Suzanne S. Eveland

Biochemical characterization of cholesteryl ester transfer protein inhibitors

Cholesteryl ester transfer protein (CETP) has been identified as a novel target for increasing HDL cholesterol levels. In this report, we describe the biochemical characterization of anacetrapib, a potent inhibitor of CETP. To better understand the mechanism by which anacetrapib inhibits CETP activity, its biochemical properties were compared with CETP inhibitors from distinct structural classes, including torcetrapib and dalcetrapib. Anacetrapib and torcetrapib inhibited CETP-mediated cholesteryl ester and triglyceride transfer with similar potencies, whereas dalcetrapib was a significantly less potent inhibitor. Inhibition of CETP by both anacetrapib and torcetrapib was not time dependent, whereas the potency of dalcetrapib significantly increased with extended preincubation. Anacetrapib, torcetrapib, and dalcetrapib compete with one another for binding CETP; however anacetrapib binds reversibly and dalcetrapib covalently to CETP. In addition, dalcetrapib was found to covalently label both human and mouse plasma proteins. Each CETP inhibitor induced tight binding of CETP to HDL, indicating that these inhibitors promote the formation of a complex between CETP and HDL, resulting in inhibition of CETP activity.

KINETIC MECHANISM OF THE ESCHERICHIA COLI UDPMURNAC-TRIPEPTIDE D-ALANYL-D-ALANINE-ADDING ENZYME : USE OF A GLUTATHIONE S-TRANSFERASE FUSION

The D-alanyl-D-alanine-adding enzyme encoded by the murF gene catalyzes the ATP-dependent formation of UDP-N-acetylmuramyl-L-gamma-D-Glu-meso-diaminopimelyl-D-Ala-D-Ala (UDP-MurNAc-tripeptide). MurF has been cloned from Escherichia coli and expressed as a glutathione S-transferase (GST) fusion using the tac promoter-based pGEX-KT vector. From induced, broken cell preparations, highly active fusion was recovered and purified in one step by affinity chromatography. The purified fusion protein was strongly inhibited by substrate UDPMurNAc-tripeptide, a response unaltered by changes in assay pH or by cleavage from the fusion partner. However, this effect was suppressed by the addition of 0.5 M NaCl. Initial velocity and dead-end inhibitor studies with the fusion enzyme were most consistent with a sequential ordered kinetic mechanism for the forward reaction in which ATP binds to free enzyme, followed by tripeptide and D-Ala-D-Ala in sequence prior to product release. Reported homologies between the MurF protein and the three preceding steps of cytoplasmic murein biosynthesis, MurC, -D, and -E, [Ikeda et al. (1990) J. Gen. Appl. Microbiol. 36, 179-187], raise the prospect that all of these enzymes will be found to proceed via this mechanism.

Biphenyl-Substituted Oxazolidinones as Cholesteryl Ester Transfer Protein Inhibitors: Modifications of the Oxazolidinone Ring Leading to the Discovery of Anacetrapib

The development of the structure-activity studies leading to the discovery of anacetrapib is described. These studies focused on varying the substitution of the oxazolidinone ring of the 5-aryloxazolidinone system. Specifically, it was found that substitution of the 4-position with a methyl group with the cis-stereochemistry relative to the 5-aryl group afforded compounds with increased cholesteryl ester transfer protein (CETP) inhibition potency and a robust in vivo effect on increasing HDL-C levels in transgenic mice expressing cynomolgus monkey CETP.

2-Arylbenzoxazoles as CETP inhibitors: Substitution of the benzoxazole moiety

A series of 2-arylbenzoxazole inhibitors of the cholesterol ester transfer protein (CETP) is described. Structure-activity studies focused on variation of the substitution of the benzoxazole moiety. Substitution at the 5- and 7-positions of the benzoxazole moiety was found to be beneficial for CETP inhibition. Compound 47 was found to be the most potent inhibitor in this series and inhibited CETP with an IC(50) of 28nM.

2-Arylbenzoxazoles as CETP inhibitors: raising HDL-C in cynoCETP transgenic mice.

We describe structure-activity studies leading to the discovery of 2-arylbenzoxazole 3, the first in a series to raise serum high-density lipoprotein cholesterol levels in transgenic mice. Replacement of the 4-piperidinyloxy moiety with piperazinyl provided a more synthetically tractable lead, which upon optimization resulted in compound 4, an excellent inhibitor of cholesteryl ester transfer protein function with good pharmacokinetic properties and in vivo efficacy.

