We have investigated the mechanism of inhibition and site of action of the novel human metabotropic glutamate receptor 5 (hmGluR5) antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP), which is structurally unrelated to classical metabotropic glutamate receptor (mGluR) ligands. Schild analysis indicated that MPEP acts in a non-competitive manner. MPEP also inhibited to a large extent constitutive receptor activity in cells transiently overexpressing rat mGluR5, suggesting that MPEP acts as an inverse agonist. To investigate the molecular determinants that govern selective ligand binding, a mutagenesis study was performed using chimeras and single amino acid substitutions of hmGluR1 and hmGluR5. The mutants were tested for binding of the novel mGluR5 radioligand [ Metabotropic glutamate receptors are G protein-coupled receptors that play important roles in regulating the activity of many synapses in the central nervous system. At present, eight mGluR 1 subtypes (mGluR1 through mGluR8) have been cloned and functionally expressed (1, 2). Based on their amino acid sequence homologies, pharmacology, and functional profiles, these subtypes are classified further into three groups. Members of group I (mGluR1 and -5) stimulate the activity of phospholipase C and mobilize intracellular Ca 2ϩ . Members of group II (mGluR2 and -3) and group III (mGluR4, -6, -7, -8) inhibit adenylyl cyclase. Despite the differences in primary structures and functional roles, all mGluRs feature a large conserved N-terminal extracellular domain, which is involved in the recognition of agonists and competitive antagonists (3-8).Most ligands for mGluRs were derived from amino acids and act at the conserved glutamate binding site (9). Recently, novel subtype-selective group I mGluR antagonists emerged that are structurally unrelated to amino acids and to each other. The first non-amino acid-like antagonist described was CPCCOEt (Fig. 1), a selective mGluR1 antagonist (10, 11). CPCCOEt inhibits receptor activity by a non-competitive mechanism which does not affect the binding affinity of glutamate (12, 13). Molecular characterization of the site of inhibition in mGluR1 revealed that CPCCOEt interacts with two non-conserved residues at the top of transmembrane (TM) helix VII (13). The first described selective mGluR5 antagonists, SIB-1757 and SIB-1893 (Fig. 1), are also unrelated to amino acids and were shown to act via a non-competitive mechanism (14).To address the question whether these structurally unrelated mGluR1 and mGluR5 antagonists interact with different sites of the mGluR subtypes or share a common binding site in the 7TM domain, we have studied the binding site and mode of action of the selective mGluR5 antagonist MPEP (15). MPEP is a novel derivative of SIB-1893 with nanomolar potency (Fig. 1); it is an effective antihyperalgesic in animal models of chronic inflammatory pain (16), a neuroprotectant in excitotoxin-induced striatal lesions (17) and an anticonvulsant in several epilepsy models (18). We generated a number of chimeric rec...

In this study we evaluate how far the scope of similarity searching can be extended to identify not only ligands binding to the same target as the reference ligand(s) but also ligands of other homologous targets without initially known ligands. This "homology-based similarity searching" requires molecular representations reflecting the ability of a molecule to interact with target proteins. The Similog keys, which are introduced here as a new molecular representation, were designed to fulfill such requirements. They are based only on the molecular constitution and are counts of atom triplets. Each triplet is characterized by the graph distances and the types of its atoms. The atom-typing scheme classifies each atom by its function as H-bond donor or acceptor and by its electronegativity and bulkiness. In this study the Similog keys are investigated in retrospective in silico screening experiments and compared with other conformation independent molecular representations. Studied were molecules of the MDDR database for which the activity data was augmented by standardized target classification information from public protein classification databases. The MDDR molecule set was split randomly into two halves. The first half formed the candidate set. Ligands of four targets (dopamine D2 receptor, opioid delta-receptor, factor Xa serine protease, and progesterone receptor) were taken from the second half to form the respective reference sets. Different similarity calculation methods are used to rank the molecules of the candidate set by their similarity to each of the four reference sets. The accumulated counts of molecules binding to the reference target and groups of targets with decreasing homology to it were examined as a function of the similarity rank for each reference set and similarity method. In summary, similarity searching based on Unity 2D-fingerprints or Similog keys are found to be equally effective in the identification of molecules binding to the same target as the reference set. However, the application of the Similog keys is more effective in comparison with the other investigated methods in the identification of ligands binding to any target belonging to the same family as the reference target. We attribute this superiority to the fact that the Similog keys provide a generalization of the chemical elements and that the keys are counted instead of merely noting their presence or absence in a binary form. The second most effective molecular representation are the occurrence counts of the public ISIS key fragments, which like the Similog method, incorporates key counting as well as a generalization of the chemical elements. The results obtained suggest that ligands for a new target can be identified by the following three-step procedure: 1. Select at least one target with known ligands which is homologous to the new target. 2. Combine the known ligands of the selected target(s) to a reference set. 3. Search candidate ligands for the new targets by their similarity to the reference set using the Similo...

