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Intuitively, it is easy to understand why we search for G protein-coupled receptor (GPCR) antagonists. It is obviously to block a functionality of a specific receptor potentially linked to some aspects of disease. Whether by focused research or by serendipity, many drugs were discovered in the last century that function as antagonist at a precise receptor. A current idea is that at least half of the drugs on the market are antagonist ligands of GPCRs. Then, why are we searching for alternative receptor agonists while the endogenous activating molecule is known? In the present commentary we try to rationalize these fields of research, since they proved to be very successful over the years, with receptor pharmacology populated with dozens of alternative agonists, particularly to bioaminergic receptors, and to a lesser extent to peptidergic ones. However, the action of such compounds is not well-characterized: are they surrogates to the endogenous agonist, and if yes in which context and for which purpose? The present essay is a reflection on this subject that leads to fundamental interrogations of our understanding of GPCR roles and functions.

La mélatonine est une molécule naturelle dérivée du tryptophane. Son rôle est de servir de relai entre la rythmicité jour/nuit et notre corps. Elle sert donc de marqueur circadien : concentration haute pendant la nuit et basse pendant la journée. Elle sert aussi de marque saisonnière : plus les nuits sont longues et plus longuement elle est produite (et vice-versa), ce qui a un rôle primordial dans les cycles reproductifs des animaux. Mais elle est aussi affublée de multiples propriétés thérapeutiques concernant la plupart des maladies humaines, du cancer à la COVID-19 en passant par l’infection par le virus Ebola, ainsi que de capacités thérapeutiques vis-à-vis de multiples toxicités (métaux, venins, produits chimiques comme l’adriamycine [doxorubicine], le méthotrexate ou le paclitaxel). Alors que l’enthousiasme à propos de cette molécule est troublant, l’assise scientifique de ces descriptions est dans le meilleur des cas faible et dans la plupart des cas, inexistante. Dans ce commentaire, les données scientifiques bien établies liées à la mélatonine sont résumées et brièvement discutées, en tâchant de redessiner les limites entre ce qui est connu et bien établi et ce qui reste du domaine du fantasme.

Compounds that simultaneously activate peroxisome proliferator-activated receptor (PPAR) subtypes α and γ have the potential to effectively treat dyslipidemia and type 2 diabetes (T2D) in a single pharmaceutically active molecule. The frequently observed side effects of selective PPARγ agonists, such as edema and weight gain, were expected to be overcome by using additive PPARα activity, leading to dual PPARα/γ agonists with balanced activity for both subtypes. Herein we report the discovery, synthesis, and optimization of a new series of α-ethoxyphenylpropionic acid bearing 5- or 6-substituted indoles. The incorporation of oxime ethers on the carbonyl portion of the benzoyl group can bring the PPARα/γ potency ratio equal to or slightly greater than one, as is the case for compounds 20 c and 21 a. Compound 20 c shows high efficacy in an ob/ob mouse model of T2D and dyslipidemia, similar to that of rosiglitazone and tesaglitazar, but with a significant increase in body weight gain. In contrast, compound 21 a, less potent as a dual PPARα/γ activator than 20 c, showed an interesting pharmacological profile, as it elicits a decrease in body weight relative to reference compounds.

