The regulation of the circadian rhythm is relayed from the central nervous system to the periphery by melatonin, a hormone synthesized at night in the pineal gland. Besides two melatonin G-coupled receptors, mt 1 and MT 2 , the existence of a novel putative melatonin receptor, MT 3 , was hypothesized from the observation of a binding site in both central and peripheral hamster tissues with an original binding profile and a very rapid kinetics of ligand exchange compared with mt 1 and MT 2 . In this report, we present the purification of MT 3 from Syrian hamster kidney and its identification as the hamster homologue of the human quinone reductase 2 (QR 2 , EC 1.6.99.2). Our purification strategy included the use of an affinity chromatography step which was crucial in purifying MT 3 to homogeneity. The protein was sequenced by tandem mass spectrometry and shown to align with 95% identity with human QR 2 . After transfection of CHO-K1 cells with the human QR 2 gene, not only did the QR 2 enzymatic activity appear, but also the melatonin-binding sites with MT 3 characteristics, both being below the limit of detection in the native cells. We further confronted inhibition data from MT 3 binding and QR 2 enzymatic activity obtained from samples of Syrian hamster kidney or QR 2 -overexpressing Chinese hamster ovary cells, and observed an overall good correlation of the data. In summary, our results provide the identification of the melatonin-binding site MT 3 as the quinone reductase QR 2 and open perspectives as to the function of this enzyme, known so far mainly for its detoxifying properties.Melatonin, a neurohormone produced at night in the pineal gland, is suspected to relay to the peripheral organs the circadian rhythm detected by the central nervous system. Several high affinity melatonin receptors have been identified to date, among which the mt 1 (1) and MT 2 (2) receptors have been cloned from human tissues. The pharmacology of these two receptors is well documented, and several compounds, including melatonin, are ligands with picomolar binding affinity (for review, see Ref.3). Another putative melatonin receptor was identified on pharmacological grounds, with lower melatonin affinity (nanomolar range), very rapid ligand association/dissociation kinetics, and an original pharmacological profile (4 -6). In line with mt 1 and MT 2 receptors, this putative receptor was named MT 3 , according to the nomenclature recommendations of the IUPHAR (7). So far, the known inhibitors of MT 3 hardly reach the nanomolar range and encompass an unusually large structural diversity of highly hydrophobic cyclic or polycyclic compounds (Refs. 5 and 6, and for review, see Ref.3).1 All pharmacological investigations on mt 1 , MT 2 , and MT 3 were performed using the radioligand [125 I]melatonin, a ligand with high affinity for mt 1 and MT 2 (K d ϭ 10 -200 pM) and with lower affinity for MT 3 (K d ϭ 3-9 nM). The hamster kidney, liver, and brain have been used as model tissues for MT 3 pharmacological studies, and our recent data con...

Escape from apoptosis is one of the major hallmarks of cancer cells. The B-cell Lymphoma 2 (BCL-2) gene family encodes pro-apoptotic and anti-apoptotic proteins that are key regulators of the apoptotic process. Overexpression of the pro-survival member BCL-2 is a well-established mechanism contributing to oncogenesis and chemoresistance in several cancers, including lymphoma and leukemia. Thus, BCL-2 has become an attractive target for therapeutic strategy in cancer, as demonstrated by the recent approval of ABT-199 (Venclexta™) in relapsed or refractory Chronic Lymphocytic Leukemia with 17p deletion. Here, we describe a novel orally bioavailable BCL-2 selective and potent inhibitor called S55746 (also known as BCL201). S55746 occupies the hydrophobic groove of BCL-2. Its selectivity profile demonstrates no significant binding to MCL-1, BFL-1 (BCL2A1/A1) and poor affinity for BCL-XL. Accordingly, S55746 has no cytotoxic activity on BCL-XL-dependent cells, such as platelets. In a panel of hematological cell lines, S55746 induces hallmarks of apoptosis including externalization of phosphatidylserine, caspase-3 activation and PARP cleavage. Ex vivo, S55746 induces apoptosis in the low nanomolar range in primary Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma patient samples. Finally, S55746 administered by oral route daily in mice demonstrated robust anti-tumor efficacy in two hematological xenograft models with no weight lost and no change in behavior. Taken together, these data demonstrate that S55746 is a novel, well-tolerated BH3-mimetic targeting selectively and potently the BCL-2 protein.

AIM: These studies were performed to test the hypothesis that endogenous neuropeptide Y (NPY) acting on the NPY Y 5 receptor subtype contributes to the control of food intake. The hypothesis was tested using S 25585Fa newly synthesized NPY Y 5 receptor antagonist. METHODS AND RESULTS: S 25585 was shown to be a high-affinity antagonist of the NPY Y 5 receptor subtype (IC 50 5 nM) with no significant affinity toward other NPY receptor subtypes and over 40 other receptors, channels or uptake systems. S 25585 (7.5 mg/kg, i.p.) did not induce a conditioned taste aversion, significantly alter need-induced sodium appetite or induce pica, suggesting that at this dose the compound did not induce illness or malaise. In satiated rats, S 25585 (5.0 and 7.5 mg/kg, i.p.) significantly decreased the overfeeding induced by i.c.v. injection of NPY (1 mg) and the highly selective NPY Y 5 receptor agonist [hPP 1À17 , Ala 31 , Aib 32 ]NPY (0.7 mg). In rats fasted for 4 h immediately before the dark phase, analysis of the microstructure of feeding behavior revealed that S 25585 significantly increased latency to eat and significantly decreased the duration and size of the meals without altering the meal number or eating rate. Analysis of the behavioral satiety sequence at this time revealed that the animals passed through the normal pattern of feeding, grooming and resting. Although S 25585 appeared to be influencing a physiological system controlling appetite, this does not involve the NPY Y 5 receptor since the antagonist also markedly reduced food intake in the NPY Y 5 knockout mouse. CONCLUSIONS:The results presented do not support a role for the NPY Y 5 receptor in the control of food intake. The results further illustrate that it is imperative that the activity of any new NPY Y 5 antagonist be assessed in the NPY Y 5 knockout mouse before assuming that its effect on food intake is due to blockade of this receptor.

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The standard method of peptide library synthesis involves coupling steps in which a single amino acid is reacted with a mixture of resin-bound amino acids. The more recently described positional scanning strategy (in which each position in the peptide sequence is occupied in turn by a single residue) is different since it involves the coupling of mixtures of amino acids to mixtures of resin-bound amino acids. In the present study, we analyze the compounds produced under these conditions measuring coupling rates and amounts of formed products, using mainly UV, HPLC, LC/MS and MS/MS techniques. Our data do not permit to conclude that the resulting libraries are complete. Indeed, our analytical data indicate that a large part of the di-, tri- and tetrapeptides synthesized with this method are not present in the final mixture. Although chemical compensation (in which poor coupling kinetics is compensated by a larger excess of the incoming amino acid) has been thought to counterbalance these biases, our experiments show that the compensation method does not take into account the crucial influence of the resin-bound amino acid and that even the dipeptide libraries obtained in this way are far from completeness. The present work provides strong evidence that the coupling of mixtures of amino acids to resin-bound residues, which is required by the positional scanning strategy, results in incomplete and/or non-equimolar libraries. It also clearly confirms that coupling rates in solid-phase peptide synthesis are dependent on the nature of both the incoming and the immobilized amino acid.