Crystal structures of cholesteryl ester transfer protein in complex with inhibitors.

Human plasma cholesteryl ester transfer protein (CETP) transports cholesteryl ester from the antiatherogenic high-density lipoproteins (HDL) to the proatherogenic low-density and very low-density lipoproteins (LDL and VLDL). Inhibition of CETP has been shown to raise human plasma HDL cholesterol (HDL-C) levels and is potentially a novel approach for the prevention of cardiovascular diseases. Here, we report the crystal structures of CETP in complex with torcetrapib, a CETP inhibitor that has been tested in phase 3 clinical trials, and compound 2, an analog from a structurally distinct inhibitor series. In both crystal structures, the inhibitors are buried deeply within the protein, shifting the bound cholesteryl ester in the N-terminal pocket of the long hydrophobic tunnel and displacing the phospholipid from that pocket. The lipids in the C-terminal pocket of the hydrophobic tunnel remain unchanged. The inhibitors are positioned near the narrowing neck of the hydrophobic tunnel of CETP and thus block the connection between the N- and C-terminal pockets. These structures illuminate the unusual inhibition mechanism of these compounds and support the tunnel mechanism for neutral lipid transfer by CETP. These highly lipophilic inhibitors bind mainly through extensive hydrophobic interactions with the protein and the shifted cholesteryl ester molecule. However, polar residues, such as Ser-230 and His-232, are also found in the inhibitor binding site. An enhanced understanding of the inhibitor binding site may provide opportunities to design novel CETP inhibitors possessing more drug-like physical properties, distinct modes of action, or alternative pharmacological profiles.

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

The CETP inhibitor, torcetrapib, was prematurely terminated from phase 3 clinical trials due to an increase in cardiovascular and noncardiovascular mortality. Because nearly half of the latter deaths involved patients with infection, we have tested torcetrapib and other CETPIs to see if they interfere with lipopolysaccharide binding protein (LBP) or bactericidal/permeability increasing protein (BPI). No effect of these potent CETPIs on LPS binding to either protein was detected. Purified CETP itself bound weakly to LPS with a Kd >or= 25 microM compared with 0.8 and 0.5 nM for LBP and BPI, respectively, and this binding was not blocked by torcetrapib. In whole blood, LPS induced tumor necrosis factor-alpha normally in the presence of torcetrapib. Furthermore, LPS had no effect on CETP activity. We conclude that the sepsis-related mortality of the ILLUMINATE trial was unlikely due to a direct effect of torcetrapib on LBP or BPI function, nor to inhibition of an interaction of CETP with LPS. Instead, we speculate that the negative outcome seen for patients with infections might be related to the changes in plasma lipoprotein composition and metabolism, or alternatively to the known off-target effects of torcetrapib, such as aldosterone elevation, which may have aggravated the effects of sepsis.

Frequency and function of CETP variants among individuals of Asian ancestry.

Genetic variation in CETP (cholesteryl ester transfer protein) has been clearly associated with HDL cholesterol levels but its association with cardiovascular disease and related phenotypes has been more controversial, possibly due to variability of polymorphisms and their frequencies across different ethnic populations. To see if there are undetected polymorphisms affecting protein sequence in individuals of Asian ancestry and to determine the functionality of such variants, all exons and adjacent intronic segments were resequenced in 96 individuals and the observed variants cloned and analyzed. Two novel SNPs, including one coding change, S332 to Y332, were identified. Y332 and all other reported variants in Asians were cloned for study in vitro. Secretion efficiency was determined by Western blotting of protein from cell lysates and media. Cholesteryl ester transfer activity was measured in vitro by following the extent of transfer of fluorescently labeled substrate. Y332, Q296 and G442 are all secreted less well than wild type protein but retain significant transfer activity. P151 is not secreted and no transfer activity was detected. These protein variants should all contribute to higher HDL cholesterol in individuals carrying them. Additionally, a splicing variation that causes a protein truncation and non-functional CETP that has been reported predominantly in Asians was also found in two individuals of European ancestry and was on the same haplotype background in the two populations, suggesting a common origin of this null variant. This improved understanding of CETP variation in Asians will allow a more effective comparison of studies across populations.

Expression of CETP and of splice variants induces the same level of ER stress despite secretion efficiency differences.