The present study describes the effect of (S)-2,3-dihydro-[3,4]cyclopentano-1,2,4-benzothiadiazine-1,1-dioxide (S18986), a positive allosteric modulator of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, on (S)-AMPA-mediated increases in brain-derived neurotrophic factor (BDNF) mRNA and protein expression in rat primary cortical neuronal cultures. (S)-AMPA (0.01-300 microM) induced a concentration-dependent increase in BDNF mRNA and protein expression (EC(50)=7 microM) with maximal increases (50-fold) compared to untreated cultures observed between 5 and 12 h, whereas for cellular protein levels, maximal expression was detected at 24 h. S18986 alone (< or =300 microM) failed to increase basal BDNF expression. However, S18986 (300 microM) in the presence of increasing concentrations of (S)-AMPA maximally enhanced AMPA-induced expression of BDNF mRNA and protein levels (3-5-fold). S18986 (100-300 microM) potentiated BDNF mRNA induced by 3 microM (S)-AMPA (2-3-fold). Under similar conditions, the AMPA allosteric modulator cyclothiazide induced a potent stimulation of (S)-AMPA-mediated BDNF expression (40-fold; EC(50)=18 microM), whereas IDRA-21 was inactive. Kinetic studies indicated that S18986 (300 microM) in the presence of 3 microM (S)-AMPA was capable of enhancing BDNF mRNA levels for up to 25 h, compared to 3 microM (S)-AMPA alone. On the other hand, S18986 only partially enhanced kainate-mediated expression of BDNF mRNA, but failed to significantly enhance N-methyl-D-aspartate-stimulated BDNF expression levels. In support of these observations, the competitive AMPA receptor antagonist NBQX (1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide) but not the selective NMDA-receptor antagonist, (+)-MK-801 [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine], abrogated S18986-induced effects on BDNF expression. S18986-mediated enhancement of (S)-AMPA-evoked BDNF protein expression was markedly attenuated in Ca(2+)-free culture conditions. Furthermore, from a series of kinase inhibitors only the Calmodulin-Kinase II/IV inhibitor (KN-62, 25 microM) significantly inhibited (-85%, P

Obesity is a common disorder in the industrialized world. The major environmental factor associated with the rising prevalence of obesity is an increasingly sedentary lifestyle, compounded by greater levels of caloric intake. Recent studies 1—12) have shown that in the central nervous system (CNS), neuropeptide Y (NPY) has been implicated in obesity and feeding, anxiety and depression, endocrine function and metabolism. 1) More particularly it was observed that food intake was inhibited by antisense oligodeoxynucleotides to the

N-myristoylation is an acylation process absolutely specific to the N-terminal amino acid glycine in proteins. This maturation process concerns about a hundred proteins in lower and higher eukaryotes involved in oncogenesis, in secondary cellular signalling, in infectivity of retroviruses and, marginally, of other virus types. Thy cytosolic enzyme responsible for this activity, N-myristoyltransferase (NMT), studied since 1987, has been purified from different sources. However, the studies of the specificities of the various NMTs have not progressed in detail except for those relating to the yeast cytosolic enzyme. Still to be explained are differences in species specificity and between various putative isoenzymes, also whether the data obtained from the yeast enzyme can be transposed to other NMTs. The present review discusses data on the various addressing processes subsequent to myristoylation, a patchwork of pathways that suggests myristoylation is only the first step of the mechanisms by which a protein associates with the membrane. Concerning the enzyme itself, there are evidences that NMT is also present in the endoplasmic reticulum and that its substrate specificity is different from that of the cytosolic enzyme(s). These differences have major implications for their differential inhibition and for their respective roles in several pathologies. For instance, the NMTs from mammalians are clearly different from those found in several microorganisms, which raises the question whether the NMT may be a new targets for fungicides. Finally, since myristoylation has a central role in virus maturation and oncogenesis, specific NMT inhibitors might lead to potent antivirus and anticancer agents.

Obesity is a common disorder in the industrialized world. The major environmental factor associated with the rising prevalence of obesity is an increasingly sedentary lifestyle, compounded by greater levels of caloric intake. Recent studies 1—12) have shown that in the central nervous system (CNS), neuropeptide Y (NPY) has been implicated in obesity and feeding, anxiety and depression, endocrine function and metabolism. 1) More particularly it was observed that food intake was inhibited by antisense oligodeoxynucleotides to the

International audience; Type-2 diabetes (T2D) is a complex metabolic disease characterized by insulin resistance in the liver and peripheral tissues accompanied by a deficiency in pancreatic beta-cells. Since their discovery, three subtypes of peroxisome proliferator activated receptors have been identified, namely PPAR alpha, PPAR gamma and PPAR beta/(delta). In this study, we were interested in designing novel PPAR gamma selective agonists and/or dual PPAR alpha/gamma agonists. Based on the typical topology of synthetic PPAR agonists, we focused our design approach on using 4,4-dimethyl-1,2,3,4-tetrahydroquinoline as a novel cyclic scaffold with oxime and acidic head group structural variations. (C) 2009 Elsevier Ltd. All rights reserved.

