Jean-Luc Fauchere

Identification of the melatonin-binding site MT3 as the quinone reductase 2.

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, mt1 and MT2, the existence of a novel putative melatonin receptor,MT3 , 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 mt1 and MT2. In this report, we present the purification of MT3 from Syrian hamster kidney and its identification as the hamster homologue of the human quinone reductase 2 (QR2, EC 1.6.99.2). Our purification strategy included the use of an affinity chromatography step which was crucial in purifying MT3 to homogeneity. The protein was sequenced by tandem mass spectrometry and shown to align with 95% identity with human QR2. After transfection of CHO-K1 cells with the human QR2 gene, not only did the QR2 enzymatic activity appear, but also the melatonin-binding sites with MT3 characteristics, both being below the limit of detection in the native cells. We further confronted inhibition data fromMT3 binding and QR2 enzymatic activity obtained from samples of Syrian hamster kidney or QR2-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 siteMT3 as the quinone reductase QR2and open perspectives as to the function of this enzyme, known so far mainly for its detoxifying properties.

Agonistic and antagonistic properties of the bradykinin B2 receptor antagonist, Hoe 140, in isolated blood vessels from different species

1 Hoe 140, a recently described bradykinin B2 antagonist, and NPC 567 from an earlier generation of bradykinin B2 antagonists, were tested in rabbit and sheep isolated blood vessels. 2 In rabbit jugular vein, a bradykinin B2 preparation, NPC 567 was an antagonist (apparent pA2: 8.67 ± 0.16) with marked residual agonistic activity (log[EC50]: −7.29 ± 0.13). Hoe 140 was a potent non‐competitive antagonist devoid of agonistic properties (slope of the Schild plot: 2.02; estimated pA2: 9.04). 3 In rabbit aorta, a bradykinin B1 preparation, NPC 567 was a competitive antagonist (pA2: 6.32 ± 0.13) but Hoe 140 was ineffective. The two antagonists did not show any agonistic properties in this tissue. 4 In sheep femoral artery without endothelium, bradykinin and Hoe 140 induced contractions with identical efficacy and similar potency (log[EC50]: −8.05 ± 0.12, −7.73 ± 0.10; maximal contraction in % of KCl [60 mm]: 59.5 ± 15.1, 62.0 ± 13.1; for bradykinin and Hoe 140, respectively). In contrast NPC 567 was an extremely weak agonist. The contractile responses to bradykinin and Hoe 140 were inhibited by NPC 567 (apparent pKB: 6.89 ± 0.22 and 6.58 ± 0.08 versus bradykinin and Hoe 140, respectively) but not by a B1 bradykinin antagonist, suggesting that the receptor involved was a bradykinin B2 receptor. 5 In sheep femoral artery with endothelium, bradykinin induced a biphasic response: an endothelium‐dependent relaxation and a contraction which were both inhibited by NPC 567 (apparent pKB: 7.10 ± 0.15) and Hoe 140 (pA2: 8.38 ± 0.12). As bradykinin B2 receptor antagonists, Hoe 140 and NPC 567 were less potent in the sheep femoral artery than in the rabbit jugular vein. Neither Hoe 140 nor NPC 567 were agonists for the endothelial receptor. 6 This study demonstrates that Hoe 140, a new bradykinin B2 receptor antagonist, is more selective and more potent than NPC 567; however, it may possess, depending on the tissue studied, marked residual agonistic properties. Furthermore, bradykinin B2 receptors are subject to important species specificity. Finally, two different bradykinin B2 receptor subtypes may coexist in the sheep femoral artery with endothelium.

[125I]-S36057: a new and highly potent radioligand for the melanin-concentrating hormone receptor.

Shortened, more stable and weakly hydrophobic analogues of melanin‐concentrating hormone (MCH) were searched as candidates for radioiodination. Starting from the dodecapeptide MCH6 – 17, we found that: (1) substitution of Tyr13 by a Phe residue; (2) addition of a 3‐iodo‐Tyr residue at the N‐terminus; and (3) addition of a hydrophilic spacer 8‐amino‐3,6‐dioxyoctanoyl between the 3‐iodo‐Tyr and MCH6 – 17 (compound S36057), led to an agonist more potent than MCH itself in stimulating [35S]‐GTPγS binding at membranes from HEK293 cells stably expressing the human MCH receptor. Specific binding of [125I]‐S36057 was found in HEK293 and CHO cell lines stably expressing the human MCH receptor. This radioligand recognized a similar number of binding sites (ca. 800 fmol mg−1) than [125I]‐[3‐iodo Tyr13]‐MCH. However, the KD for [125I]‐S36057 obtained from saturation studies (0.037 nM) or from binding kinetics (0.046 nM) was at least 10 fold higher to that of [125I]‐[3‐iodo Tyr13]‐MCH (0.46 nM). Affinities determined for a series of MCH analogues were similar with both radioligands, S36057 being the most potent compound tested (Ki=0.053 nM). Finally, [125I]‐S36057 also potently labelled the MCH receptor in membranes from whole rat brain (KD 0.044 nM, Bmax=11 fmol mg−1). In conclusion, [125I]‐S36057 is a more potent and more stable radioligand than [125I]‐[3‐iodo Tyr13]‐MCH that will represent a reliable tool for binding assays in the search of novel MCH ligands. It should also provide great help for autoradiographic studies of the MCH receptor distribution in the central nervous system.

