Cholesteryl ester transfer between lipoproteins does not require a ternary tunnel complex with CETP.

The cholesteryl ester transfer protein (CETP) enables the transfer of cholesteryl ester (CE) from high-density lipoproteins (HDL) to low-density lipoproteins (LDL) in the plasma compartment. CETP inhibition raises plasma levels of HDL cholesterol; a ternary tunnel complex with CETP bridging HDL and LDL was suggested as a mechanism. Here, we test whether the inhibition of CETP tunnel complex formation is a promising approach to suppress CE transfer from HDL to LDL, for potential treatment of cardio-vascular disease (CVD). Three monoclonal antibodies against different epitopes of CETP are assayed for their potential to interfere with CE transfer between HDL and/or LDL. Surprisingly, antibodies that target the tips of the elongated CETP molecule, interaction sites sterically required to form the suggested transfer complexes, do not interfere with CETP activity, but an antibody binding to the central region does. We show that CETP interacts with HDL, but not with LDL. Our findings demonstrate that a ternary tunnel complex is not the mechanistic prerequisite to transfer CE among lipoproteins.

Evidence for a role of CETP in HDL remodeling and cholesterol efflux: role of cysteine 13 of CETP.

Cholesteryl ester transfer protein (CETP), a key regulator of high-density lipoprotein (HDL) metabolism, induces HDL remodeling by transferring lipids between apolipoprotein B-containing lipoproteins and HDL, and/or by promoting lipid transfer between HDL subparticles. In this study, we investigated the mechanism as to how CETP induces the generation of lipid-poor particles (pre-ß-HDL) from HDL, which increases ATP-binding cassette transporter 1-mediated cholesterol efflux. This CETP-dependent HDL remodeling is enhanced by the CETP modulator dalcetrapib both in plasma and isolated HDL. The interaction of dalcetrapib with cysteine 13 of CETP is required, since this effect was abolished when using mutant CETP in which cysteine 13 was substituted for a serine residue. Other thiol-containing compounds were identified as CETP modulators interacting with cysteine 13 of CETP. In order to mimic dalcetrapib-bound CETP, mutant CETP proteins were prepared by replacing cysteine 13 with the bulky amino acid tyrosine or tryptophan. The resultant mutants showed virtually no CETP-dependent lipid transfer activity but demonstrated preserved CETP-dependent pre-ß-HDL generation. Overall, these data demonstrate that the two functions of CETP i.e., cholesteryl ester transfer and HDL remodeling can be uncoupled by interaction of thiol-containing compounds with cysteine 13 of CETP or by introducing large amino acid residues in place of cysteine 13.

Lipid and apoprotein composition of HDL in partial or complete CETP deficiency.

Hyperalphalipoproteinemia, as observed in patients who are either homozygous or heterozygous for cholesteryl ester transfer protein (CETP) deficiency, has been shown to be associated with striking changes in apolipoprotein size distribution, namely, of high-density lipoprotein (HDL) and HDL-like particles. We compared the effect of varying degrees of CETP activity on the HDL apolipoprotein profile in Caucasian CETP-deficient subjects and following pharmacological decrease in CETP activity, using Size Exclusion Chromatography followed by Reverse Phase Protein Array (SEC RPA). The main HDL-associated apolipoproteins (Apo), i.e. ApoA-I, ApoA-II, ApoC-I, and ApoC-III, co-eluted with the HDL peak. The presence of a HDL-like peak migrating between the ApoB-LDL and ApoA-I-HDL was identified in a Caucasian patient with homozygosity for a point mutation in exon 2 of the CETP gene (c.109 C > T) resulting in a premature termination codon (R37X) and complete CETP deficiency. This HDL-like peak was not observed either in healthy volunteers treated with the CETP modulator dalcetrapib, patients heterozygous for the same mutation, or in patients heterozygous with G165X mutations. SEC RPA offers the possibility to investigate the distribution of a large number of apolipoproteins simultaneously under non-denaturing separation in normal and dyslipidemic subjects. This is only limited by the availability of antibodies against specific apolipoproteins to be investigated.

