Targeting PCSK9 to tackle cardiovascular disease.

Lowering blood cholesterol levels efficiently reduces the risk of developing atherosclerotic cardiovascular disease (ASCVD), including coronary artery disease (CAD), which is the main cause of death worldwide. CAD is caused by plaque formation, comprising cholesterol deposits in the coronary arteries. Proprotein convertase subtilisin kexin/type 9 (PCSK9) was discovered in the early 2000s and later identified as a key regulator of cholesterol metabolism. PCSK9 induces lysosomal degradation of the low-density lipoprotein (LDL) receptor in the liver, which is responsible for clearing LDL-cholesterol (LDL-C) from the circulation. Accordingly, gain-of-function PCSK9 mutations are causative of familial hypercholesterolemia, a severe condition with extremely high plasma cholesterol levels and increased ASCVD risk, whereas loss-of-function PCSK9 mutations are associated with very low LDL-C levels and protection against CAD. Since the discovery of PCSK9, extensive investigations in developing PCSK9 targeting therapies have been performed. The combined delineation of clear biology, genetic risk variants, and PCSK9 crystal structures have been major drivers in developing antagonistic molecules. Today, two antibody-based PCSK9 inhibitors have successfully progressed to clinical application and shown to be effective in reducing cholesterol levels and mitigating the risk of ASCVD events, including myocardial infarction, stroke, and death, without any major adverse effects. A third siRNA-based inhibitor has been FDA-approved but awaits cardiovascular outcome data. In this review, we outline the PCSK9 biology, focusing on the structure and nonsynonymous mutations reported in the PCSK9 gene and elaborate on PCSK9-lowering strategies under development. Finally, we discuss future perspectives with PCSK9 inhibition in other severe disorders beyond cardiovascular disease.

PCSK9 deficiency alters brain lipid composition without affecting brain development and function.

PCSK9 induces lysosomal degradation of the low-density lipoprotein (LDL) receptor (LDLR) in the liver, hereby preventing removal of LDL cholesterol from the circulation. Accordingly, PCSK9 inhibitory antibodies and siRNA potently reduce LDL cholesterol to unprecedented low levels and are approved for treatment of hypercholesterolemia. In addition, PCSK9 inactivation alters the levels of several other circulating lipid classes and species. Brain function is critically influenced by cholesterol and lipid composition. However, it remains unclear how the brain is affected long-term by the reduction in circulating lipids as achieved with potent lipid lowering therapeutics such as PCSK9 inhibitors. Furthermore, it is unknown if locally expressed PCSK9 affects neuronal circuits through regulation of receptor levels. We have studied the effect of lifelong low peripheral cholesterol levels on brain lipid composition and behavior in adult PCSK9 KO mice. In addition, we studied the effect of PCSK9 on neurons in culture and in vivo in the developing cerebral cortex. We found that PCSK9 reduced LDLR and neurite complexity in cultured neurons, but neither PCSK9 KO nor overexpression affected cortical development in vivo. Interestingly, PCSK9 deficiency resulted in changes of several lipid classes in the adult cortex and cerebellum. Despite the observed changes, PCSK9 KO mice had unchanged behavior compared to WT controls. In conclusion, our findings demonstrate that altered PCSK9 levels do not compromise brain development or function in mice, and are in line with clinical trials showing that PCSK9 inhibitors have no adverse effects on cognitive function.

Crystal and solution structures of fragments of the human leucocyte common antigen-related protein.

Leucocyte common antigen-related protein (LAR) is a post-synaptic type I transmembrane receptor protein that is important for neuronal functionality and is genetically coupled to neuronal disorders such as attention deficit hyperactivity disorder (ADHD). To understand the molecular function of LAR, structural and biochemical studies of protein fragments derived from the ectodomain of human LAR have been performed. The crystal structure of a fragment encompassing the first four FNIII domains (LARFN1-4) showed a characteristic L shape. SAXS data suggested limited flexibility within LARFN1-4, while rigid-body refinement of the SAXS data using the X-ray-derived atomic model showed a smaller angle between the domains defining the L shape compared with the crystal structure. The capabilities of the individual LAR fragments to interact with heparin was examined using microscale thermophoresis and heparin-affinity chromatography. The results showed that the three N-terminal immunoglobulin domains (LARIg1-3) and the four C-terminal FNIII domains (LARFN5-8) both bound heparin, while LARFN1-4 did not. The low-molecular-weight heparin drug Innohep induced a shift in hydrodynamic volume as assessed by size-exclusion chromatography of LARIg1-3 and LARFN5-8, while the chemically defined pentameric heparin drug Arixtra did not. Together, the presented results suggest the presence of an additional heparin-binding site in human LAR.

Heparan sulfate proteoglycans present PCSK9 to the LDL receptor.

Coronary artery disease is the main cause of death worldwide and accelerated by increased plasma levels of cholesterol-rich low-density lipoprotein particles (LDL). Circulating PCSK9 contributes to coronary artery disease by inducing lysosomal degradation of the LDL receptor (LDLR) in the liver and thereby reducing LDL clearance. Here, we show that liver heparan sulfate proteoglycans are PCSK9 receptors and essential for PCSK9-induced LDLR degradation. The heparan sulfate-binding site is located in the PCSK9 prodomain and formed by surface-exposed basic residues interacting with trisulfated heparan sulfate disaccharide repeats. Accordingly, heparan sulfate mimetics and monoclonal antibodies directed against the heparan sulfate-binding site are potent PCSK9 inhibitors. We propose that heparan sulfate proteoglycans lining the hepatocyte surface capture PCSK9 and facilitates subsequent PCSK9:LDLR complex formation. Our findings provide new insights into LDL biology and show that targeting PCSK9 using heparan sulfate mimetics is a potential therapeutic strategy in coronary artery disease.PCSK9 interacts with LDL receptor, causing its degradation, and consequently reduces the clearance of LDL. Here, Gustafsen et al. show that PCSK9 interacts with heparan sulfate proteoglycans and this binding favors LDLR degradation. Pharmacological inhibition of this binding can be exploited as therapeutic intervention to lower LDL levels.

Structural basis of head to head polyketide fusion by CorB.

Corallopyronin A is a polyketide derived from the myxobacterium Corallococcus coralloides with potent antibiotic features. The gene cluster responsible for the biosynthesis of corallopyronin A has been described recently, and it was proposed that CorB acts as a ketosynthase to interconnect two polyketide chains in a rare head-to-head condensation reaction. We determined the structure of CorB, the interconnecting polyketide synthase, to high resolution and found that CorB displays a thiolase fold. Site-directed mutagenesis showed that the catalytic triad consisting of a cysteine, a histidine and an asparagine is crucial for catalysis, and that this triad shares similarities with the triad found in HMG-CoA synthases. We synthesized a substrate mimic to derivatize purified CorB and confirmed substrate attachment by ESI-MS. Structural analysis of the complex yielded an electron density-based model for the polyketide chain and showed that the unusually wide, T-shaped active site is able to accommodate two polyketides simultaneously. Our structural analysis provides a platform for understanding the unusual head-to-head polyketide-interconnecting reaction catalyzed by CorB.