associated muscle symptoms (SAMS) are one of the principal reasons for statin non-adherence and/or discontinuation, contributing to adverse cardiovascular outcomes. This European Atherosclerosis Society (EAS) Consensus Panel overviews current understanding of the pathophysiology of statin-associated myopathy, and provides guidance for diagnosis and management of SAMS. Statin-associated myopathy, with significant elevation of serum creatine kinase (CK), is a rare but serious side effect of statins, affecting 1 per 1000 to 1 per 10 000 people on standard statin doses. Statin-associated muscle symptoms cover a broader range of clinical presentations, usually with normal or minimally elevated CK levels, with a prevalence of 7-29% in registries and observational studies. Preclinical studies show that statins decrease mitochondrial function, attenuate energy production, and alter muscle protein degradation, thereby providing a potential link between statins and muscle symptoms; controlled mechanistic and genetic studies in humans are necessary to further understanding. The Panel proposes to identify SAMS by symptoms typical of statin myalgia (i.e. muscle pain or aching) and their temporal association with discontinuation and response to repetitive statin re-challenge. In people with SAMS, the Panel recommends the use of a maximally tolerated statin dose combined with non-statin lipid-lowering therapies to attain recommended low-density lipoprotein cholesterol targets. The Panel recommends a structured work-up to identify individuals with clinically relevant SAMS generally to at least three different statins, so that they can be offered therapeutic regimens to satisfactorily address their cardiovascular risk. Further research into the underlying pathophysiological mechanisms may offer future therapeutic potential.

Ongoing challenges for pharmacotherapy for dyslipidemia.

INTRODUCTION: While increasing evidence has led to lipid-modifying therapy achieving an important role in the treatment guidelines for the prevention of cardiovascular disease, these agents are suboptimally used and there remains a considerable risk of clinical events. Accordingly, there is a need to develop more effective lipid-modifying approaches in many patients. AREAS COVERED: A literature search was performed of topical manuscripts focusing on factors influencing use of established therapies and new agents in development that target a range of lipid factors. EXPERT OPINION: More intensive efforts are required to ensure that statin use is maximized in higher risk patients. A range of novel therapies, including proprotein convertase subtilisin kexin-type 9 and cholesteryl ester transfer protein inhibitors, may provide additional protection, although this remains to be established by clinical trials.

PCSK9 and its modulation.

Proprotein convertase subtilisin/kexin type 9 (PCSK9), a newly-recognized protein, plays a key role in regulating cholesterol homeostasis. PCSK9 reduces hepatic low-density lipoprotein receptors (LDLRs) thereby increasing LDL-cholesterol (LDL-C). Recently, biologic and genetic research proposed several approaches to inhibit or reduce PCSK9 to improve lipid profile and cardiovascular performance in patients with dyslipidemia, particularly hypercholesterolemia. Of note, PCSK9 is a secreted protein under tight control by multiple modulators. Therefore, elucidating the factors that influence PCSK9 would enhance our understanding of PCSK9 and potentially day-to-day management of these patients at high cardiovascular risk. This review will focus on genetic variants, physiologic processes, pharmacologic agents and pathologic conditions related to PCSK9 in order to assess current and future therapeutic strategies targeting this molecule.

Inhibition of proprotein convertases abrogates processing of the middle eastern respiratory syndrome coronavirus spike protein in infected cells but does not reduce viral infectivity.

Middle East respiratory syndrome coronavirus (MERS-CoV) infection is associated with a high case-fatality rate, and the potential pandemic spread of the virus is a public health concern. The spike protein of MERS-CoV (MERS-S) facilitates viral entry into host cells, which depends on activation of MERS-S by cellular proteases. Proteolytic activation of MERS-S during viral uptake into target cells has been demonstrated. However, it is unclear whether MERS-S is also cleaved during S protein synthesis in infected cells and whether cleavage is required for MERS-CoV infectivity. Here, we show that MERS-S is processed by proprotein convertases in MERS-S-transfected and MERS-CoV-infected cells and that several RXXR motifs located at the border between the surface and transmembrane subunit of MERS-S are required for efficient proteolysis. However, blockade of proprotein convertases did not impact MERS-S-dependent transduction of target cells expressing high amounts of the viral receptor, DPP4, and did not modulate MERS-CoV infectivity. These results show that MERS-S is a substrate for proprotein convertases and demonstrate that processing by these enzymes is dispensable for S protein activation. Efforts to inhibit MERS-CoV infection by targeting host cell proteases should therefore focus on enzymes that process MERS-S during viral uptake into target cells.

A 52-week placebo-controlled trial of evolocumab in hyperlipidemia.