Type-2 diabetes (T2D) is a complex metabolic disease characterized by insulin resistance in the liver and peripheral tissues accompanied by a defect in pancreatic beta-cell. Since their discovery three subtypes of Peroxisomes Proliferators Activated Receptors were identified namely PPARalpha, PPARgamma and PPARbeta/(delta). We were interested in designing novel PPARgamma selective agonists and/or dual PPARalpha/gamma agonists. Based on the typical topology of synthetic PPAR agonists, we focused our design approach on 4,4-dimethyl-1,2,3,4-tetrahydroquinoline as novel cyclic tail.

Retinoic acid receptor-related orphan receptor alpha (RORalpha) is a member of the nuclear receptor superfamily. Using RT-PCR, RORalpha mRNA was identified in human aortic smooth muscle cells (hASMC), endothelial cells (EC), as well as in human mammary arteries and atherosclerotic plaques. We found a predominant expression of RORalpha1 in hASMC, and RORalpha4 in EC. RORalpha2 and RORalpha3 were not detected. In arteries, RORalpha4 was predominant compared with RORalpha1. In atherosclerotic plaques, RORalpha expression was significantly decreased. In hASMC stimulated with cytokines, RORalpha expression was increased by 2.5-fold. RORalpha mRNA was also significantly increased (approximately 2-fold) in hASMC and EC cultured under hypoxia.

Using a genomics-based approach for screening orphan G-protein-coupled receptors, we have identified and cloned a novel high-affinity, melanin-concentrating hormone (MCH) receptor. This receptor, named S643b, displays the greatest overall identity (32%) with the previously reported human SLC-1 receptor (MCH1) and to a lesser extent with the somatostatin receptor subtypes. The gene encoding the S643b receptor spans more than 23 kilobase pairs (kb) and was mapped, by radiation hybrid experiments, on chromosome 6q14.3-q15. Comparison of the S643b cDNA with human genomic sequence reveals that the 340-amino-acid receptor is encoded by five exons. Its tissue distribution, as determined by Northern blot and reverse transcription-polymerase chain reaction analysis, indicates that a 4-kb transcript is predominantly expressed in the brain. When expressed in Chinese hamster ovary (CHO) cells, the S643b receptor displays a strong, dose-dependent, transient elevation of intracellular calcium in response to MCH (EC(50) = 9.5 nM). During the present study, we isolated a splice variant, designated S643a, encoding for a receptor that was not activated by MCH in a cellular calcium mobilization assay. Comparative pharmacological studies using CHO cells stably expressing either SLC-1 or S643b receptors demonstrated that similar structural features of MCH are required to stimulate intracellular Ca(2+) mobilization at both receptors. The identification and localization of this new MCH receptor (MCH2) provides further insight into the physiological implication of MCH in modulating behavioral responses, including food intake.

