Monitoring Cyp2b10 mRNA expression at cessation of 2-year carcinogenesis bioassay in mouse liver provides evidence for a carcinogenic mechanism devoid of human relevance: the dalcetrapib experience.

INTRODUCTION: Dalcetrapib is a cholesteryl ester transfer protein (CETP) modulator in clinical assessment for cardiovascular outcome benefits. In compliance with regulatory requirements, dalcetrapib was evaluated in rodent 2-year carcinogenesis bioassays. In the mouse bioassay, male mice demonstrated increased liver weight and statistically increased incidences of hepatocellular adenoma/carcinoma. Hepatic cytochrome p450 (Cyp) 2b10 mRNA induction and increased Cyp2b10 enzyme activity signify activation of hepatic nuclear receptor constitutive androstane receptor (CAR), a widely established promoter of rodent-specific hepatic tumors. We therefore monitored hepatic Cyp2b10 mRNA and its enzyme activity in a subset of dalcetrapib-treated male mice from the bioassay. METHODS: Liver samples were obtained from ~1/3 of male mice from each dose group including vehicle-controls (mean and earliest study day of death 678 and 459 respectively). Quantitative real time PCR (qRT-PCR) was performed to determine Cyp2b10 mRNA expression and Cyp1a-, Cyp2b10- and Cyp3a-selective activities were monitored. RESULTS: Cyp2b10 mRNA was strongly induced by dalcetrapib with an expected wide inter-individual variation (5-1421-fold). Group average fold-induction versus vehicle-controls showed a dose-related increase from 48-fold (250mg/kg/day) to 160-fold (750mg/kg/day), which declined slightly at 2000mg/kg/day (97-fold). Cyp enzyme activities showed approximate doubling of total Cyp P450 content per milligram protein and a 9-fold increase in Cyp2b10-selective pentoxyresorufin O-dealkylase activity (750mg/kg/day). DISCUSSION: These data from hepatic Cyp2b10 monitoring are strongly suggestive of CAR activation by dalcetrapib, a mechanism devoid of relevance towards hepatocarcinogenesis in humans; results show feasibility of Cyp2b10 as a surrogate marker for this mechanism at cessation of a carcinogenesis bioassay.

The role of poly(ADP-ribose) metabolism in response to active oxygen cytotoxicity.

These experiments are a continuation of our work describing the effect of H2O2 and O2- on DNA strand breaks, NAD pools and poly(ADP-ribose) synthesis in C3H10T1/2 cells (Lautier et al. (1990) Biochem. Cell Biol. 68, 602-608). The current experiments were carried out firstly to evaluate the polymer synthesis in C3H10T1/2 cells exposed to benzamide, oxygen radicals and hyperthermia. Secondly, using four different protocols for the time of addition and removal of benzamide, the lowest benzamide levels shown to inhibit polymer synthesis were used to study the effect on plating efficiency and colony-forming ability of cells exposed to H2O2 and O2(-). Plating efficiency and colony-forming ability were affected by the active oxygen-species-generating system xanthine-xanthine oxidase and 100 microM benzamide. With higher levels of benzamide, this effect disappeared, and 0.5 to 1 mM benzamide were actually protective against the effects of xanthine-xanthine oxidase, suggesting the involvement of other processes in addition to poly(ADP-ribosyl)ation in response to oxygen radical damage.

Characterization of anti-peptide antibodies directed towards the automodification domain and apoptotic fragment of poly (ADP-ribose) polymerase.

Poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) is a highly conserved nuclear enzyme present in higher eukaryotes. PARP is activated following DNA damage, is implicated in DNA repair, and its proteolysis has been shown to be an early marker of programmed cell death or apoptosis. In order to better understand the role of PARP in apoptosis and DNA repair and also to study PARP automodification, we have developed anti-peptide sera directed against four peptides from the conserved automodification domain of PARP. Four peptides were synthesized according to the four branched Multiple Antigenic Peptide (MAP) system and injected into rabbits. Immune sera were titrated by ELISA and analysed in Western blotting experiments on cell lines. The sera were also analysed for their capacity to inhibit PARP activity in an in vitro assay. Of the eight sera developed (two for each peptide), a serum directed against a peptide localized at the C-terminal part of the automodification domain of PARP (#422) appeared to be the best antibody to detect PARP from different species. All antipeptide antibodies were efficient in detecting the apoptotic fragment of PARP during programmed cell death in HL-60 apoptotic cells. None of the serum alone was able to completely inhibit PARP activity but combinations of the sera could significantly reduce automodification of PARP consistent with the localization of half of the automodification sites on bovine PARP. Sera were also used to map proteolysed purified PARP and to immunoprecipitate purified bovine PARP.

