Helix stabilization of amphipathic peptides by hydrocarbon stapling increases cholesterol efflux by the ABCA1 transporter

Abstract

Apolipoprotein mimetic peptides are short amphipathic peptides that efflux cholesterol from cells by the ABCA1 transporter and are being investigated as therapeutic agents for cardiovascular disease. We examined the role of helix stabilization of these peptides in cholesterol efflux. A 23-amino acid long peptide (Ac-VLEDSFKVSFLSALEEYTKKLNTQ-NH2) based on the last helix of apoA-I (A10) was synthesized, as well as two variants, S1A10 and S2A10, in which the third and fourth and third and fifth turn of each peptide, respectively, were covalently joined by hydrocarbon staples. By CD spectroscopy, the stapled variants at 24 °C were more helical in aqueous buffer than A10 (A10 17%, S1A10 62%, S2A10 97%). S1A10 and S2A10 unlike A10 were resistant to proteolysis by pepsin and chymotrypsin. S1A10 and S2A10 showed more than a 10-fold increase in cholesterol efflux by the ABCA1 transporter compared to A10. In summary, hydrocarbon stapling of amphipathic peptides increases their helicity, makes them resistant to proteolysis and enhances their ability to promote cholesterol efflux by the ABCA1 transporter, indicating that this peptide modification may be useful in the development of apolipoprotein mimetic peptides.

Introduction

Apolipoprotein mimetic peptides are being investigated as possible therapeutic agents for the treatment of cardiovascular diseases [1], [2], as well as disorders associated with inflammation [1], [3]. They reduce atherosclerosis in animal models and appear to be safe in early stage clinical trials [4], [5], [6]. Apolipoprotein mimetic peptides have similar biological properties as full length apolipoproteins, such as apoA-I, the main protein on high density lipoproteins (HDL). Weekly intravenous infusions of recombinant or purified apoA-I reconstituted with phospholipids for 4–5 weeks have been shown to reduce plaque volume in patients with acute coronary syndrome comparable to what has been achieved with several years of statin treatment [7], [8]. A major limitation of the use of full length apoA-I is the cost to produce the large quantities that are needed for this type of treatment and hence the interest in the use of short synthetic mimetic peptides [1]. Another potential advantage of apolipoprotein mimetic peptides is that when they are synthesized with d-amino acids, such as the D4F peptide, they are resistant to proteolysis and can reduce atherosclerosis in animal models when given orally [9], [10]. Clinical development of D4F, however, has been halted because of concerns related to long-term tissue accumulation [4].

ApoA-I and apolipoprotein mimetic peptides potentially have several different beneficial effects in preventing or reducing atherosclerosis [1], but the best understood and possibly the central mechanism behind many of their properties is based on their ability to increase the reverse cholesterol transport pathway [11], [12]. Recently, it was shown that the ability of HDL in serum to efflux excess cholesterol from macrophages was, in fact, a better predictor of the atheroprotective effect of HDL than the cholesterol content of HDL [13], the current diagnostic test for assessing atheroprotection by HDL. An early step in this process involves the interaction of apoA-I with the ABCA1 transporter, which by a detergent-like extraction step removes cholesterol and phospholipid from cells and forms small nascent HDL [14]. Only proteins, such as apoA-I and other apolipoproteins that contain amphipathic alpha helices, can remove lipid from the plasma membrane microdomain created by the ABCA1 transporter [2], [15].

In the absence of associated phospholipids, apolipoproteins do not as readily form amphipathic alpha helices [16], [17]. This is particularly true for short synthetic amphipathic peptides, which largely form random coils when present in aqueous solutions, because water more effectively competes with the intermolecular hydrogen bonds that stabilize alpha helices. Whether this less conformational constrained state for apolipoproteins or their mimetic peptides is beneficial or detrimental in their interaction with the ABCA1 transporter in the cholesterol efflux process is not known. Recently, it was shown that the hydrocarbon stapling of short synthetic peptides markedly increases their ability to form helices and has been used to improve the immunogenicity of synthetic peptide vaccines when an antigenic epitope is present in an alpha helical region of an intact protein [18], [19]. Because of the hydrophobicity of the hydrocarbon staple, these modified peptides also readily cross cell membranes, enabling their use for blocking intracellular protein–protein interactions [18], [19]. Increased membrane permeability may also account for the overall improved oral availability of stapled peptides, including relatively long peptides containing multiple helices [20]. In addition, because most proteases preferentially degrade unfolded proteins, the increased helical structure of stapled peptides reduces their degradation in the digestive tract and may increase their half-life in the plasma compartment [20].

In this study, we investigate the biophysical properties of the last helix of apoA-I and two hydrocarbon stapled variants of this peptide. The last helix of apoA-I has been shown to be critical in the ability of the full length protein to promote cholesterol efflux, but when synthesized as a single helical peptide, it is unable to promote cholesterol efflux [21]. In this study, we, therefore, also examined the effect of hydrocarbon stapling of the last helix of apoA-I on cholesterol efflux from cells by the ABCA1 transporter, as well as by several other mechanisms.