Abstract 677: Short Hydrocarbon-Stapled Apolipoprotein E Mimetic Peptides Enhance Lipoprotein Uptake

Abstract

ApoE facilitates the clearance of apoB-containing lipoproteins by binding to heparan sulfate proteoglycans (HSPG), low density lipoprotein receptor (LDLR) and LDLR-related protein 1 (LRP1). ApoE has a receptor binding and a lipid binding domain, which are both needed for its biological function. Several apoE-based peptides have been developed that lower plasma lipid levels in animal models and in early-stage clinical trials. However, these peptides are long (28-61 residues), costly to synthesize and are potentially immunogenic as they often contain many amino acid substitutions that vary from the native apoE sequence. We developed novel short (10-12 residues) apoE mimetic peptides based on the apoE receptor binding region (residues 141-150) containing only 2 amino acid substitutions. We used hydrocarbon stapling to stabilize the native alpha helical structure of this region and to increase the lipid binding affinity of the peptides. In addition, octanoic or myristic acid was attached to the N-terminus of the peptides to further increase lipid binding affinity. Helical conformation and lipid binding of the peptides were confirmed by circular dichroism spectroscopy and phospholipid vesicle solubilization assay. Our peptides increased the uptake of fluorescent tagged VLDL and LDL in a concentration dependent manner (0.625-20 μM peptides) in both wild-type (WT) and LDLR knockout (KO) HepG2 cells as measured by flow cytometry. In addition, an all D-amino acid version of one of the peptides had a similar effect on LDL uptake as its all L-amino acid version. Altogether, this suggests that peptide-mediated uptake is LDLR independent. Treatment of WT HepG2 cells with heparinases diminished the low-dose effect (up to 2.5 μM) of the peptides on LDL uptake, indicating that HSPGs plays a role in peptide-mediated LDL uptake. Finally, the peptides (4.6 mg/kg, IV) lowered plasma total cholesterol (37% at 3h; 29% at 6h) and triglycerides (33% at 1h) in apoE-KO mice on a standard chow diet. In summary, the novel apoE peptides are shorter than previously reported peptides, are more similar to the native sequence of apoE, and are likely resistant to proteolysis, because of hydrocarbon stapling and, therefore, could potentially be developed into an oral therapy.