Complete inhibition of poly(ADP-ribose) polymerase activity prevents the recovery of C3H10T1/2 cells from oxidative stress.

Activation of the poly(ADP-ribose) polymerase after oxidative damage is implicated in different responses of the cells, for example, cell recovery after sublethal damage or cell death after lethal damage. However, the extent and mechanism of involvement of the enzyme in these two processes appear to be different. Inhibitors of this polymerase, such as benzamides, which do not completely inhibit PARP have been shown to protect the cells from killing by massive oxidant damage, could neither reduce the cellular recovery after mild oxidant damage nor completely inhibit DNA repair in vitro. We report here that 1,5-dihydroxyisoquinoline, which was earlier shown to be a strong inhibitor of this polymerase in vitro, is also its potent inhibitor in vivo. Using sensitive techniques for measuring low levels of cellular poly(ADP-ribose) polymer, we show that this inhibitor can completely abolish oxidant-induced activation of the polymerase in C3H10T1/2 cells. We show that only a minor fraction of the poly(ADP-ribose) polymerase activity is sufficient in cellular recovery after sublethal oxidant damage. We also demonstrate that cells are unable to recover from oxidant damage in the complete absence of polymerase activity.

This is not a G protein-coupled receptor.

On his canvas entitled 'La trahison des Images' ('The Perfidy of Images'), René Magritte painted a tobacco pipe in a very realistic manner and added the words: 'Ceci n'est pas une pipe' ('This is not a pipe'). In similar style, it is of prime importance to state that the first three-dimensional (3D) models of G protein-coupled receptors (GPCRs) that have been defined and displayed are definitely not GPCRs and never will be. However, they probably represent a very important breakthrough in our understanding of GPCR structure and function as well as being a source of novel working hypotheses that can be experimentally explored. Several generations of scientists have elaborated their research on the concept of hormone-receptor interaction without having access to any structural representation of this molecular complex. Thus, for many years receptors remained only indirectly characterized through their functions or their binding properties. As a consequence, a number of important issues are still to be addressed. How does a hormone bind and activate its receptor? How does an antagonist inactivate a receptor? What is a partial agonist? What are the molecular mechanisms of signal transduction and modulation? Obviously, answers to these questions are of prime importance for medicinal chemists in their attempts to rationalize drug design.

1H-NMR study of endothelin, sequence-specific assignment of the spectrum and a solution structure.

The solution conformation of the recently discovered bi-cyclic, 21 amino acid vasoconstrictor peptide, Endothelin I, has been examined by 1H-NMR in deuterated dimethyl sulphoxide. A full sequential assignment has been achieved. In addition, 19 long range NOEs were detected which were employed as distance constraints in molecular dynamics calculations to yield a possible solution structure for this new peptide.

Two aromatic residues regulate the response of the human oxytocin receptor to the partial agonist arginine vasopressin.

We investigated the mechanisms that regulate the efficacy of agonists in the arginine-vasopressin (AVP)/oxytocin (OT) receptor system. In this paper, we present evidence that AVP, a full agonist of the vasopressin receptors, acts as a partial agonist on the oxytocin receptor. We also found that AVP becomes a full agonist when two aromatic residues of the oxytocin receptor are replaced by the residues present at equivalent positions in the vasopressin receptor subtypes. Our results indicate that these two residues modulate the response of the oxytocin receptor to the partial agonist AVP.

Biochemical properties and function of poly(ADP-ribose) glycohydrolase.

