Abstract
Cholesteryl ester transfer protein (CETP) inhibitors are a new class of therapeutics for dyslipidemia that simultaneously improve two major cardiovascular disease (CVD) risk factors: elevated low-density lipoprotein (LDL) cholesterol and decreased high-density lipoprotein (HDL) cholesterol. However, the detailed molecular mechanisms underlying their efficacy are poorly understood, as are any potential mechanistic differences among the drugs in this class. Herein, we used electron microscopy (EM) to investigate the effects of three of these agents (Torcetrapib, Dalcetrapib and Anacetrapib) on CETP structure, CETP-lipoprotein complex formation and CETP-mediated cholesteryl ester (CE) transfer. We found that although none of these inhibitors altered the structure of CETP or the conformation of CETP-lipoprotein binary complexes, all inhibitors, especially Torcetrapib and Anacetrapib, increased the binding ratios of the binary complexes (e.g., HDL-CETP and LDL-CETP) and decreased the binding ratios of the HDL-CETP-LDL ternary complexes. The findings of more binary complexes and fewer ternary complexes reflect a new mechanism of inhibition: one distal end of CETP bound to the first lipoprotein would trigger a conformational change at the other distal end, thus resulting in a decreased binding ratio to the second lipoprotein and a degraded CE transfer rate among lipoproteins. Thus, we suggest a new inhibitor design that should decrease the formation of both binary and ternary complexes. Decreased concentrations of the binary complex may prevent the inhibitor was induced into cell by the tight binding of binary complexes during lipoprotein metabolism in the treatment of CVD.
Highlights
Elevated plasma low-density lipoprotein cholesterol (LDL-C) and decreased high-density lipoprotein cholesterol (HDL-C) levels are two major risk factors for cardiovascular disease (CVD) [1]
Given that Cholesteryl ester transfer protein (CETP) is an approximately 53 kDa asymmetric molecule (~74 kDa for fully glycosylated CETP) that is too small for cryo-electron microscopy (EM), optimized negative staining (OpNS) [33,34] was used to investigate how CETP inhibitors influence the CETP structure
The statistical analyses of ternary complexes showed that the CETP binding on HDL and LDL increased by ~20% and 50% respectively when CETP was bound to two species of lipoproteins simultaneously (Fig. 4F, control row)
Summary
Elevated plasma low-density lipoprotein cholesterol (LDL-C) and decreased high-density lipoprotein cholesterol (HDL-C) levels are two major risk factors for cardiovascular disease (CVD) [1]. The difficulty in studying the mechanism of CETP lies in the heterogeneity of its lipoprotein substrates and in the softness and high flexibility of their complex three-dimensional (3D) structure [17,18,19]. These properties limit the application of the other experimental procedures. Our early EM studies have shown that CETP bridges HDL and LDL together, thereby forming a ternary complex [25] in which the N-terminal β-barrel domain inserts into the surface lipid monolayer of HDL. We used EM techniques, including cryo-electron microscopy (cryo-EM) and optimized negative staining (OpNS), to investigate the effects of CETP inhibitors on the CETP-lipoprotein structure and their conformations under various incubation times
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More From: Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids
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