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Structural requirements for PCSK9-mediated degradation of the low-density lipoprotein receptor.

ABSTRACT: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that controls plasma LDL cholesterol levels by posttranslational regulation of the LDL receptor (LDLR). Previously, we showed that PCSK9 binds specifically to an EGF-like repeat (EGF-A) in LDLR and reroutes the receptor from endosomes to lysosomes rather than to the cell surface. Here, we defined the regions in LDLR and PCSK9 that are required for receptor degradation and examined the relationship between PCSK9 binding and LDLR conformation. Addition of PCSK9 to cultured hepatocytes promoted degradation of WT LDLR and of receptors lacking up to four ligand binding domains, EGF-B or the clustered O-linked sugar region. In contrast, LDLRs lacking the entire ligand binding domain or the beta-propeller domain failed to be degraded, although they bound and internalized PCSK9. Using gel filtration chromatography, we assessed the effects of PCSK9 binding on an acid-dependent conformational change that happens in the extracellular domain of the LDLR. Although PCSK9 prevented the reduction in hydrodynamic radius of the receptor that occurs at a reduced pH, the effect was not sufficient for LDLR degradation. A truncated version of PCSK9 containing the prodomain and the catalytic domain, but not the C-terminal domain, bound the receptor but did not stimulate LDLR degradation. Thus, domains in both the LDLR and PCSK9 that are not required for binding (or internalization) are essential for PCSK9-mediated degradation of the LDLR.

SUBMITTER: Zhang DW

PROVIDER: S-EPMC2526098 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundInhibition of PCSK9 (proprotein convertase subtilisin/kexin type 9)-low density lipoprotein receptor interaction with injectable monoclonal antibodies or small interfering RNA lowers plasma low density lipoprotein-cholesterol, but despite nearly 2 decades of effort, an oral inhibitor of PCSK9 is not available. Macrocyclic peptides represent a novel approach to target proteins traditionally considered intractable to small-molecule drug design.MethodsNovel mRNA display screening technology was used to identify lead chemical matter, which was then optimized by applying structure-based drug design enabled by novel synthetic chemistry to identify macrocyclic peptide (MK-0616) with exquisite potency and selectivity for PCSK9. Following completion of nonclinical safety studies, MK-0616 was administered to healthy adult participants in a single rising-dose Phase 1 clinical trial designed to evaluate its safety, pharmacokinetics, and pharmacodynamics. In a multiple-dose trial in participants taking statins, MK-0616 was administered once daily for 14 days to characterize the safety, pharmacokinetics, and pharmacodynamics (change in low density lipoprotein cholesterol).ResultsMK-0616 displayed high affinity (Ki = 5pM) for PCSK9 in vitro and sufficient safety and oral bioavailability preclinically to enable advancement into the clinic. In Phase 1 clinical studies in healthy adults, single oral doses of MK-0616 were associated with >93% geometric mean reduction (95% CI, 84-103) of free, unbound plasma PCSK9; in participants on statin therapy, multiple-oral-dose regimens provided a maximum 61% geometric mean reduction (95% CI, 43-85) in low density lipoprotein cholesterol from baseline after 14 days of once-daily dosing of 20 mg MK-0616.ConclusionsThis work validates the use of mRNA display technology for identification of novel oral therapeutic agents, exemplified by the identification of an oral PCSK9 inhibitor, which has the potential to be a highly effective cholesterol lowering therapy for patients in need.

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Structural requirements for PCSK9-mediated degradation of the low-density lipoprotein receptor.

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