Philippe Pflieger

Modulating cholesteryl ester transfer protein activity maintains efficient pre-β-HDL formation and increases reverse cholesterol transport

The mechanism by which cholesteryl ester transfer protein (CETP) activity affects HDL metabolism was investigated using agents that selectively target CETP (dalcetrapib, torcetrapib, anacetrapib). In contrast with torcetrapib and anacetrapib, dalcetrapib requires cysteine 13 to decrease CETP activity, measured as transfer of cholesteryl ester (CE) from HDL to LDL, and does not affect transfer of CE from HDL3 to HDL2. Only dalcetrapib induced a conformational change in CETP, when added to human plasma in vitro, also observed in vivo and correlated with CETP activity. CETP-induced pre-β-HDL formation in vitro in human plasma was unchanged by dalcetrapib ≤3 µM and increased at 10 µM. A dose-dependent inhibition of pre-β-HDL formation by torcetrapib and anacetrapib (0.1 to 10 µM) suggested that dalcetrapib modulates CETP activity. In hamsters injected with [3H]cholesterol-labeled autologous macrophages, and given dalcetrapib (100 mg twice daily), torcetrapib [30 mg once daily (QD)], or anacetrapib (30 mg QD), only dalcetrapib significantly increased fecal elimination of both [3H]neutral sterols and [3H]bile acids, whereas all compounds increased plasma HDL-[3H]cholesterol. These data suggest that modulation of CETP activity by dalcetrapib does not inhibit CETP-induced pre-β-HDL formation, which may be required to increase reverse cholesterol transport.

Mechanisms underlying off-target effects of the cholesteryl ester transfer protein inhibitor torcetrapib involve L-type calcium channels.

Objective The increased mortality observed with the cholesteryl ester transfer protein inhibitor torcetrapib is partly due to increased aldosterone production and blood pressure. The mechanisms underlying these effects were investigated. Methods Cytochrome P450 subunit 11B2 (aldosterone synthase), extracellular signal-regulated kinase (p44/42) and voltage-gated Ca2+channel alpha subunit mRNA profiling, aldosterone production, cytosolic calcium and RNA interference were assessed in adrenocarcinoma human cells (H295R). Telemetry was conducted in spontaneously hypertensive rats. Results Torcetrapib and angiotensin II (Ang II) but not dalcetrapib (a structurally different cholesteryl ester transfer protein inhibitor) elevated both cytochrome P450 subunit 11B2 mRNA and aldosterone production in H295R cells at 6 h. At days 1–5, torcetrapib produced a sustained increase of cytochrome P450 subunit 11B2 mRNA, unlike Ang II. Although torcetrapib and Ang II potentiated the effect of 25-OH cholesterol and raised pregnenolone levels, torcetrapib increased neither cytosolic Ca2+ at 5 min nor extracellular signal-regulated kinase1/2 phosphorylation, suggesting initially divergent pathways. Unlike Ang II, torcetrapib steroidogenesis was not affected by Ang II type 1 receptor antagonism or voltage-gated T-type Ca2+ channel antagonism, but was blocked by several L-type Ca2+channel antagonists. In unbiased genome-wide screening, Ang II and torcetrapib modulated an overlapping but distinct set of genes in H295R cells. Torcetrapib, but not Ang II, upregulated mRNA levels of the L-type Ca2+ channel alpha 1C subunit. In spontaneously hypertensive rat, torcetrapib had a potent hypertensive effect mediated by the L-type Ca2+ channel. Conclusion The unique steroidogenic and hypertensive side effects of torcetrapib may be linked and involve voltage-gated L-type Ca2+ channels. Structurally unrelated cholesteryl ester transfer protein inhibitors such as dalcetrapib do not share this effect.

Evidence for a role of CETP in HDL remodeling and cholesterol efflux: role of cysteine 13 of CETP.

Cholesteryl ester transfer protein (CETP), a key regulator of high-density lipoprotein (HDL) metabolism, induces HDL remodeling by transferring lipids between apolipoprotein B-containing lipoproteins and HDL, and/or by promoting lipid transfer between HDL subparticles. In this study, we investigated the mechanism as to how CETP induces the generation of lipid-poor particles (pre-β-HDL) from HDL, which increases ATP-binding cassette transporter 1-mediated cholesterol efflux. This CETP-dependent HDL remodeling is enhanced by the CETP modulator dalcetrapib both in plasma and isolated HDL. The interaction of dalcetrapib with cysteine 13 of CETP is required, since this effect was abolished when using mutant CETP in which cysteine 13 was substituted for a serine residue. Other thiol-containing compounds were identified as CETP modulators interacting with cysteine 13 of CETP. In order to mimic dalcetrapib-bound CETP, mutant CETP proteins were prepared by replacing cysteine 13 with the bulky amino acid tyrosine or tryptophan. The resultant mutants showed virtually no CETP-dependent lipid transfer activity but demonstrated preserved CETP-dependent pre-β-HDL generation. Overall, these data demonstrate that the two functions of CETP i.e., cholesteryl ester transfer and HDL remodeling can be uncoupled by interaction of thiol-containing compounds with cysteine 13 of CETP or by introducing large amino acid residues in place of cysteine 13.