Combinatorial chemistry for the generation of molecular diversity and the discovery of bioactive leads

Abstract The principles of the combinatorial synthesis of large compound libraries are described. It is shown that these technologies can afford unprecedented numbers of chemical entities thus increasing the molecular diversity of the universe of organic compounds. Library design is considered in view of the generation of a maximized diversity for a given set of synthetic products. The advantages of solid phase organic chemistry are stressed and compared to the achievements of solution procedures for the production of large compound collections. The trend from the truly combinatorial synthesis of millions of biooligomers towards the parallel synthesis of individual small organics is stated and exemplified. Finally, an overview is given of the methods developed for the structural identification of the active component(s) in compound libraries and arrays, including iterative deconvolution via re-synthesis, positional scanning or structural analysis of the ligand on a single polymer bead. Strategies are suggested for both lead discovery and lead optimization.

Venous and arterial endothelial cells respond differently to thrombin and its endogenous receptor agonist

The effects of thrombin and a peptide mimicking the amino terminus of its receptor, Res (42-55), on vascular reactivity were compared in isolated canine blood vessels. In saphenous veins contracted with endothelin-1, both thrombin and Res (42-55) caused relaxation in rings with endothelium and contraction in rings without endothelium. In coronary arteries, thrombin caused similar responses while Res (42-55) only caused contraction. These data suggest that different thrombin receptors are present on venous and arterial endothelial cells.

Hormone--receptor interactions: [4-carboranylalanine, 5-leucine]-enkephalin as a structural probe for the opiate receptor.

The new phenylalanine analogue, L-carboranylalanine (Car) [l] was recently found to be a good probe for investigating some special properties of the aromatic recognition site of chymotrypsin [2] . The icosahedral side chain of Car is somewhat larger than the phenyl ring of Phe, rotating about its 1,4 axis. Yet it is easily accomodated by chymotrypsin; the concomitant deformation of the recognition site propagated to the mechanistic site (especially Ser 195) is probably the reason that Car esters are not hydrolysed by the enzyme. One of the aims of our laboratory is to study hormone-receptor interactions. After the promising results with chymotrypsin as a model, we are investigating the use of Car replacements of Phe, Tyr, and Trp in polypeptide hormones. Phenylalanine in position 4 seems to be quite important for enkephalin action, because a number of analogues with replacements of this amino acid by others all show reduced or even missing opiate receptor affinity and biological activity (review [3] ; see also [4-61). An introduction of Car into this position seemed ideally suitable to see whether or not carboranylalanine analogues of peptide hormones can be expected to have exceptional features. We have therefore prepared [4carboranylalanine, S-leucine] enkephalin, H.Tyr-Gly-Gly-Car-Leu.OH and investigated its ability to displace [3H]naloxone from rat brain opiate receptor preparations according to the technique developed in the laboratory of Snyder [7] . [S-Methionine] -enkephalin, [S-leucine] enke-

Selection of a histidine-containing inhibitor of gelatinases through deconvolution of combinatorial tetrapeptide libraries

SummaryA fully automated peptide synthesizer was used to generate tetrapeptide sublibraries from 24 natural and nonnatural amino acids, from which new inhibitors of gelatinases (matrix metalloproteinases MMP-2 and MMP-9) were selected as potential anticancer drugs. MMP-2 and MMP-9 from mouse Balbc/3T3 fibroblasts conditioned media were assayed in their linear range response by zymography to quantify inhibition at each step of the tetrapeptide library deconvolution. The histidine-ɛ-amino caproic acid-βalanine-histidine (His-ɛAhx-βAla-His) sequence was found to yield optimal inhibition of both MMP-2 and MMP-9. Inhibition by selected tetrapeptides was also evaluated with two other techniques, a native type IV collagen degradation assay and a fluorogenic enzymatic assay, confirming the tetrapeptide potency. The His-ɛAhx-βAla-His tetrapeptide also inhibited purified human MMP-2 and MMP-9 and the corresponding enzymes present in conditioned media from human tumour cells. Finally, the length of the spacer between the two terminal histidines was found to be crucial to the inhibitory potential. This approach may thus be considered as a successful strategy to yield specific peptide or pseudopeptide inhibitors, although their potency remains moderate, since it was measured before any chemical optimization was undertaken.