We describe here the latest observations on poly(ADP-ribose) glycohydrolase. There is now extensive evidence that this nuclear enzyme is an endo-exoglycosidase which has a key role to perform in the removal of polymers which interact with proteins through covalent and non-covalent interactions. Also, we have developed a zymogram which will permit the isolation of the various isoforms of the glycohydrolase and the eventual cloning of this enzyme. Finally, we have evidence that very short oligomers and even monomers of ADP-ribose covalently bound to proteins can be removed by poly(ADP-ribose) glycohydrolase.

Modeling of G-protein-coupled receptors: application to dopamine, adrenaline, serotonin, acetylcholine, and mammalian opsin receptors.

Hydropathicity analysis of 39 G-protein-coupled receptors (GPCR) reveals seven hydrophobic stretches corresponding to membrane spanning alpha-helices. The alignment of the primary sequences shows a high degree of homology in the GPCR transmembrane regions. 3D models of 39 GPCRs were generated using the refined model of bacteriorhodopsin as a template. Five cationic neurotransmitter receptors (serotonergic 5-HT2, dopaminergic D2, muscarinic m2, adrenergic alpha 2 and beta 2 receptors) were taken as prototypes and studied in detail. The 3D models of the cationic neurotransmitter receptors, together with their primary structure comparison, indicate that the agonist binding site is located near the extracellular face of the receptor and involves residues of the membrane-spanning helices 3, 4, 5, 6, and 7. The binding site consists of a negatively-charged Asp located at the middle of transmembrane helix 3 and a hydrophobic pocket containing conserved aromatic residues on helices 4, 5, 6, and 7. To define the precise receptor-ligand interactions, the natural neurotransmitters were docked into the binding sites. Residues responsible for the affinity, selectivity, and eventually stereospecificity of dopamine, adrenaline, noradrenaline, serotonin, and acetylcholine for their receptors were identified. The ligands are involved in electrostatic interactions as well as hydrogen bonds and specific hydrophobic aromatic interactions. All the GPCRs possess invariant hinge residues, which might be responsible for a conformational change during agonist binding and therefore influence dissociation and association of G-proteins to the receptors. The role of hydrophobic interactions and hydrogen bonds in the conformational change of the receptors, modulating the coupling to the G-protein, is discussed with regard to these residues. The models are in agreement with published data obtained from mutagenesis and labeling studies and represent important working hypotheses to direct future mutagenesis studies. They also enable structure-activity relationship studies and more rational drug design. The 3D models of other G-protein-coupled receptors have been generated in a similar way.

Activation of neurotensin receptors and purinoceptors in human colonic adenocarcinoma cells detected with the microphysiometer.

Activation of endogenous neurotensin (NT) receptors and P2-purinoceptors expressed by human colonic adenocarcinoma HT-29 cells increased extracellular acidification rates that were detected in the microphysiometer. NT (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu), NT[8-13] (Arg-Arg-Pro-Tyr-Ile-Leu), NT[9-13] (Arg-Pro-Tyr-Ile-Leu), and NT1 (N alpha methyl-Arg-Lys-Pro-Trp-Tle-Leu [Tle = tert-leucine]) were full agonists, whereas XL 775 (N-[N-[2-[3-[[6-amino-1-oxo-2-[[(phenylmethoxy)carbonyl]-amino]hex yl]amino]phenyl]-3-(4-hydroxyphenyl)-1-oxo-2-propenyl]-L-isoleucyl]-L-le ucine) was a partial agonist for activating NT receptors expressed by HT-29 cells. Desensitization induced by NT was rapid and monophasic with 85% of the initial response lost by a 30-s exposure. Once initiated, the rate and extent of desensitization were similar for different concentrations of a given agonist, for agonists of different potencies, and for agonists of different efficacies, which suggests that desensitization may be independent of receptor occupancy or agonist efficacy. Resensitization was a much slower process, requiring 60 min before the full agonist response to NT was recovered. ATP, via P2-purinoceptors, also activated cellular acidification rates in a concentration-dependent manner. ATP induced a biphasic desensitization of purinoceptors with a loss of ca. 50% of the initial stimulation detectable between 30 and 90 s of exposure to the agonist. Desensitization of NT receptors did not influence the activation of P2-purinoceptors by ATP, suggesting there was no heterologous desensitization between the two types of receptors. Superfusion with NT receptor agonists for 15 min at concentrations that did not elicit changes in extracellular acidification rates blocked, in a concentration-dependent manner, the agonist response induced by 100 nM NT. This may reflect sequestration of the receptor. These results suggest that the high agonist affinity state of NT receptors may modulate receptor sequestration, whereas activation of the low agonist affinity state may be linked to cellular metabolism. Comparison of our results with published data found differences as well as similarities of NT responses among three lines of HT-29 cells.

