Development of Innovative Antiatherosclerotic Peptides through the Combination of Molecular Modeling and a Dual (Biochemical‐Cellular) Screening System

Abstract

Cardiovascular disease (CVD) is a leading cause of death worldwide. Approximately 60% of patients treated with low‐density lipoprotein (LDL)‐lowering drug treatments, with on‐target plasma cholesterol levels, are still suffering clinical acute ischemic events. Mechanisms, such as LDL aggregation, underlie extracellular and intracellular cholesterol accumulation in the vasculature. A peptide sequence (P3) of the low‐density lipoprotein receptor‐related protein 1 (LRP1) efficiently protects LDL from sphingomyelinase (SMase‐) and phospholipase A2 (PLA2)‐induced LDL aggregation. The aim is to design families of peptide derivatives from P3 with enhanced potency and proteolytic stability. New peptides are designed through in silico conformational sampling and ApoB‐100 molecular docking, and are tested in dual (biochemical‐cellular) screening assays. A total of 46 new peptides including linear, fragment, cyclic, and alanine scanning derivatives are generated through two consecutive optimization rounds. Structurally and functionally optimized peptides contain hotspot residues that are replaced by alanine. This strategy confers an increased capacity to form prone alpha‐helix conformations crucial for the electrostatic interaction with ApoB‐100. These new compounds are highly efficient at inhibiting LDL aggregation and human coronary vascular smooth muscle cell‐cholesteryl ester loading and should be studied in preclinical models of atherosclerosis.

What’s in a name? From “fluctuation fit” to “conformational selection”: rediscovery of a concept

“…Papers listed in Table S1 cite Straub’s and Szabolcsi’s work in the text (in some cases only indirectly or mistakenly) and the concept of the term “fluctuation fit”. The most recent one has been published recently (Benitez‐Amaro et al, 2020 ).…”

Apolipoprotein and LRP1-Based Peptides as New Therapeutic Tools in Atherosclerosis

“…In particular, the sequence Gly 1127 -Cys 1140 , which covers the C-terminal half of the CR9 domain, is crucial for agLDL interaction and the cellular uptake of cholesteryl esters from agLDL [58]. From the sequence Gly 1127 -Cys 1140 , the LP3 peptide and other stable retroenantiomer peptides, such as DP3, were structurally optimized to achieve maximal functionality [32,59].…”

“…Structurally and functionally optimized peptides contain hotspot residues that were replaced by alanine, because this strategy confers an increased capacity to form prone α-helix conformations that are crucial for electrostatic interaction with ApoB-100. These new compounds were proven to efficiently inhibit LDL aggregation promoted by SMase and PLA2 [59].…”

Peptides against Low Density Lipoprotein (LDL) Aggregation Inhibit Intracellular Cholesteryl Ester Loading and Proliferation of Pancreatic Tumor Cells

“…The sequence (Gly 1127 –Cys 1140 ) (P3) located in the CR9 domain of cluster II of the LRP1 α-chain interacts with the apolipoprotein B100 (ApoB100) of modified lipoproteins [ 28 ]. Peptides based on this P3 sequence electrostatically interact with ApoB100 and maintain ApoB100 conformation, preserving LDL against aggregation induced by phospholipolytic enzymes present in the ECM [ 29 , 30 ]. In the vascular scenario, innovative therapeutic attempts, such as peptides anti-LDL aggregation [ 29 , 30 , 31 , 32 ] and their antibodies [ 33 , 34 , 35 ], have successfully inhibited the formation of lipid droplet cell phenotypes and atherosclerosis.…”

“…Peptides based on this P3 sequence electrostatically interact with ApoB100 and maintain ApoB100 conformation, preserving LDL against aggregation induced by phospholipolytic enzymes present in the ECM [ 29 , 30 ]. In the vascular scenario, innovative therapeutic attempts, such as peptides anti-LDL aggregation [ 29 , 30 , 31 , 32 ] and their antibodies [ 33 , 34 , 35 ], have successfully inhibited the formation of lipid droplet cell phenotypes and atherosclerosis. The retro-enantio (e.g., reversed d-amino acid order) peptide of P3, termed DP3 (Ac-dN·dE·dE·dD·dS·dN·dD·dE·dS·dD·dN·dD·G-NH 2 ) and an ApoB-100 based peptide, termed IP321 (H-RLTRKRGLK-NH 2 ), have been previously validated by our group as positive and negative controls, respectively, in biochemical and cell assays developed to test the anti-LDL aggregation and anti-cholesteryl ester loading properties of a new family of optimized peptides [ 30 ].…”