Solid-phase synthesis of α-substituted 3-bisarylthio N-Hydroxy propionamides as Specific MMP Inhibitors

A novel series of potent and specific MMP-2,3,9,13 inhibitors has been obtained by modulation on solid phase by alpha and aryl substitutions on 3-arylthio-N-hydroxy-propionamides starting from itaconic acid.

A Three Binding Site Hypothesis for the Interaction of Ligands with Monoamine G Protein-coupled Receptors: Implications for Combinatorial Ligand Design

Three-dimensional models of ligand-receptor complexes based on site-directed mutagenesis experiments of the monoamine G protein-coupled receptors reveal the existence of three distinct drug binding sites inside the receptors. Here, we develop this “three-site” hypothesis and outline its implications for the modular design of ligands for monoamine GPCRs. Molecular models of receptor-ligand complexes are built for the 5-HT1A receptor where mutagenesis studies map three spatially distinct binding regions which correspond to the binding sites of the “small, one site-filling” ligands 5-HT, propranolol and 8-OH-DPAT, respectively. The models of the 5-HT1A ligand-receptor complexes provide a frame for the discussion of other ligand-receptor interactions, including α1 and β2 adrenoceptors, D1 and D2 dopamine, and 5-HT1D and 5-HT2A receptors, where mutagenesis and modelling studies also showed occupation of the corresponding three binding locations. All three binding sites are located within the highly conserved seven helix transmembrane domain of the receptor and overlap partially at the prominent Asp residue in TM3 which constitutes the benchmark anchor site for monoamine ligands. The analysis of the sequence similarity, for each binding site, among the monoamine GPCR superfamily shows that the three loci display different degrees of evolutionary conservation. This result suggests different roles for each of the binding sites in intrinsic receptor functions and provides additional insights for the design of ligand functionality and selectivity. The existence of three distinct binding sites is also reflected by the architecture of known high affinity ligands which crosslink two or three “one site-filling” fragments around a basic amino group. Typical ligands reported in the Cipsline/MDDR portfolio illustrate this point despite the occasional difficulty of attributing the individual ligand fragments to a specific receptor site. The database exploration illustrates the binding site promiscuity of some fragments which is particularly evident for symmetrical ligands and which has implications for 3D QSAR methods dependent on alignments. We propose to generate by deconvolution of known ligands three distinct databases of site-specific bioisosters which should provide keystones for the design of novel recomposed monoamine GPCR ligands. The systematic exploration of the “three site” hypothesis should open novel perspectives for the understanding of ligand recognition for this class of therapeutically important receptors.

In vitro and in vivo pharmacology of S 16474, a novel dual tachykinin NK1 and NK2 receptor antagonist

Since tachykinins released from lung sensory nerve endings are thought to play a role in inflammatory diseases of airways via NK1 and NK2 receptors, dual tachykinin NK1 and NK2 receptor antagonists may have a great therapeutic potential. In vitro, the cyclopeptide S 16474 (cyclo-[Abo-Asp(D-Trp(Suc0Na)-Phe-N-(Me)Bzl)]) bound to both human tachykinin NK1 and NK2 receptors expressed in two lines of transfected Chinese hamster ovary cells (IC50 values 85 nM and 129 nM, respectively), while showing a poor affinity for the rat tachykinin NK1 receptor. S 16474 inhibited the contractions induced by substance P in isolated rabbit vena cava (pA2 7.0) and by neurokinin A in rabbit pulmonary artery (pA2 5.6). In vivo in anaesthetized guinea-pigs, S 16474 was found to dose dependently inhibit the bronchoconstrictions induced by intravenously administered substance P, neurokinin A and capsaicin. Plasma extravasation evoked in bronchi by endogenously released tachykinins under vagus nerve stimulation was abolished by S 16474 (10 mu mol/kg i.v.). These results demonstrate clearly that S 16474 is a tachykinin receptor antagonist exhibiting, in vitro and in vivo, a dual inhibitory effect on NK1 and NK2 receptors.