S18616 ((S)-spiro[(1-oxa-2-amino-3-azacyclopent-2-ene)-4, 2'-(8'-chloro-1',2',3',4'-tetrahydronaphthalene)]) displayed high affinity at native rat alpha(2)-adrenoceptors (AR)s (pK(i), 9.8), native human (h)alpha(2A)-ARs (9.6), and cloned halpha(2A)- (9.5), halpha(2B)- (9.2), and halpha(2C)- (9.0) ARs. It showed 40-fold lower affinity for halpha(1A)-ARs (8.4) and/=100-fold lower affinity for rat alpha(1)-ARs (7.1), halpha(1B)-ARs (7.7), halpha(1D)-ARs (7.6), imidazoline(1) (7.4), and imidazoline(2) (7.4) sites and100-fold lower affinity for all other (50) sites. At halpha(2A)-ARs, in guanosine-5'-O-(3-[(35)S]thio)triphosphate binding studies, S18616 was a potent (partial) agonist: log effective concentration (pEC(50)), 9.3/maximal effect, 51%. This observation was corroborated employing a halpha(2A)-Gi1alpha fusion protein/GTPase assay (9.0/40%) in which the actions of S18616 were blocked by pertussis toxin. Employing guanosine-5'-O-(3-[(35)S]thio)triphosphate binding assays, S18616 was also a partial agonist at halpha(2C)-ARs (8.2/63%) but a full agonist (8.4/124%) at halpha(2B)-ARs. At halpha(2A)-, halpha(2B)-, and halpha(2C)-ARs, the selective alpha(2)-AR antagonist, atipamezole, abolished the actions of S18616: pK(b) values of 9.1, 9. 1, and 9.4, respectively. As determined by depletion of membrane-bound [(3)H]phosphatidyl inositols, S18616 behaved as a (less potent) agonist (7.8/79%) at halpha(1A)-ARs, an action abolished by prazosin (pK(b), 8.9). Reflecting alpha(2)-AR agonist properties, S18616 potently (/=1 microg/kg, s.c.) and dose dependently elicited hypothermia and antinociception (nine diverse models) in rodents. These actions were dose dependently inhibited by chemically diverse alpha(2)- versus alpha(1)-AR antagonists, atipamezole, idazoxan, RX821,002, and BRL44418 (a preferential alpha(2A)-AR ligand). In contrast, the actions of S18616 were unaffected by the alpha(1)-AR antagonists, ARC239 and prazosin (which preferentially block alpha(2B/2C)- versus alpha(2A)-ARs). Although the affinity of dexmedetomidine at alpha(2)-ARs was lower than S18616; it displayed a similar receptor and functional profile. Clonidine displayed lower efficacy than S18616, was substantially less potent, and had marked affinity for imidazoline(1) sites and alpha(1)-ARs. In conclusion, S18616 is a novel, selective, and highly potent agonist at alpha(2)-ARs.

S 12363 is a new vinca alkaloid derivative obtained by grafting an optically active alpha-aminophosphonate at the C23 position of O4-deacetyl vinblastine. This compound was as potent as Vincristine (VCR), and less potent than Vinblastine (VLB), in inhibiting in vitro tubulin polymerization. However, S 12363 was found to be 7 to 553 and 12 to 74-fold more cytotoxic than VCR and VLB, respectively, when tested on a panel of 2 murine and 6 human tumor cell lines using the Microculture Tetrazolium Assay. S 12362, which differs only by the configuration of the asymmetric carbon atom of the side chain, was 18 to 59-fold less cytotoxic. At equitoxic doses, all these compounds induced a "G2 + M" phase accumulation of L1210 cells, suggesting a similar mechanism of action. S 12363, administered i.p. or i.v., was at least as active as reference compounds on two murine transplantable tumors (P388 leukemia and B16 melanoma) while the optimal dosage was 20-fold lower: 0.15-0.20 mg/kg versus 2-5 mg/kg, respectively. S 12362 was practically inactive at 1-3 mg/kg. The hematological toxicity of S 12363 (0.1 mg/kg) was similar to that of VLB (4 mg/kg). The exceptionally high potency of S 12363 did not appear to be due to a better interaction with tubulin, its intracellular target, but rather to some properties conferred by the alpha-aminophosphonic acid, such as a facilitated uptake and/or a better cellular retention.

A tyrosine protein kinase (TPK) partially purified and two co-purified proteins (pp39 and pp44) from HL-60 cells were used as a basis for a SDS-PAGE electrophoresis screening test of inhibitors of the activity catalyzed by this enzyme. Such inhibitors may constitute a new class of anticancer agents. TPK is a protein of about 32 to 35 kDa as measured by gel-sizing exclusion. This enzyme is apparently the main tyrosine protein kinase from the cytosol of HL-60 cells. The test was assessed using known inhibitors of various types of protein kinases. Erbstatin and Cibcron Blue--a nucleotide analog--were shown to be potent inhibitors of TPK activity.

We studied the in vitro capability of hepatic microsomal UDP-glucuronosyltransferase (UDPGT) in male rats to conjugate 22 natural xenobiotics which are known to be excreted as glucuronides in vivo. We clearly demonstrated that the Vmax can range in a decreasing scale for the following families of aglycones: 7-hydroxylated coumarins greater than 2-naphthol and phenols greater than monoterpenoid alcohols greater than 4-hydroxylated coumarins. The Km app. cannot be arranged in the same scale. This suggests that the catalytic mechanism of UDPGT is dependent on the hydroxyl group reactivity rather than on the binding interaction at the active site expressed by the Km app. The effects of various concentrations of detergent (Triton X-100) were determined on specificity (apparent Km) and activity (Vmax). For the 22 aglycones we showed that activation caused a variation in the Vmax which was a function of the concentration in detergent. The maximum of this activation did not always correspond to the same detergent/protein weight ratio. The impact of activation on Km app. was less clear since the variations observed were slightly different.

