Biophysical and Biochemical Approach to Locating an Inhibitor Binding Site on Cholesteryl Ester Transfer Protein

Abstract

Cholesteryl ester transfer protein (CETP) transfers neutral lipids between different types of plasma lipoprotein. Inhibitors of CETP elevate the fraction of plasma cholesterol associated with high-density lipoproteins and are being developed as new agents for the prevention and treatment of cardiovascular disease. The molecular basis of their function is not yet fully understood. To aid in the study of inhibitor interactions with CETP, a torcetrapib-related compound was coupled to different biotin-terminated spacer groups, and the binding of CETP to the streptavidin-bound conjugates was monitored on agarose beads and in a surface plasmon resonance biosensor. CETP binding was poor with a 2.0 nm spacer arm, but efficient with polyethyleneglycol spacers of 3.5 or 4.6 nm. The conjugate based on a 4.6 nm spacer was used for further biosensor experiments. Soluble inhibitor blocked the binding of CETP to the immobilized drug, as did preincubation with a disulfide-containing covalent inhibitor. To provide a first estimate of the binding site for torcetrapib-like inhibitors, CETP was modified with a disulfide-containing agent that modifies Cys-13 of CETP. Mass spectrometry of the modified protein indicated that a single half-molecule of the disulfide was covalently bound to CETP, and peptide mapping after digestion with pepsin confirmed previous reports based on mutagenesis that Cys-13 was the site of modification. Modified CETP was unable to bind to the biosensor-mounted torcetrapib analog, indicating that the binding site on CETP for torcetrapib is in the lipid-binding pocket near the N-terminus of the protein. The crystal structure of CETP shows that the sulfhydryl group of Cys-13 resides at the bottom of this pocket.

Biochemical characterization of cholesteryl ester transfer protein inhibitors

“…Dalcetrapib is an inhibitor of CETP transfer activity and is currently in phase III clinical studies ( 35 ). Inhibition of CETP by dalcetrapib requires formation of a covalent disulfi de bond with Cys13 of CETP ( 35,56 ). Treatment of humans with dalcetrapib results in increases in HDL-C (19-37%) and a modest decrease ( ‫ف‬ 6%) in LDL-C levels ( 57 ).…”

“…Anacetrapib is an inhibitor of CETP transfer activity and is currently in phase III clinical studies ( 58 ). Treatby guest, on May 10, 2018 www.jlr.org Downloaded from upon bound ligand was separated from free by processing 80 l on a Pierce Zeba TM 96-well Desalt Spin Plate (Pierce) according to the manufacturer's instructions. The 50 l of the column eluate containing CETP and bound inhibitor was quantitated in 5 ml Perkin Elmer Ultima Gold MV using a Perkin Elmer TriCarb 2900TR liquid scintillation analyzer.…”

“…Binding of torcetrapib to CETP has previously been demonstrated using either a [ 3 H]torcetrapib-CETP binding assay ( 50 ) or by surface plasmon resonance (Biacore) ( 56 ). To measure the specifi c binding of anacetrapib to CETP, we used a similar assay with purifi ed CETP protein and either [ 3 H]anacetrapib or [ 3 H]torcetrapib.…”

Assessment of cholesteryl ester transfer protein inhibitors for interaction with proteins involved in the immune response to infection

“…For this study, CETP inhibitors (CETPIs) representing two series were tested, including torcetrapib and its close analog CP-532623 ( 1,13 ), and the Merck inhibitor anacetrapib ( 3 ). Orlistat, a nonselective lipase inhibitor, was included as a control nonCETPI compound with similar high lipophilicity (ElogD = 8.95) to that of the CETPIs (ElogD = 7.4-10.1).…”

“…LBP is predicted to be much more similar to BPI than to CETP, both in sequence homology (LBP:BPI 44% identity, LBP:CETP 25% identity) and ability to bind lipid. The binding site for torcetrapib has recently been defi ned to the N-terminal side of the CETP tunnel ( 13 ), which is about twice the size of a BPI pocket and shares no apparent similarity in lipid binding modes.…”