The cholesteryl ester transfer protein (CETP) gene has been associated with a variety of phenotypes, including HDL-cholesterol levels and, more sporadically, with cardiovascular disease, obesity, and extreme longevity. Alterations of CETP activity levels can be caused by single-base polymorphisms as well as by alternative splicing. In addition to the previously characterized alternative splicing that skips exon 9, we found additional minor variants and characterized the activity of the resultant proteins. The novel variants skipped exon 9 sequences and inserted one of two in-frame exons from Alu-derived intronic sequences. None of the alternatively spliced variants are efficiently secreted, and coexpression of them inhibits wild-type CETP secretion. Expression of the alternative spliced variants causes an induction of genes linked to the endoplasmic reticulum (ER) stress response, including the neighboring HERPUD1 (homocysteine- and ER stress-inducible protein, ubiquitin-like domain-containing) gene. Unexpectedly, even though wild-type CETP is secreted much more efficiently than spliced variants, it induces the same degree of stress response as spliced variants, whereas a control secreted protein does not. CETP plays a complex role in modulating ER stress, with its expression inducing the response and its cholesteryl ester transfer activity and differential splicing modulating the response in other ways.

Crystal structure of cholesteryl ester transfer protein reveals a long tunnel and four bound lipid molecules.

Cholesteryl ester transfer protein (CETP) shuttles various lipids between lipoproteins, resulting in the net transfer of cholesteryl esters from atheroprotective, high-density lipoproteins (HDL) to atherogenic, lower-density species. Inhibition of CETP raises HDL cholesterol and may potentially be used to treat cardiovascular disease. Here we describe the structure of CETP at 2.2-A resolution, revealing a 60-A-long tunnel filled with two hydrophobic cholesteryl esters and plugged by an amphiphilic phosphatidylcholine at each end. The two tunnel openings are large enough to allow lipid access, which is aided by a flexible helix and possibly also by a mobile flap. The curvature of the concave surface of CETP matches the radius of curvature of HDL particles, and potential conformational changes may occur to accommodate larger lipoprotein particles. Point mutations blocking the middle of the tunnel abolish lipid-transfer activities, suggesting that neutral lipids pass through this continuous tunnel.

Protein fusions of BPI with CETP retain functions inherent to each.

Cholesteryl ester transfer protein (CETP), bactericidal/permeability inducing protein (BPI), and lipopolysaccharide binding protein (LBP) are members of the lipid transfer/lipopolysaccharide binding protein (LT/LBP) family of proteins that share a common secondary/tertiary structure. Despite this commonality of structure, very different patterns of lipid binding and protein-protein interactions are observed among the family members. BPI was previously shown to retain aspects of its own function when part of it was fused with LBP to form a chimeric protein. We have extended those observations to CETP. Some aspects of cholesteryl ester transfer function can be maintained in a chimeric protein even when over 40% of the sequence is from BPI. Further replacement of an additional 60 amino acids resulted in a complete loss of CETP function even though the chimera was able to retain some BPI-like properties. These artificial fusions retain BPI functions such as lipopolysaccharide (LPS) binding and protein-protein interactions that are not observed with native CETP. BPI-CETP chimeras are inhibited by LPS but cannot be inhibited by small molecule CETP inhibitors as effectively as native CETP. These results localize the site of LPS binding in BPI to a region no larger than the amino terminal 155 amino acids. This region can participate in some protein-protein interactions similar to intact BPI. Chimeras containing the amino terminus of CETP and the carboxy terminus of BPI did not retain any observable CETP function. These results further confirm the modular nature of the LT/LBP family of proteins but also highlight the discrete nature of their individual functions.

Novel variants in human and monkey CETP.

Variation in CETP has been shown to play an important role in HDL-C levels and cardiovascular disease. To better characterize this variation, the promoter and exonic DNA for CETP was resequenced in 189 individuals with extreme HDL-C or age. Two novel amino acid variants were found in humans (V-12D and Y361C) and an additional variant (R137W) not previously studied in vitro were expressed. D-12 was not secreted and had no detectable activity in cells. C361 and W137 retained near normal amounts of cholesteryl ester transfer activity when purified but were less well secreted than wild type. Torcetrapib, a CETP inhibitor in clinical development with atorvastatin, was found to have a uniform effect on inhibition of wild type CETP versus W137 or C361. In addition, the level of variation in other species was assessed by resequencing DNA from nine cynomolgus monkeys. Numerous intronic and silent SNPs were found as well as two variable amino acids. The amino acid altering SNPs were genotyped in 29 monkeys and not found to be significantly associated with HDL-C levels. Three SNPs found in monkeys were identical to three found in humans with these SNPs all occurring at CpG sites.

Pentamidine reduces hERG expression to prolong the QT interval.