The neurohormone melatonin is a key agent in synchronizing the circadian rhythms. At least three types of binding sites have been described for melatonin: the G-coupled, seven-transmembrane domain receptors mt1 and MT2 and a putative binding site called MT3. The latter has been described in hamster brain membranes, and its binding capacity is optimum at 4 degrees C. We further characterized this binding site on other peripheral hamster tissues, including intestine, liver, kidney, lung, muscle, and heart. We found a high level of binding sites (30 fmol/mg of protein) in intestine and kidney. Furthermore, we completed the existing pharmacological profile of this site, which can now be described as 2-iodomelatonin6-chloromelatoninmethy-isobutyl-amilorideacridine orange5-methylcarbonylamino-N-acetyltryptamineprazosinN-acetylserotoninmelatonin. This profile was found in all the hamster organs tested that had a large number of binding sites, namely, brain, intestine, kidney and liver. Furthermore, when comparisons were possible, the MT3 pharmacological characteristics were similar to those described in the literature for hamster brain and testis. This profile was compared to the pharmacology obtained on human cloned mt1 and MT2 receptors and proved to be completely different, as expected. We provide new evidence for an alternate melatonin binding site not only in hamster brain but also in some peripheral organs.

S18327 displayed modest affinity for human (h)D(2) and hD(3) receptors and high affinity for hD(4) receptors. At each, S18327 antagonized stimulation of [(35)S]guanosine-5'-O-(3-thio)triphosphate binding by dopamine (DA). It also blocked activation of mitogen-activated protein kinase at hD(3) receptors. The affinity of S18327 at hD(1) and hD(5) sites was modest. S18327 showed pronounced affinity for human serotonin (h5-HT)(2A) receptors and human alpha(1A)-adrenergic receptors (hARs), at which it antagonized increases in intracellular Ca(2+) concentration levels elicited by 5-HT and norepinephrine (NE), respectively. S18327 presented significant affinity for halpha(2A)-ARs and antagonized NE-induced[(35)S]guanosine-5'-O-(3-thio)triphosphate binding both at these sites and at alpha(2)-ARs in rat amygdala. Reflecting blockade of alpha(2)-autoreceptors, S18327 enhanced firing of adrenergic neurons in locus ceruleus, accelerated hippocampal synthesis of NE, and increased dialysate levels of NE in hippocampus, accumbens, and frontal cortex. S18327 abolished inhibition of ventrotegmental area-localized dopaminergic neurons by apomorphine. However, S18327 alone did not affect their activity and only modestly enhanced cerebral turnover of DA and dialysate levels of DA in striatum and accumbens. In contrast, S18327 markedly increased dialysate levels of DA in frontal cortex, an action abolished by the selective alpha(2)-AR agonist, S18616. Finally, S18327 reduced synthesis and dialysate levels of 5-HT in striatum and suppressed firing of dorsal raphe-localized serotonergic neurons, an action attenuated by the alpha(1)-AR agonist cirazoline. In conclusion, S18327 possesses marked antagonist activity at alpha(1)-ARs and D(4) and 5-HT(2A) receptors and less potent antagonist activity at alpha(2)-ARs and D(1) and D(2) receptors. Antagonism by S18327 of alpha(2)-ARs enhances adrenergic transmission and reinforces frontocortical dopaminergic transmission, whereas blockade of alpha(1)-ARs inhibits dorsal raphe-derived serotonergic pathways. As further described in the accompanying paper, this profile of activity may contribute to the potential antipsychotic properties of S18327.