Binding is good, displacing is better. By adding a weakly binding reporter ligand to a mixture of protein and test compounds, NMR screening can identify strongly binding ligands by observing the displacement of the reporter ligand. For example, NMR screening identified, as a reporter ligand, a small fragment, 2‐acetylbenzofuran (1, gray skeleton), which binds to the active site of 3α‐HSD. A molecular model of the complex between 3α‐HSD and 1 is shown.

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The optimization of GS39783 into potent, selective and safe positive allosteric modulators of GABA B receptors is presented.The receptors for the major inhibitory neurotransmitter in the central nervous system, GABA, are subdivided into ionotropic GABA A and GABA C receptors and metabotropic GABA B receptors. Whereas GABA A and GABA C receptors form chloride-permeable ion channels, GABA B receptors are G-protein coupled receptors (GPCRs). These receptors were discovered in 1980 by Norman G. Bowery 1 and act post and pre-synaptically to inhibit neuronal excitability and neurotransmitter release, respectively. A possible role of GABA B receptors in a large number of CNS disorders such as cognition deficits, anxiety, depression, epilepsy, pain and drug addiction has been discussed. 2 Some of these diseases like anxiety, pain and drug addiction could potentially be treated by activation of GABA B receptors, which can be achieved by administration of either agonists or positive allosteric modulators. Whereas benzodiazepines are well known positive allosteric modulators of GABA A receptors, the first examples of allosteric enhancers for GABA B receptors have been described only recently. 3 One of the most interesting compound found was GS39783 (Figure 1). 3b However, despite an interesting in vitro and in vivo profile, 3b,4 GS39783 was found to be genotoxic probably because of its aromatic nitro group (Figure 1) 5 5.This communication describes our efforts towards the identification of a novel, drug-like class of compounds acting as positive allosteric modulators for GABA B receptors.In order to introduce molecular diversity in position 5 of the pyrimidine ring, a 4 steps procedure depicted in Scheme 1 was optimized in order to obtain compounds with a chlorine or with a hydrogen in position 6. 4,6-dichloro-2-methylpyrimidine was first substituted by cyclopentylamine and then iodinated to lead to compound 6. This scaffold was then used in a Suzuki cross coupling 6 to give very efficiently a small focused library of substituted 4-amino-6-chloro-5-phenylpyrimidines (Cpds 7a-15a) which were then hydrogenated under standard conditions to give the desired 4-amino-5-phenylpyrimidines (Cpds 7b-15b). As a Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript means to introduce molecular diversity at the last step in position 4 of the pyrimidine ring, a versatile way of synthesis was designed (Scheme 2). Starting from the commercially available 5-bromo-2,4-dichloropyrimidine, a regioselective n...

We disclose herein the discovery of estrogen receptor alpha (ERalpha) selective estrogen receptor modulators (SERMs) of the tetrahydroisoquinoline series that incorporate novel conformationally restricted side chains as replacement of the aminoethoxy residue typical of SERMs. Molecular modeling studies used in conjunction with the X-ray crystal structure of the ERalpha ligand binding domain (LBD) with raloxifene (7) suggested a diazadecaline moiety as a viable mimic of the SERM side chain. On the basis of this knowledge, the piperidinylethoxy moiety of our lead compound 60 was replaced by a diazadecaline subunit, providing the novel tetrahydroisoquinoline 29. In addition to exhibiting a binding affinity to ERalpha and antagonistic properties in the estrogen response element and MCF-7 assays similar to those of the parent compound 60, ligand 29 showed a reduced agonist behavior in the MCF-7 assay in the absence of 17beta-estradiol. These data point toward the fact that 29 may have a potential for breast cancer prevention/treatment in vivo, a feature which is particularly attractive in the quest for safe alternatives to hormone replacement therapy. In a pharmacokinetic experiment carried out in rats, 29 displayed an interesting profile, with a bioavailability of 49%. We also disclose the X-ray crystal structure of 29 in complex with ERalpha-LBD, which reveals the preferred configurations of 29 at the two chiral centers and the details of its interactions with the receptor. Finally, our structure-activity relationship studies show that other analogues bearing constrained side chains retain potency and antagonist activity and that a 3-OH substituted phenyl D-ring increases the selectivity of a set of piperazinyl-containing ligands in favor of ERalpha over ERbeta.

In an effort to identify selective ligands for the estrogen receptor subtype ERbeta, a series of aryl benzthiophenes was synthesized. In a radioligand binding assay and reporter gene assays in HeLa and SH-SY5Y cells, compounds were characterized as ERbeta-selective agonists. By targeting ERbeta in the brain, these compounds could lead to drugs able to separate the beneficial effects of estrogens on mood, learning, and memory from side effects such as the stimulation of endometrial and breast cancer.

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