Solution conformation of endothelin-3 by (1)H NMR and distance geometry calculations.

Endothelin-3 dissolved in 10% aqueous acetic acid was studied by nuclear magnetic resonance spectroscopy. A total of 363 distances (143 intra-residue, 108 sequential and 112 long range) was compiled from the nuclear Overhauser effect spectra and used in distance geometry calculations. The molecule assumes a compact conformation stabilized by hydrophobic interactions of the side chains. There is a helix-like structure between the residues 9-15 and an extended strand at the N-terminus. The C-terminus is in close proximity to the bicyclic ring.

Modelling and modification of the binding site of endothelin and other receptors.

From three-dimensional models of its receptors, residues which bind the carboxy-terminus of endothelin were predicted. This site is in a pocket consisting of five putative transmembrane helices and includes a histidine in the sixth helix. This residue is either phenylalanine or asparagine in cationic neurotransmitter receptors. The histidine alkylating agent diethylpyrocarbonate potently inhibited binding of [125I]endothelin-1 to its receptors in bovine cerebellum, where a single population of endothelin ETB receptors was shown to exist. From the absence of pH sensitivity of inhibition above pH 5 and the reversal by hydroxylamine of inhibition, diethylpyrocarbonate is concluded to inhibit by histidine modification. Diethylpyrocarbonate inhibited ligand binding to several receptors with the potency order endothelin ETB > or = bombesin > or = dopamine D2 > or = m2 muscarinic > alpha 1-adrenoceptor > or = m 1 muscarinic > 5-HT2. This is consistent with histidine in the binding site of endothelin (and some other peptidergic) receptors and the proposed model.

Mutagenicity of ethylene glycol ethers and of their metabolites in Salmonella typhimurium his-.

Ethylene glycol ethers, their aldehyde and their acid metabolites were evaluated for their mutagenicity with the Ames test. The Salmonella typhimurium his- tester strains TA 97a, TA 98, TA 100 and TA 102 were used with and without rat S9 mix. Ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol n-butyl ether and their corresponding aldehyde and acid derivatives were tested up to 10(-4) mol/plate (around 10 mg/plate) or up to cytotoxic concentrations. All tested substances gave negative results with TA 98, TA 100 and TA 102 either with or without S9 mix. In contrast, ethylene glycol n-butyl ether (EGBE) and the aldehyde metabolite of ethylene glycol monomethyl ether, methoxyacetaldehyde (MALD), displayed mutagenic potency in strain TA 97a with and without S9 mix at high concentrations. A significant number of revertants was obtained from 19 mumol/plate EGBE (2.2 mg/plate) and from 34 mumol/plate MALD (2.5 mg/plate). At these concentrations the level of revertants reached up to 7-fold and 3-fold the control values for EGBE and MALD respectively.

Identification of agonist binding sites of vasopressin and oxytocin receptors.

The present study aims at delineating residues in the vasopressin/oxytocin receptor family responsible for the high affinity binding of the hormone. Therefore, we have constructed a computer-generated 3 dimensional model of the rat V1a vasopressin receptor subtype which allowed us to propose residues likely to be involved in agonist binding. Among these residues, several are highly conserved in the receptor family. They were selected for site-directed mutagenesis on the basis of putative direct interaction with bound ligands. The present model and experimental results led us to conclude that the hormone is docked in a pocket completely buried in the transmembrane core of the receptor. Large polar residues, such as glutamine and lysine, located in transmembrane regions 2,3,4 and 6 are involved in the binding of the neurohypophysial hormone. Since all the mutated residues are highly conserved in AVP and OT receptors, we propose that the agonist binding site is similar in all members of the receptor family; only minor changes were found in antagonist potencies, suggesting that agonist and antagonist binding sites do not completely overlap.

