Abstract 1092: Dalcetrapib Binds to and Changes the Conformation of CETP in a Unique Manner (Differing to That Observed With Torcetrapib)
Objectives: Dalcetrapib (DAL), which decreases cholesteryl ester transfer protein (CETP) activity, has a unique chemical structure. The molecular site involved in binding DAL to CETP and associated conformational changes were evaluated and compared with those for torcetrapib (TOR).
Methods: Binding sites were evaluated using 14C-radiolabelled DAL-thiol (a derivative rapidly formed in plasma) and TOR in co-incubation studies on CETP immobilized on sepharose; binding responses were determined using Surface Plasmon Resonance (SPR). A series of monoclonal antibodies (mAb) raised against recombinant human CETP were used to develop an enzyme-linked immunosorbent assay (ELISA) specific towards detecting DAL-induced changes in CETP epitopes. Size exclusion chromatography/reverse phase arrays (SEC/RPA) was also employed.
Results 14C-DAL de-esterified active form (2.5 μM) binding to sepharose immobilized CETP was displaced by excess (25 μM) cold DAL or DAL-thiol in the presence of reducing agents such as DTT or TCEP but not by TOR. 14C-TOR (0.25 μM) was displaced by 25 μM TOR, but not by DAL or DAL-thiol. SPR studies confirmed that binding of DAL-thiol to immobilized CETP occurs in a saturable manner and is noncompetitive to TOR. JHC-1, a specific mAb raised against CETP, showed reduced reactivity when DAL or DAL-thiol (0.05 to 10 μM) was incubated with CETP; at 1 μM, OD was decreased by −85% (DAL) and −74% (DAL-thiol). In contrast, epitope recognition by JHC-1 was unchanged when CETP was incubated with TOR (+6% at 10 μM). Recognition of CETP epitopes by several other mAb including TP2 remained unchanged in the presence of DAL, DAL-thiol or TOR. SEC/RPA experiments demonstrated differential detection of “free” and lipoprotein-bound CETP.
Conclusions: DAL binds to a unique site of CETP identified previously as Cys13, which is different from that occupied by TOR. DAL’s binding may modulate Cys13 interaction with other free Cys residues of CETP. SEC/RPA experiments supported the concept that important conformational changes are linked to CETP activity. Antibodies recognizing different epitopes of CETP suggest DAL induces a conformational change in CETP not observed with TOR and which is most likely associated with dalcetrapib’s mechanism of CETP modulation.