BACKGROUND: Evolocumab, a monoclonal antibody that inhibits proprotein convertase subtilisin/kexin type 9 (PCSK9), significantly reduced low-density lipoprotein (LDL) cholesterol levels in phase 2 studies. We conducted a phase 3 trial to evaluate the safety and efficacy of 52 weeks of treatment with evolocumab. METHODS: We stratified patients with hyperlipidemia according to the risk categories outlined by the Adult Treatment Panel III of the National Cholesterol Education Program. On the basis of this classification, patients were started on background lipid-lowering therapy with diet alone or diet plus atorvastatin at a dose of 10 mg daily, atorvastatin at a dose of 80 mg daily, or atorvastatin at a dose of 80 mg daily plus ezetimibe at a dose of 10 mg daily, for a run-in period of 4 to 12 weeks. Patients with an LDL cholesterol level of 75 mg per deciliter (1.9 mmol per liter) or higher were then randomly assigned in a 2:1 ratio to receive either evolocumab (420 mg) or placebo every 4 weeks. The primary end point was the percent change from baseline in LDL cholesterol, as measured by means of ultracentrifugation, at week 52. RESULTS: Among the 901 patients included in the primary analysis, the overall least-squares mean (±SE) reduction in LDL cholesterol from baseline in the evolocumab group, taking into account the change in the placebo group, was 57.0±2.1% (P<0.001). The mean reduction was 55.7±4.2% among patients who underwent background therapy with diet alone, 61.6±2.6% among those who received 10 mg of atorvastatin, 56.8±5.3% among those who received 80 mg of atorvastatin, and 48.5±5.2% among those who received a combination of 80 mg of atorvastatin and 10 mg of ezetimibe (P<0.001 for all comparisons). Evolocumab treatment also significantly reduced levels of apolipoprotein B, non-high-density lipoprotein cholesterol, lipoprotein(a), and triglycerides. The most common adverse events were nasopharyngitis, upper respiratory tract infection, influenza, and back pain. CONCLUSIONS: At 52 weeks, evolocumab added to diet alone, to low-dose atorvastatin, or to high-dose atorvastatin with or without ezetimibe significantly reduced LDL cholesterol levels in patients with a range of cardiovascular risks. (Funded by Amgen; DESCARTES ClinicalTrials.gov number, NCT01516879.).

[HDL, or non-HDL: that is the question. Possibilities of pharmacological treatment in residual dyslipidaemia]. / HDL vagy non-HDL: az itt a kérdés. A residualis dyslipidaemia gyógyszeres kezelésének lehetoségei.

Instead of LDL-cholesterol, non-HDL-cholesterol is proposed as a secondary lipid target when triglyceride level is above 2.3 mmol/L. Non-HDL-cholesterol target values are 0.8 mmol/L higher than those for LDL-cholesterol in the same cardiovascular risk category. Currently, the main issue of lipidology is the degree by which the cardiovascular risk can be reduced with the treatment of residual dyslipidemia that exists under statin therapy. In such a role the examined agents have essentially failed despite their more or less profound effect on HDL-cholesterol and/or non-HDL-cholesterol. The largest loser has been the nicotinic acid. The results of cardiovascular, otherwise controversial fish oil studies cannot be considered convincing because of the administered low doses. In a combination with statin (i) ezetimibe may have role if the LDL-cholesterol target cannot be reached with statin monotherapy, or (ii) fibrates, in case of large increase of triglyceride level, or in less severe hypertriglyceridemia if it is associated with considerable decrease in HDL-cholesterol level. Potential further possibilities are: (i) cholesterol ester transfer protein inhibitors that dramatically raise HDL-cholesterol, while reduce LDL-cholesterol, or (ii) proprotein convertase subtilisin/kexin 9 inhibitors that markedly decrease LDL-cholesterol even on the top of statin.

Identification of a small molecule that selectively inhibits mouse PC2 over mouse PC1/3: a computational and experimental study.

The calcium-dependent serine endoproteases prohormone convertase 1/3 (PC1/3) and prohormone convertase 2 (PC2) play important roles in the homeostatic regulation of blood glucose levels, hence implicated in diabetes mellitus. Specifically, the absence of PC2 has been associated with chronic hypoglycemia. Since there is a reasonably good conservation of the catalytic domain between species translation of inhibitory effects is likely. In fact, similar results have been found using both mouse and human recombinant enzymes. Here, we employed computational structure-based approaches to screen 14,400 compounds from the Maybridge small molecule library towards mouse PC2. Our most remarkable finding was the identification of a potent and selective PC2 inhibitor. Kinetic data showed the compound to be an allosteric inhibitor. The compound identified is one of the few reported selective, small-molecule inhibitors of PC2. In addition, this new PC2 inhibitor is structurally different and of smaller size than those reported previously. This is advantageous for future studies where structural analogues can be built upon.

Proprotein convertase inhibitory activities of flavonoids isolated from Oroxylum indicum.

BACKGROUND: Proprotein Convertases (PCs) or Proprotein Convertase Subtilisin/Kexins (PCSKs) belong to a family of calcium-dependent endoproteases that are structurally related to bacterial subtilisin and yeast kexin. These enzymes play major roles in the processing of inactive precursor proteins producing their bioactive mature forms that are implicated in a wide variety of diseases including cancer, viral and bacterial infections. As a result, PCs are major targets for intervention of these diseases. OBJECTIVE: Our objective in this study is to find non-peptide inhibitors of PC-enzymes from a potential natural source. RESULTS: Herein we describe several natural flavonoid compounds as inhibitors of PC-enzymes including furin, a key member. These compounds were isolated from the medicinal plant Oroxylum indicum, fully characterized and tested in vitro for their PC-inhibitory property against the fluorogenic peptide substrate, Boc-RVRR-MCA (Boc = tert-butyloxy carbonyl, MCA = 4-methyl coumarin7-amide). The measured Ki and IC50 were found to be in low microM ranges. A comparative analysis of inhibition against furin, PC4, PC5 and PC7 suggested a partial selectivity towards PC4. These flavonoids also blocked efficiently the PC4-mediated processing of a fluorogenic peptide derived from the processing site of its substrate, pro-Insulin Growth Factor-1 (proIGF-1). This anti-protease activity may provide a rationale for the observed anticancer and anti-HIV properties of some of these flavonoid compounds. This is the first demonstration of anti-PC activity of flavonoid compounds.