Multidimensional profiling of plasma lipoproteins by size exclusion chromatography followed by reverse-phase protein arrays.

The composition of lipoproteins and the association of proteins with various particles are of much interest in the context of cardiovascular disease. Here, we describe a technique for the multidimensional analysis of lipoproteins and their associated apolipoproteins. Plasma is separated by size exclusion chromatography (SEC), and fractions are analyzed by reverse-phase arrays. SEC fractions are spotted on nitrocellulose slides and incubated with different antibodies against individual apolipoproteins or antibodies against various apolipoproteins. In this way, tens of analytes can be measured simultaneously in 100 µl of plasma from a single SEC separation. This methodology is particularly suited to simultaneous analysis of multiple proteins that may change their distribution to lipoproteins or alter their conformation, depending on factors that influence circulating lipoprotein size or composition. We observed changes in the distribution of exchangeable apolipoproteins following addition of recombinant apolipoproteins or interaction with exogenous compounds. While the cholesteryl ester transfer protein (CETP)-dependent formation of pre-ß-HDL was inhibited by the CETP inhibitors torcetrapib and anacetrapib, it was not reduced by the CETP modulator dalcetrapib. This finding was elucidated using this technique.

Modulating cholesteryl ester transfer protein activity maintains efficient pre-ß-HDL formation and increases reverse cholesterol transport.

The mechanism by which cholesteryl ester transfer protein (CETP) activity affects HDL metabolism was investigated using agents that selectively target CETP (dalcetrapib, torcetrapib, anacetrapib). In contrast with torcetrapib and anacetrapib, dalcetrapib requires cysteine 13 to decrease CETP activity, measured as transfer of cholesteryl ester (CE) from HDL to LDL, and does not affect transfer of CE from HDL3 to HDL2. Only dalcetrapib induced a conformational change in CETP, when added to human plasma in vitro, also observed in vivo and correlated with CETP activity. CETP-induced pre-ß-HDL formation in vitro in human plasma was unchanged by dalcetrapib ≤3 µM and increased at 10 µM. A dose-dependent inhibition of pre-ß-HDL formation by torcetrapib and anacetrapib (0.1 to 10 µM) suggested that dalcetrapib modulates CETP activity. In hamsters injected with [³H]cholesterol-labeled autologous macrophages, and given dalcetrapib (100 mg twice daily), torcetrapib [30 mg once daily (QD)], or anacetrapib (30 mg QD), only dalcetrapib significantly increased fecal elimination of both [³H]neutral sterols and [³H]bile acids, whereas all compounds increased plasma HDL-[³H]cholesterol. These data suggest that modulation of CETP activity by dalcetrapib does not inhibit CETP-induced pre-ß-HDL formation, which may be required to increase reverse cholesterol transport.

Synthesis and structure-activity studies of novel orally active non-terpenoic 2,3-oxidosqualene cyclase inhibitors.

New orally active non-terpenoic inhibitors of human 2,3-oxidosqualene cyclase (hOSC) are reported. The starting point for the optimization process was a set of compounds derived from a fungicide project, which in addition to showing high affinity for OSC from Candida albicans showed also high affinity for human OSC. Common structural elements of these inhibitors are an amine residue and an electrophilic carbonyl C atom embedded in a benzophenone system, which are at a distance of about 10.7 A. Considering that the keto moiety is in a potentially labile position, modifications of the substitution pattern at the benzophenone as well as annelated heteroaryl systems were explored. Our approach combined testing of the compounds first for increased binding affinity and for increased stability in vitro. Most promising compounds were then evaluated for their efficacy in lowering plasma total cholesterol (TC) and plasma low-density lipoprotein cholesterol (LDL-C) in hyperlipidemic hamsters. In this respect, the most promising compounds are the benzophenone derivative 1.fumarate and the benzo[d]isothiazol 24.fumarate, which lowered TC by 40% and 33%, respectively.