Torcetrapib
Biosensor mapping places the torcetrapib binding site at Cys-13 in the CETP lipid-binding pocket (Bioconjug Chem 2008)
Original title: Biophysical and biochemical approach to locating an inhibitor binding site on cholesteryl ester transfer protein
To locate where CETP inhibitors bind cholesteryl ester transfer protein (CETP), researchers coupled a torcetrapib-related compound to biotin-terminated spacer arms of varying length and measured CETP binding to the immobilized conjugates using agarose beads and surface plasmon resonance. CETP bound poorly to a 2.0 nm spacer but efficiently to 3.5 or 4.6 nm polyethylene glycol spacers, and both soluble inhibitor and a disulfide-containing covalent inhibitor blocked this binding. Modifying CETP with a disulfide reagent that covalently labels Cys-13, previously implicated by mutagenesis, abolished binding to the immobilized torcetrapib analog, and mass spectrometry and peptide mapping confirmed Cys-13 as the modification site. The authors conclude the torcetrapib binding site lies in the lipid-binding pocket near the N-terminus of CETP, consistent with the crystal structure showing the Cys-13 sulfhydryl group at the bottom of this pocket.
Original 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.
Summary written by cetpinhibition.org from the published abstract; figures as published. Page updated 18 August 2026. Methods.