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[Image: see text] Biased ligands that selectively confer activity in one pathway over another are pharmacologically important because biased signaling may reduce on-target side effects and improve drug efficacy. Here, we describe an N-terminal modification in the incretin hormone glucagon-like peptide (GLP-1) that alters the signaling capabilities of the GLP-1 receptor (GLP-1R) by making it G protein biased over internalization but was originally designed to confer DPP-4 resistance and thereby prolong the half-life of GLP-1. Despite similar binding affinity, cAMP production, and calcium mobilization, substitution of a single amino acid (Ala8 to Val8) in the N-terminus of GLP-1(7–36)NH(2) (GLP-1 Val8) severely impaired its ability to internalize GLP-1R compared to endogenous GLP-1. In-depth binding kinetics analyses revealed shorter residence time for GLP-1 Val8 as well as a slower observed association rate. Molecular dynamics (MD) displayed weaker and less interactions of GLP-1 Val8 with GLP-1R, as well as distinct conformational changes in the receptor compared to GLP-1. In vitro validation of the MD, by receptor alanine substitutions, confirmed stronger impairments of GLP-1 Val8-mediated signaling compared to GLP-1. In a perfused rat pancreas, acute stimulation with GLP-1 Val8 resulted in a lower insulin and somatostatin secretion compared to GLP-1. Our study illustrates that profound differences in molecular pharmacological properties, which are essential for the therapeutic targeting of the GLP-1 system, can be induced by subtle changes in the N-terminus of GLP-1. This information could facilitate the development of optimized GLP-1R agonists.

Herein, we report the development of bitopic ligands aimed at targeting the orthosteric binding site (OBS) and a metastable binding site (MBS) within the same receptor unit. Previous molecular dynamics studies on ligand binding to the β2-adrenergic receptor (β2AR) suggested that ligands pause at transient, less-conserved MBSs. We envisioned that MBSs can be regarded as allosteric binding sites and targeted by homobivalent bitopic ligands linking two identical pharmacophores. Such ligands were designed based on docking of the antagonist (S)-alprenolol into the OBS and an MBS and synthesized. Pharmacological characterization revealed ligands with similar potency and affinity, slightly increased β2/β1AR-selectivity, and/or substantially slower β2AR off-rates compared to (S)-alprenolol. Truncated bitopic ligands suggested the major contribution of the metastable pharmacophore to be a hydrophobic interaction with the β2AR, while the linkers alone decreased the potency of the orthosteric fragment. Altogether, the study underlines the potential of targeting MBSs for improving the pharmacological profiles of ligands.

N-(1-Benzyl-4-piperidinyl)-2,4-dichlorobenzamide 5 (BPDBA) is a noncompetitive inhibitor of the betaine/GABA transporter 1 (BGT1). We here report the synthesis and structure-activity relationship of 71 analogues. We identify 26m as a more soluble 2,4-Cl substituted 3-pyridine analogue with retained BGT1 activity and an improved off-target profile compared to 5. We performed radioligand-based uptake studies at chimeric constructs between BGT1 and GAT3, experiments with site-directed mutated transporters, and computational docking in a BGT1 homology model based on the newly determined X-ray crystal structure of the human serotonin transporter (hSERT). On the basis of these experiments, we propose a binding mode involving residues within TM10 in an allosteric site in BGT1 that corresponds to the allosteric binding pocket revealed by the hSERT crystal structure. Our study provides first insights into a proposed allosteric binding pocket in BGT1, which accommodates the binding site for a series of novel noncompetitive inhibitors.

GPR139 is an orphan G protein-coupled receptor expressed mainly in the central nervous system. We developed a pharmacophore model based on known GPR139 surrogate agonists which led us to propose aromatic-containing dipeptides as potential ligands. Upon testing, the dipeptides demonstrated agonism in the Gq pathway. Next, in testing all 20 proteinogenic l-α-amino acids, L-tryptophan and l-phenylalanine were found to have EC50 values of 220 and 320 μM, respectively, making them the first putative endogenous agonists for GPR139.

We describe a new class of subunit-selective antagonists of N-methyl D-Aspartate (NMDA)-selective ionotropic glutamate receptors that contain the (E)-3-phenyl-2-styrylquinazolin-4(3H)-one backbone. The inhibition of recombinant NMDA receptor function induced by these quinazolin-4-one derivatives is non-competitive and voltage-independent, suggesting that this family of compounds does not exert action on the agonist binding site of the receptor or block the channel pore. The compounds described here resemble CP-465,022 ((S)-3-(2-chlorophenyl)-2-[2-(6-diethylaminomethyl-pyridin-2-yl)-vinyl]-6-fluoro-3H-quinazolin-4-one), a non-competitive antagonist of AMPA-selective glutamate receptors. However, modification of ring substituents resulted in analogues with greater than 100-fold selectivity for recombinant NMDA receptors over AMPA and kainate receptors. Furthermore, within this series of compounds, analogues were identified with 50-fold selectivity for recombinant NR2C/D-containing receptors over NR2A/B containing receptors. These compounds represent a new class of non-competitive subunit-selective NMDA receptor antagonists.