Pentamidine, an antiprotozoal agent, has been traditionally known to cause QT prolongation and arrhythmias; however, its ionic mechanism has not been illustrated. In a stable HEK-293 cell line, we observed a concentration-dependent inhibition of the hERG current with an IC50 of 252 microM. In freshly isolated guinea-pig ventricular myocytes, pentamidine showed no effect on the L-type calcium current at concentrations up to 300 microM, with a slight prolongation of the action potential duration at this concentration. Since the effective concentrations of pentamidine on the hERG channel and APD were much higher than clinically relevant exposures (approximately 1 microM free or lower), we speculated that this drug might not prolong the QT interval through direct inhibition of I(Kr) channel. We therefore incubated hERG-HEK cells in 1 and 10 microM pentamidine-containing media (supplemented with 10% serum) for 48 h, and examined the hERG current densities in the vehicle control and pentamidine-treated cells. In all, 36 and 85% reductions of the current densities were caused by 1- and 10-microM pentamidine treatment (P<0.001 vs control), respectively. A similar level of reduction of the hERG polypeptides and a reduced intensity of the hERG protein on the surface membrane in treated cells were observed by Western blot analysis and laser-scanning confocal microscopy, respectively. Taken together, our data imply that chronic administration of pentamidine at clinically relevant exposure reduces the membrane expression of the hERG channel, which may most likely be the major mechanism of QT prolongation and torsade de pointes reported in man.

Cholesteryl ester transfer protein variants have differential stability but uniform inhibition by torcetrapib.

Cholesteryl ester transfer protein (CETP) is an important modulator of high density lipoprotein cholesterol in humans and thus considered to be a therapeutic target for preventing cardiovascular disease. The gene encoding CETP has been shown to be highly variable, with multiple single nucleotide polymorphisms responsible for altering both its transcription and sequence. Examining nine missense variants of CETP, we found some had significant associations with CETP mass and high density lipoprotein cholesterol levels. Two variants, Pro-373 and Gln-451, appear to be more stable in vivo, an observation mirrored by partial proteolysis studies performed in vitro. Because these naturally occurring variant proteins are potentially present in clinical populations that will be treated with CETP inhibitors, all commonly occurring haplotypes were tested to determine whether the proteins they encode could be inhibited by torcetrapib, a compound currently in clinical trials in combination with atorvastatin. Torcetrapib behaved similarly with all variants, with no significant differences in inhibition.

Role of a KCNH2 polymorphism (R1047 L) in dofetilide-induced Torsades de Pointes.

Various drugs are reported to prolong the QT-interval on the surface ECG, thereby increasing the risk of developing a potentially fatal arrhythmia known as Torsades de Pointes (TdP). TdP case reports for these drugs have often been associated with risk factors such as overdosing, concomitant drugs and/or existing pathophysiological conditions. A few cases appear to be devoid of these factors. To determine what role genetic variation in the hERG gene plays in drug-induced arrhythmias, we screened DNA samples collected from 105 atrial-fibrillation patients treated with dofetilide for polymorphisms, seven of whom developed TdP. An uncommon missense change, R1047L, was identified in two of seven patients who experienced TdP as compared with five of 98 individuals who were free of TdP. Included in the affected individuals was the only subject homozygous for this SNP. Cellular electrophysiological studies revealed a 10-mV positive shift in the steady-state activation curve of the 1047L hERG channel stably expressed in HEK-293 cells as compared with the wild-type (WT) channel. The activation and inactivation kinetics of the 1047L current were significantly slower than the WT (P < 0.05) at given membrane potentials. A computer simulation using a rabbit ventricular myocyte model indicated that same extent of changes in the I(Kr) channel may result in an approximately 15% prolongation in the action potential duration. Our study suggests that 1047L leads to a functional impairment of the hERG channel, which may contribute to the higher incidence of TdP in 1047L carriers when challenged with a channel blocker.

Highly polymorphic repeat region in the CETP promoter induces unusual DNA structure.

Genetic variation in the human cholesteryl ester transfer protein (CETP) promoter is associated with HDL cholesterol levels and cardiovascular disease with much of the genetic variation in CETP attributed to the promoter region. In this region, there are several single nucleotide polymorphisms as well as a variable length tandem repeat located 1946 base pairs upstream of the CETP transcription start that is highly polymorphic with respect to both length and sequence. There are more than 10 different long alleles and these vary in their repeat structure. We find that the short allele of this repeat is associated with high HDL cholesterol levels in vivo (P<0.0001). In males, this association is independent of the nearby -629 polymorphism. In addition, the variable length GAAA repeat can stimulate an adjacent GGGGA repeat to form a structure that hinders DNA amplification and sequencing. This structure also has an effect in vivo as shown by orientation effects and cloning efficiency in Escherichia coli.