Biochemical characterization of cholesteryl ester transfer protein inhibitors

Abstract

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.

Fig. 1.

Inhibition of CETP-dependent neutral lipid transfer using a fluorogenic transfer assay. Increasing amounts of the indicated compounds were assayed in the fluorogenic assay using purified recombinant N341Q CETP as described in “Methods.” A: Dose response of inhibitors reducing CETP-mediated CE transfer. Inhibitors were preincubated with CETP and HDL donor particles for 1 h before addition of acceptor particles. B: Dose response of inhibitors reducing CETP-mediated TG transfer. Inhibitors were preincubated with CETP and HDL donor particles for 1 h before addition of acceptor particles. C: The effect of 24 h preincubation on the potency of inhibitors in reducing CETP-mediated CE transfer. Inhibitors were preincubated with CETP and HDL donor particles for 24 h before addition of acceptor particles. Typical results representative of at least three independent experiments are shown and are fit to a sigmoidal dose-response curve by nonlinear regression.

Fig. 2.

Inhibition of CETP-dependent CE transfer using a radioactive neutral lipid transfer assay. Increasing amounts of the indicated compounds were assayed in the in vitro radioactive assay using either 2% human serum with additional (30 nM) purified N341Q CETP protein added, or in 95% human serum as described in “Methods.” Inhibitors were preincubated for either 1 or 24 h before addition of LDL containing labeled tracer neutral lipids. A: Dose response of inhibitors reducing CETP-mediated CE transfer in 2% human serum. Inhibitors were preincubated with CETP in 2% human serum for 1 h before addition of ³H-labeled LDL. B: Dose response of inhibitors reducing CETP-mediated CE transfer in 95% human serum. Inhibitors were preincubated in 95% human serum for 1 h before addition of ³H-labeled LDL. C: The effect of 24 h preincubation on the potency of inhibitors in reducing CETP-mediated CE transfer in 95% human serum. Inhibitors were preincubated in 95% human serum for 24 h before addition of ³H-labeled LDL. Typical results representative of at least three independent experiments are shown and are fit to a sigmoidal dose-response curve by nonlinear regression.

Fig. 3.

Binding of [³H]anacetrapib to purified CETP. A: Silver-stained gel of purified N341Q CETP protein. Media from a stable S2 cell line overexpressing N341Q mutant CETP protein was used for purification of secreted protein as described in “Methods.” The results shown are from 3 μg of recombinant N341Q CETP protein loaded and are run next to a standard protein molecular weight ladder (L). B: Dose response of [³H]anacetrapib and [³H]torcetrapib binding to CETP. Increasing amounts of [³H]anacetrapib or [³H]torcetrapib were combined with purified N341Q CETP protein (20 nM) and samples were processed as described in “Methods.” Specific binding activity was calculated as the difference between the amount of inhibitor bound in the presence of CETP minus the amount of inhibitor bound in the absence of CETP.

Fig. 4.

Competition of inhibitors for binding CETP and reversibility of binding. A: Competition between [³H]torcetrapib and unlabeled inhibitors for binding CETP. Increasing concentrations of indicated unlabeled compounds were incubated with 15 nM [³H]torcetrapib and CETP and CETP-bound [³H]torcetrapib was measured as detailed in “Methods.” B: Competition between [³H]anacetrapib and unlabeled inhibitors for binding CETP. Increasing concentrations of indicated unlabeled compounds were incubated with 15 nM [³H]anacetrapib and CETP and CETP-bound [³H]anacetrapib was measured as detailed in “Methods.” C: Chemical structures of dalcetrapib. Dalcetrapib is represented both as a disulfide-linked dimer of 637 Da molecular weight and as a half-molecule of 318 Da molecular weight. D: MS of CETP. i) CETP control; ii) CETP incubated with anacetrapib; iii) CETP with dalcetrapib disulfide inhibitor showed a modification of 318 Da corresponding to a half-molecule of the inhibitor. This modification was reversed by DTT (iv). Incubation with iodoacetamide (v) illustrated the susceptibility of a free cysteine in CETP for modification; the appearance of a peak with a difference of 57 Da corresponding to iodoacetamide modification was detected.

Fig. 5.

Covalent binding of dalcetrapib to human and mouse plasma proteins in vitro. Human and mouse plasma was incubated with either [¹⁴C]anacetrapib or [¹⁴C]dalcetrapib and the degree of protein labeling was determined after various times of incubation as described in “Methods.” A: Labeling of human plasma proteins. B: Analysis of human plasma proteins labeled by [¹⁴C]dalcetrapib. Following incubation of 50 μM [¹⁴C]anacetrapib or [¹⁴C]dalcetrapib, proteins were separated via nonreducing SDS-PAGE. Analysis of total protein loaded (Coomassie), [¹⁴C]labeled proteins, or CETP (anti-CETP immunoblot) was carried out as described in “Methods.” C: Labeling of mouse plasma proteins. Plasma from either wild-type mice lacking CETP (WT) or CETP^Tg mice was incubated with 3 μM [¹⁴C]anacetrapib or [¹⁴C]dalcetrapib, and the degree of protein labeling was determined after various times of incubation as described in “Methods.” D: Following incubation of 50 μM [¹⁴C]anacetrapib or [¹⁴C]dalcetrapib, proteins were separated via nonreducing SDS-PAGE. Analysis of total protein loaded (Coomassie), [¹⁴C]labeled proteins, or CETP (anti-CETP immunoblot) was carried out as described in “Methods.”

Fig. 6.

Effect of inhibitors on formation of a stable CETP-HDL complex. Binding assay conditions were designed to mimic the neutral lipid fluorescence transfer assay in order to compare inhibition of transfer activity with formation of a CETP-HDL complex. Each panel includes representative dose responses for inhibition of neutral lipid transfer by each compound in the fluorogenic neutral lipid transfer assay and native-PAGE/CETP Western-blot analysis as described in “Methods.” A: Dose response of anacetrapib. B: Dose response of torcetrapib. C: Dose response of dalcetrapib. CETP-HDL binding assay reactions consisted of CETP alone or with HDL and increasing concentrations of each inhibitor.

Fig. 7.

Effect of inhibitors on CETP-HDL complex formation in human plasma. FPLC fractionation of human plasma after treatment with either vehicle or CETP inhibitor was carried out as described in “Methods.” Samples were treated with 1 μM anacetrapib or torcetrapib or 10 μM dalcetrapib. Shown are representative FPLC profiles from three independent experiments.