Establishment of robust functional assays for the characterization of neuropeptide Y (NPY) receptors: identification of 3-(5-benzoyl-thiazol-2-ylamino)-benzonitrile as selective NPY type 5 receptor antagonist.

The human Neuropeptide Y (NPY) receptors 1 (hY1), 2 (hY2), 4 (hY4), and the mouse type 5 (mY5) receptor were expressed in human embryonic kidney 293 (HEK293) cells. The receptors bound a radioiodinated NPY ligand with high affinity and various NPY analogs competed for binding in a receptor selective-manner. Similarly, cAMP-inhibition and GTPgammaS binding assays were established. The four NPY receptors were further tested in the fluorimetric imaging plate reader (FLIPR) format, a cellular high-throughput assay, in the absence and presence of chimeric G proteins, Gqo5, Gqi5 and Gqi9. The receptors stimulated transient calcium release only in the presence of chimeric G proteins. While hY1, hY2 and hY4 receptors coupled to Gqo5, Gqi5 and Gqi9, the mY5 receptor stimulated transient calcium release only when co-expressed with Gqi9. Using an in silico screening approach we identified a small molecule 3-(5-benzoyl-thiazol-2-ylamino)-benzonitrile (compound 1), which bound to the mY5 receptor with high affinity (Ki=32.1+/-1.8 nM), competitively antagonized NPY-mediated GTPgammaS binding and calcium stimulation with high potency, and had no affinity for other NPY receptors. These data show that NPY receptors can be functionally coupled to the FLIPR readout, allowing for high throughput compound testing and identification of novel molecules.

Aminothiazole Derivatives as Neuropeptide Y5 Receptor Ligands: Finding the Balance between Affinity and Physicochemical Properties

Neuropetide Y is a peptide of 36 amino acids that is widely distributed in the central and peripheral nervous systems. It mediates a number of physiological effects through its various receptor subtypes. Studies in animals have shown that neuropeptide Y is a powerful stimulus of food intake, and it has been demonstrated that the activation of neuropeptide Y5 (NPY5) receptors results in hyperphagia and decreased thermogenesis. Therefore, compounds that antagonize neuropeptide Y at the Y5 receptor subtype might represent an approach to the treatment of eating disorders such as obesity and hyperphagia. Although it has been suggested that the antagonism of the NPY5 receptor does have a major effect on feeding in rats, there is evidence to support the NPY receptor system in humans as a valuable target for the treatment of metabolic disorders. In the course of a medicinal chemistry program designed to identify novel NPY5 receptor antagonists, thiazole derivatives had been described as interesting scaffolds. Our research group previously reported specific substitution patterns on the thiazole scaffold that proved favorable for the compounds to bind the NPY5 receptor with IC50 values in the low nanomolar range, yet these molecules also had poor physicochemical properties. However, a favorable C=O···S interaction was identified that yielded promising, potent thiazole derivatives. As this scaffold offers ample opportunities for optimization, we turned our interest toward the influence of the nature and length of the linker that connects the thiazole to the sulfonamide moiety, and thus the potential of the resulting compounds to function as antagonists of the NPY5 receptor (Scheme 1). As mentioned above, low solubility was identified as a major disadvantage of these thiazole compounds. Therefore, an optimization cycle was added to improve the physicochemical property profile. This was monitored by measuring solubility and permeability by using the parallel artificial membrane permeation assay (PAMPA). A correlation analysis of pIC50 values and physicochemical properties was carried out to determine whether these two parameters could be optimized independently, or if the optimization of affinity would simultaneously yield an inferior (or superior) physicochemical profile. The results indicated that solubility and membrane permeability were positively correlated, whereas no direct correlation between pIC50 and solubility/membrane permeability was observed (Table 1). However, a principal components analysis

Synthesis of enantiomerically pure [14C]-labelled morpholine derivatives for a class of trace amine-associate receptor 1 agonists

Various agonists of the trace amine-associate receptor 1, under consideration as potential clinical development candidates, were labelled with carbon-14 for use in preclinical in vitro and in vivo drug metabolism studies. Herein, the [14 C]-radiosynthesis of 2-phenyl-substituted morpholines 1 is described. After evaluating and optimizing different synthetic routes, 4-iodonitrobenzene 3 was selected as starting material for the 14-step synthesis. Incorporation of carbon-14 into the acetyl moiety allowed a safe and efficient synthesis of [14 C]-labelled 4-nitroacetophenone 2 in five steps and 38% yield. Further transformation of 2 to the target compounds 1 was achieved in a 9-step synthesis. In a representative example, [14 C]-labelled 1 was obtained in an overall yield of 11% and was isolated in >99% radiochemical purity and a specific activity of 47 mCi/mmol.