Atherosclerosis is a causative factor in nearly one fifth of all human deaths worldwide. A promising strategy for combating atherosclerosis involves modulating high-density lipoproteins (HDLs) to facilitate the process of reverse cholesterol transport (RCT) and thereby remove excess cholesterol from peripheral tissues for elimination. However, recent clinical trial failures of compounds that elevate total plasma HDL levels highlight the need for a better understanding of the atheroprotective mechanisms of HDLs, and for new approaches to improve HDL function. It is becoming increasingly clear that improving HDL quality (i.e., function) is more important than simply increasing HDL quantity. The proposed research program is built on our recent success in advancing a new class of apolipoprotein A-I (apoA-I) mimetics that enhance the RCT function of HDLs both in vitro and in vivo. ApoA-I plays a key role in initiating RCT and its anti-atherogenic properties have been documented in numerous studies, including human clinical trials. However, the high production costs and lack of oral bioavailability of apoA-I have made it impractical for chronic use in the management of atherosclerosis. We have designed novel apoA-I mimetics based on multivalent presentation of short, synthetic, amphiphilic, helical peptides that can be fashioned into discoidal HDL-like nanolipopeptide constructs. These constructs interact with and remodel human and mouse plasma HDLs, increase the level of pre-beta HDL particles (the subspecies of HDLs considered to be the most anti-atherogenic), and enhance cholesterol efflux from macrophages. Moreover, a trimeric construct is remarkably effective in vivo in preventing the progression of atherosclerotic lesions in a 10-week study in LDLr-/- mice (an animal model of atherosclerosis that resembles hyperlipidemia in humans). Importantly, our apoA-I mimetics exhibit surprisingly high oral bioavailability in mice, thus providing a promising path toward the development of future therapeutic agents. In short, our studies to date provide a strong basis for the proposed research aimed at developing safe, orally bioavailable apoA-I mimetics to treat atherosclerosis. Our first objective is to optimize the lead constructs for synthetic efficiency, oral bioavailabiliy, pharmacokinetic profile, and in vivo efficacy (Aims 1 and 2). These aims will be achieved through a series of new designs, structural optimizations, and functional analyses. Our second objective is to better understand the basic mechanisms by which apolipoprotein mimetic peptides modulate HDL function by performing a series of proposed biophysical and enzymological analyses with native and reconstituted HDL particles (Aim 3). Our assembled team, which includes experts in bioactive peptide design and chemistry, mouse models of atherosclerosis, and drug development, is uniquely suited to carry out the proposed studies. We hope that these studies will result in a more detailed knowledge of how best to modulate HDLs and identify drug candidates to develop as orally bioavailable anti-atherosclerotic agents.