Different Binding Modes of Compounds Affecting CETP Activity: Dalcetrapib and Torcetrapib

Abstract

Cholesteryl ester transfer protein (CETP) is a hydrophobic glycoprotein in plasma and facilitates the exchange of neutral lipids between various lipoproteins resulting in a net transport of cholesteryl ester from HDL to LDL. Decrease in CETP activity leads to an increase of HDL cholesterol which potentially reduces cardiovascular risk. Human recombinant CETP (rhCETP) was purified to homogeneity by hydrophobic interaction chromatography and size exclusion chromatography (SEC). The protein showed an extraordinarily high thermodynamic stability against thermal as well as denaturant-induced unfolding. The effects of CETP on lipoprotein profiles were characterized after in-vitro incubation of plasma with rhCETP followed by SEC and reverse phase protein arrays. Monitoring the distribution of apolipoproteins and the cholesterol profiles confirmed the transfer of cholesteryl ester from HDL to LDL induced by CETP. In the presence of the potent CETP inhibitor torcetrapib (1 µM) added CETP is bound to lipoproteins while free CETP can still be detected in presence of dalcetrapib (3 µM), a compound from a different chemical class. 14C-torcetrapib and 14C-dalcetrapib were bound to CETP immobilized on sepharose and incubated with excess of unlabelled compounds. 14C-dalcetrapib bound as Dalcetrapib-thiol could only be displaced in the presence of reducing agent by an excess of unlabeled dalcetrapib but not by torcetrapib. Dalcetrapib did not compete for binding of 14C-torcetrapib. Interaction studies with Surface Plasmon Resonance confirmed reversible covalent binding of dalcetrapib-thiol and different binding sites for dalcetrapib and torcetrapib. In agreement with literature cysteine 13 of CETP was identified as attachment point of dalcetrapib-thiol. All data suggested that torcetrapib and dalcetrapib bind to different sites on CETP which may be related to differences observed in their pharmacological profiles.