2-Arylbenzoxazoles as CETP inhibitors: Substitution and modification of the α-alkoxyamide moiety

The development of a series of 2-arylbenzoxazole alpha-alkoxyamide and beta-alkoxyamine inhibitors of cholesteryl ester transfer protein (CETP) is described. Highly fluorinated alpha-alkoxyamides proved to be potent inhibitors of CETP in vitro, and the highly fluorinated 2-arylbenzoxazole beta-alkoxyamine 4 showed a desirable combination of in vitro potency (IC(50)=151 nM) and oral bioavailability in the mouse.

ApoA-I mimetic peptides promote pre-β HDL formation in vivo causing remodeling of HDL and triglyceride accumulation at higher dose.

Reverse cholesterol transport promoted by HDL-apoA-I is an important mechanism of protection against atherosclerosis. We have previously identified apoA-I mimetic peptides by synthesizing analogs of the 22 amino acid apoA-I consensus sequence (apoA-I(cons)) containing non-natural aliphatic amino acids. Here we examined the effect of different aliphatic non-natural amino acids on the structure-activity relationship (SAR) of apoA-I mimetic peptides. These novel apoA-I mimetics, with long hydrocarbon chain (C(5-8)) amino acids incorporated in the amphipathic α helix of the apoA-I(cons), have the following properties: (i) they stimulate in vitro cholesterol efflux from macrophages via ABCA1; (ii) they associate with HDL and cause formation of pre-β HDL particles when incubated with human and mouse plasma; (iii) they associate with HDL and induce pre-β HDL formation in vivo, with a corresponding increase in ABCA1-dependent cholesterol efflux capacity ex vivo; (iv) at high dose they associate with VLDL and induce hypertriglyceridemia in mice. These results suggest our peptide design confers activities that are potentially anti-atherogenic. However a dosing regimen which maximizes their therapeutic properties while minimizing adverse effects needs to be established.

Reconstituted HDL Elicits Marked Changes in Plasma Lipids Following Single-Dose Injection in C57Bl/6 Mice:

High-density lipoprotein (HDL)-targeting therapies, including reconstituted HDL (rHDL), are attractive agents for treating dyslipidemia and atherosclerosis, as they may increase HDL levels and enhance therapeutic activities associated with HDL, including reverse cholesterol transport (RCT). Using CSL-111, a rHDL consisting of native human apolipoprotein AI (hApoAI) and phospholipids, we characterized the acute effects of rHDL administration in C57Bl/6 mice to (i) further our understanding of the mechanism of action of rHDL, and (ii) evaluate the usefulness of the mouse as a preclinical model for HDL-targeting therapies. After a single injection of CSL-111, there was a dose- and time-dependent increase of hApoAI, human pre-β HDL, total cholesterol, and triglycerides in serum, consistent with the effects of CSL-111 in humans. However, unlike in humans, there was no measurable increase in cholesteryl esters. Evaluated ex vivo, the ATP binding cassette A1 (ABCA1)- and scavenger receptor type BI (SR-BI)-dependent cholesterol efflux capacity of serum from CSL-111-treated mice was increased compared with serum from vehicle-treated animals. Fractionation by size exclusion chromatography of lipoproteins in serum from treated mice revealed hApoAI in particles the size of endogenous HDL and slightly larger, cholesterol-enriched particles of all sizes, including sizes distinct from endogenous HDL or CSL-111 itself, and triglyceride-enriched particles the size of very-low-density lipoprotein (VLDL). These results suggest that in mouse blood CSL-111 is remodeled and generates enhanced cholesterol efflux capacity which increases mobilization of free cholesterol from peripheral tissues. Our findings complement the previous reports on CSL-111 in human participants and provide data with which to evaluate the potential utility of mouse models in mechanistic studies of HDL-targeting therapies.

Design of a novel class of biphenyl CETP inhibitors

A new class of CETP inhibitors was designed and prepared. These compounds are potent both in vitro and in vivo. The most active compound (12d) has shown an ability to raise HDL significantly in transgenic mouse PD model.

2-(4-carbonylphenyl)benzoxazole inhibitors of CETP: scaffold design and advancement in HDLc-raising efficacy.

The development of 2-phenylbenzoxazoles as inhibitors of cholesteryl ester transfer protein (CETP) is described. Initial efforts aimed at engineering replacements for the aniline substructures in the benchmark molecule. Reversing the connectivity of the central aniline lead to a new class of 2-(4-carbonylphenyl)benzoxazoles. Structure-activity studies at the C-7 and terminal pyridine ring allowed for the optimization of potency and HDLc-raising efficacy in this new class of inhibitors. These efforts lead to the discovery of benzoxazole 11v, which raised HDLc by 24 mg/dl in our transgenic mouse PD model.