Molecular basis for agonist selectivity in the vasopressin/oxytocin receptor family.

Vasopressin (AVP) and oxytocin (OT) are two nonapeptides differing at position 3, in the cyclic part of the peptide, and at position 8, in the C-terminal tripeptide. In this study, we have evaluated the interactions between these two positions of the hormones and the oxytocin receptor (OTR), the V1a and the V2 vasopressin receptors. The contribution of these two positions to receptor selectivity was analyzed by using several peptide analogues bearing substitutions at either position 3 or 8. The putative interactions between receptor residues and hormone residues at position 3 and 8 were then deduced by using a three dimensional model of the neurohypophysial hormones docked into their respective receptors. On the basis of this model, we found that the lateral chain of residue 8 might interact with residues located in the first extracellular loop. By using site-directed mutagenesis on the cloned receptors, we identified a non-conserved residue in the first extracellular loop that interacts with the lateral chain of residue 8 in the hormone. We demonstrated that this interaction is crucial for receptor selectivity to the different agonists.

[Modeling of hormonal receptors. Application to drug design]. / Modélisation des récepteurs hormonaux. Application à la conception de médicaments.

A number of three-dimensional models of serotonin receptor recognition sites which were derived from a conformational analysis of their ligands and successfully used for drug design are reviewed. Models of the complete three-dimensional structure of sequenced G-protein coupled receptors are defined from primary sequence analysis, published experimental data, the crystal structure of bacteriorhodopsin and energy minimizations. Adrenaline, serotonin, dopamine and acetylcholine can thus be docked in their respective binding sites. There is an excellent convergence between the two approaches: modelling of the recognition site from its ligands and modelling of the receptor from its primary sequence.

Serum cardiac troponin I concentrations transiently increase in rats given rosiglitazone.

Rosiglitazone, a peroxisome proliferator-activated receptor γ (PPARγ) agonist of the thiazolidinedione class, is a major insulin-sensitizing drug widely used to treat type-2 diabetes. Rosiglitazone causes myocardial hypertrophy in rodents and increases the risk of cardiac events in man. To better characterize its cardiac effects, male Wistar rats were orally administered 0, 10 or 80 mg/kg/day rosiglitazone. Myocardial gene expression profiling, hematology, histopathology and clinical chemistry, including measurement of serum cardiac troponin (cTn) I concentration with the ultrasensitive assay, were evaluated after 6 and 24h and 7 and 14 days of dosing. Heart weight was increased 10% after 7 days and 16% after 14 days of dosing at 80 mg/kg/day in the absence of microscopic changes. At the transcriptomic level, the number of differentially expressed probes was small: it was most at 24h in rats given 80 mg/kg rosiglitazone with 356 differentially regulated probes (fold change >1.3 fold, p<0.05). Also, gene categories typically associated with myocardial damage were not over-represented. Most importantly, serum cTnI concentrations in 5/9 rats after 7 days of dosing at 80 mg/kg/day were above the upper limit of serum cTnI concentration. cTnI concentrations after 14 days of dosing were similar between rats given the vehicle and rosiglitazone at 80 mg/kg. This is the first study to detect increases of serum cTnI concentrations in rats administered rosiglitazone. In light of reported cardiac events in patients chronically dosed with PPARγ agonists, our results support serum cTnI concentrations as an early biomarker of cardiac liability.

Solution conformation of endothelin-1 by 1H NMR, CD, and molecular modeling.

The solution conformation of Endothelin-1, a recently discovered bicyclic, 21 amino acid peptide, has been examined by 1H NMR in deuterated dimethylsulphoxide and circular dichroism in aqueous and organic solvents. A total of 158 NOEs were detected, which were used as distance constraints in the distance geometry program DISGEO. Two families of structures were obtained, both characterized by a helix-like region extending from Lys9 to Cys15, but with opposite "handedness". Circular dichroism studies of the peptide in both aqueous and trifluoroethanol solutions show a negative shoulder at 224 nm, characteristic of right-handed helices. Molecular dynamics and energy minimization yielded a solution structure for this new peptide compatible with all experimental observations.