Increased Valency Improves Inhibitory Activity of Peptides Targeting Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9)

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a clinically validated target for treating hypercholesterolemia. Peptide-based PCSK9 inhibitors have attracted pharmaceutical interest, but the effect of multivalency on bioactivity is poorly understood. Here we designed bivalent and tetravalent dendrimers, decorated with the PCSK9 inhibitory peptides Pep2-8[RRG] or P9-38, to study relationships between peptide binding affinity, peptide valency, and PCSK9 inhibition. Increased valency resulted in improved PCSK9 inhibition for both peptides, with activity improvements of up to 100-fold achieved for the P9-38-decorated dendrimers compared to monomeric P9-38 in in vitro competition binding assays. Furthermore, the P9-38-decorated dendrimers showed improved potency at restoring functional low-density lipoprotein (LDL) receptor levels and internalizing LDL in the presence of PCSK9, demonstrating significant cell-based activity at picomolar concentrations. This study demonstrates the potential of increasing valency as a strategy for increasing the efficacy of peptide-based PCSK9 therapeutics.     

Docosahexaenoic Acid Attenuates Cardiovascular Risk Factors via a Decline in Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Plasma Levels

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a circulating protein that regulates cholesterol metabolism by promoting LDL receptor degradation in the liver and has recently been proposed as a therapeutic target in the management of hyperlipidaemia. We investigated the impact of dietary fat on the metabolism of sterols and on plasma PCSK9 concentrations to explore likely clinical usefulness. In a post hoc analysis of a double‐blind randomised crossover controlled feeding trial, the Canola Oil Multicenter Intervention Trial (COMIT), volunteers (n = 54) with at least one condition related to metabolic syndrome consumed diets with one of the following treatment oils in beverages: (1) conventional canola oil (Canola); (2) canola oil rich in docosahexanoic acid (DHA) (CanolaDHA); and (3) high‐oleic acid canola oil (CanolaOleic). The enrichment in oleic acid resulted in lower plasma cholesterol concentration compared with diets enriched in DHA. Contrarily, DHA‐enriched oil significantly decreased plasma PCSK9 and triacylglycerols levels, but increased circulating levels of sterols. The variations in lathosterol, sitosterol, and campesterol indicate that plasma PCSK9 levels are sensitive to changes in cholesterol synthesis and/or absorption. There was a significant correlation between plasma PCSK9 levels and plasma triacylglicerol and apolipoprotein B levels, which was not affected by dietary fat. Therefore, our results suggest that the impact of dietary fats should not be discarded as complementary treatment in the management of patients with hyperlipidaemia. These findings should be considered in the analysis of ongoing studies and may represent a cautionary note in the treatment of patients with cardiovascular risk.     

Exploring Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) Autoproteolysis Process by Molecular Simulations: Hints for Drug Design

Proprotein convertase subtilisin/kexin 9 (PCSK9) is a notable target for the treatment of hypercholesterolemia because it regulates the population of the low-density lipoprotein receptor (LDLR) on liver cells. The PCSK9 zymogen is a serine protease that spontaneously undergoes a double self-cleavage step. Available X-ray structures depict the PCSK9 mature state, but the atomic details of the zymogen state of the enzyme are still unknown. Additionally, why the protease activity of PCSK9 is blocked after the second autoprocessing step remains unclear, as this deviates from other members of the PCSK family. By performing constant-pH molecular dynamics (MD) simulations, we investigated the protonation state of the catalytic triad of PCSK9 and found that it strongly influences the catalytic properties of the enzyme. Moreover, we determined the final step of the maturation process by classical and steered MD simulations. This study could facilitate the identification of ligands capable of interfering with the PCSK9 maturation process.     

Proprotein Convertase Subtilisin Kexin Type 9 (PCSK9) Deletion but Not Inhibition of Extracellular PCSK9 Reduces Infarct Sizes Ex Vivo but Not In Vivo

Hypoxia upregulates PCSK9 expression in the heart, and PCSK9 affects the function of myocytes. This study aimed to investigate the impact of PCSK9 on reperfusion injury in rats and mice fed normal or high-fat diets. Either the genetic knockout of PCSK9 (mice) or the antagonism of circulating PCSK9 via Pep2-8 (mice and rats) was used. Isolated perfused hearts were exposed to 45 min of ischemia followed by 120 min of reperfusion. In vivo, mice were fed normal or high-fat diets (2% cholesterol) for eight weeks prior to coronary artery occlusion (45 min of ischemia) and reperfusion (120 min). Ischemia/reperfusion upregulates PCSK9 expression (rats and mice) and releases it into the perfusate. The inhibition of extracellular PCSK9 does not affect infarct sizes or functional recovery. However, genetic deletion largely reduces infarct size and improves post-ischemic recovery in mice ex vivo but not in vivo. A high-fat diet reduced the survival rate during ischemia and reperfusion, but in a PCSK9-independent manner that was associated with increased plasma matrix metalloproteinase (MMP)9 activity. PCSK9 deletion, but not the inhibition of extracellular PCSK9, reduces infarct sizes in ex vivo hearts, but this effect is overridden in vivo by factors such as MMP9.     

PCSK9 Affects Astrocyte Cholesterol Metabolism and Reduces Neuron Cholesterol Supplying In Vitro: Potential Implications in Alzheimer’s Disease

The Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) involvement in Alzheimer’s disease (AD) is poorly investigated. We evaluated the in vitro PCSK9 modulation of astrocyte cholesterol metabolism and neuronal cholesterol supplying, which is fundamental for neuronal functions. Moreover, we investigated PCSK9 neurotoxic effects. In human astrocytoma cells, PCSK9 reduced cholesterol content (−20%; p < 0.05), with a greater effect in presence of beta amyloid peptide (Aβ) (−37%; p < 0.01). PCSK9 increased cholesterol synthesis and reduced the uptake of apoE-HDL-derived cholesterol (−36%; p < 0.0001), as well as the LDL receptor (LDLR) and the apoE receptor 2 (ApoER2) expression (−66% and −31%, respectively; p < 0.01). PCSK9 did not modulate ABCA1- and ABCG1-cholesterol efflux, ABCA1 levels, or membrane cholesterol. Conversely, ABCA1 expression and activity, as well as membrane cholesterol, were reduced by Aβ (p < 0.05). In human neuronal cells, PCSK9 reduced apoE-HDL-derived cholesterol uptake (−41%; p < 0.001) and LDLR/apoER2 expression (p < 0.05). Reduced cholesterol internalization occurred also in PCSK9-overexpressing neurons exposed to an astrocyte-conditioned medium (−39%; p < 0.001). PCSK9 reduced neuronal cholesterol content overall (−29%; p < 0.05) and increased the Aβ-induced neurotoxicity (p < 0.0001). Our data revealed an interfering effect of PCSK9, in cooperation with Aβ, on brain cholesterol metabolism leading to neuronal cholesterol reduction, a potentially deleterious effect. PCSK9 also exerted a neurotoxic effect, and thus represents a potential pharmacological target in AD.