Farnesylation is a key maturation step involved in the ras-dependent transformation of cells. This acylation step is catalyzed by protein: farnesyltransferase, a soluble enzyme. The present work describes the use of a new HPLC method of measurement of this enzymatic activity using the K-ras-derived CVIM tetrapeptide as substrate. The method is used to check the activity catalyzed by cytosols issued from various types of cancer cells. J82, a human bladder cancer cell line was retained for measurement of the inhibitory potency of a few peptide sequences and will be used as starting biological material for the purification of the enzyme. This HPLC method presented herein has the main advantages over other published methods of being automatisable and versatile, because it can be used with a wide spectrum of peptide substrates. Results presented herein are only first studies and need some more structural observations. The obtention of the cancer cell line-derived, partially purified farnesyltransferase will hopefully lead us to the discovery of specific inhibitors with potential non-cytotoxic anti-cancer activities.

Flavonoids form an important family of compounds widely present in plants and therefore in food, sometimes as substitutes for synthetic antioxidants. Although the excretion routes of flavonoids in animals has been explored, little is known about the details of their conjugation in the xenobiotic metabolism pathway. In this study, we investigate the metabolism of diosmetin as a model compound in the rat, particularly its level in blood after treatment (100 mg/kg, po) and the presence of its glucuronide(s) in both blood and urine. We demonstrate that after po treatment of the rat, a rapid glucuronidation takes place and that diosmetin circulates as glucuronides, whereas no free diosmetin is present in blood and urine. The glucuronides formed are present in the blood plasma at a high level (approximately 10 micrograms/ml), for at least 6 hr after the treatment and the conjugates are excreted in urine. We have detected four different glucuronides in blood and characterized the two major ones after purification by a combination of MS, NMR, and UV spectroscopy: diosmetin-7,3'-diglucuronide and diosmetin-3'-glucuronide. A brief characterization of the in vitro glucuronidation of some flavonoid compounds using liver microsomal preparations suggests that this important class of natural compounds might be conjugated by a specific isoform of UDP-glucuronosyltransferase. Thus, this work brings evidence that diosmetin is rapidly glucuronoconjugated in the rat and provides an explanation for the low po bioavailability of the unchanged compound that can be generalized to this important class of natural compounds.

Living in the wild involves to cope with a variable seasonal environment availability. When winter is coming, animals use various strategies to adapt to hostile environment by limiting energy expenditure such as hibernation. In this study, expression of 21 selected genes was compared at different states of the hibernation cycle of the true hibernator European hamster. Level of mRNA encoding proteins involved in seasonal timing (melatonin receptors, thyroid metabolism, clock) and energy homeostasis were measured by digital droplet PCR in eight central and peripheral organs. During the arousal phase, Periods genes expression is increased in all organs indicating a possible resetting of body’s clocks at the beginning of the active period. The brown adipose tissue displays a specific regulation of deiodinases leading to increased synthesis of thyroxine during both torpor and arousal. The melatonin receptor MT2 of the European hamster had been partially cloned and pharmacologically characterized. While in most hamster species, MT2 is a natural knock out, the studied receptor seems to be functional and could be critical during hibernation., Afin de faire face aux conditions environnementales défavorables, certains animaux réduisent drastiquement leur activité métabolique et leur température grâce à des phases de torpeur hivernal. L’objectif de cette étude est d’établir une signature moléculaire de chacune des phases d’hibernation. Pour cela, les variations d’expression de 21 gènes impliqués dans le contrôle des fonctions saisonnières (horloge circadienne, hormones thyroïdiennes, récepteurs à la mélatonine) et le métabolisme ont été étudiées dans 8 organes. Les résultats ont mis en évidence une augmentation ubiquitaire de l’expression des gènes Périodes indiquant un possible réajustement de l’horloge au début de la phase de réveil. Ainsi qu’une régulation spécifique des déiodinases induisant une augmentation de la synthèse de thyroxine dans le tissu adipeux brun et l’hypothalamus pendant la torpeur et le réveil. Le récepteur MT2 du hamster d’Europe a été partiellement caractérisé génétiquement et pharmacologiquement. A la différence d’autres espèces de hamster dont le récepteur MT2 est tronqué, le récepteur étudié semble être fonctionnel pour la mélatonine et pourrait être critique durant l’hibernation.