The design, synthesis, and pharmacological characterization of a highly potent and selective glutamate GluR5 agonist is reported. (S)-2-Amino-3-((RS)-3-hydroxy-8-methyl-7,8-dihydro-6H-cyclohepta[d]isoxazol-4-yl)propionic acid (5) is the 8-methyl analogue of (S)-2-amino-3-(3-hydroxy-7,8-dihydro-6H-cyclohepta[d]isoxazol-4-yl)propionic acid ((S)-4-AHCP, 4). Compound 5 displays an improved selectivity profile compared to 4. A versatile stereoselective synthetic route for this class of compounds is presented along with the characterization of the binding affinity of 5 to ionotropic glutamate receptors (iGluRs). Functional characterization of 5 at cloned iGluRs using a calcium imaging assay and voltage-clamp recordings show a different activation of GluR5 compared to (S)-glutamic acid (Glu), kainic acid (KA, 1), and (S)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl)propionic acid ((S)-ATPA, 3) as previously demonstrated for 4. An X-ray crystallographic analysis of 4 and computational analyses of 4 and 5 bound ...

Four 2-substituted Tet-AMPA [Tet = tetrazolyl, AMPA = 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid] analogues were characterized functionally at the homomeric AMPA receptors GluR1i, GluR2Qi, GluR3i, and GluR4i in a Fluo-4/Ca2+ assay. Whereas 2-Et-Tet-AMPA, 2-Pr-Tet-AMPA, and 2-iPr-Tet-AMPA were nonselective GluR agonists, 2-Bn-Tet-AMPA exhibited a 40-fold higher potency at GluR4i than at GluR1i. Examination of homology models of the S1-S2 domains of GluR1 and GluR4 containing 2-Bn-Tet-AMPA suggested four nonconserved residues in a region adjacent to the orthosteric site as possible determinants of the GluR4i/GluR1i selectivity. In a mutagenesis study, doubly mutating M686V/I687A in GluR1i in combination with either D399S or E683A increased both the potency and the maximal response of 2-Bn-Tet-AMPA at this receptor to levels similar to those elicited by the agonist at GluR4i. The dependence of the novel selectivity profile of 2-Bn-Tet-AMPA upon residues located outside of the orthosteric site underlines the potential for developing GluR subtype selective ligands by designing compounds with substituents that protrude beyond the (S)-Glu binding pocket.

The four stereoisomers of 5-(2-amino-2-carboxyethyl)-4,5-dihydroisoxazole-3-carboxylic acid(+)-4, (-)-4, (+)-5, and (-)-5 were prepared by stereoselective synthesis of two pairs of enantiomers, which were subsequently resolved by enzymatic procedures. These four stereoisomers and the four stereoisomers of the bicyclic analogue 5-amino-4,5,6,6a-tetrahydro-3aH-cyclopenta[d]isoxazole-3,5-dicarboxylic acid (+)-2, (-)-2, (+)-3, and (-)-3 were tested at ionotropic and metabotropic glutamate receptor subtypes. The most potent NMDA receptor antagonists [(+)-2, (-)-4, and (+)-5] showed a significant neuroprotective effect when tested in an oxygen glucose deprivation (OGD) cell culture test. The same compounds were preliminarily assayed using Xenopus oocytes expressing cloned rat NMDA receptors containing the NR1 subunit in combination with either NR2A, NR2B, NR2C, or NR2D subunit. In this assay, all three derivatives showed high antagonist potency with preference for the NR2A and NR2B subtypes, with derivative (-)-4 behaving as the most potent antagonist. The biological data are discussed on the basis of homology models reported in the literature for NMDA receptors and mGluRs.

The two diastereomeric pairs of acidic amino acids 5-(2-amino-2-carboxyethyl)-4,5-dihydroisoxazole-3-carboxylic acid (8A/8B) and 4-(2-amino-2-carboxyethyl)-5,5-dimethyl-4,5-dihydroisoxazole-3-carboxylic acid (10A/10B) were prepared via a strategy based on a 1,3-dipolar cycloaddition. The four amino acids were tested at ionotropic and metabotropic glutamate receptors. None of the compounds was active, neither as agonists nor as antagonists, at 1 mM on metabotropic receptors (mGluR1, -2, -4, and -5 expressed in CHO cell lines). Conversely, the pair of stereoisomers 8A/8B showed a remarkable affinity, antagonist potency, and selectivity for NMDA receptors, when tested on ionotropic glutamate receptors. The affinity of 8A proved to be 5 times higher than that of diastereomer 8B (K(i) values 0.21 and 0.96 microM, respectively). Furthermore, compounds 8A and 8B exhibited a noteworthy anticonvulsant activity in in vivo tests on DBA/2 mice. Derivative 10A was inactive at all ionotropic glutamate receptors, whereas its stereoisomer 10B displayed a seizable binding to both NMDA and AMPA receptors.

On the basis of structural studies, chloro-homoibotenic acid (Cl-HIBO) was designed and synthesized. Cl-HIBO was characterized in binding and electrophysiology experiments on native and cloned subtypes of GluRs. Electrophysiological selectivities ranged from 275 to 1600 for GluR1/2 over GluR3/4. The potent AMPA receptor activity was strongly desensitizing and the neurotoxicity similar to AMPA. Thus, Cl-HIBO is the most subtype selective agonist reported to date on GluR1/2, and offers a new standard for selectively studying subtypes of AMPA receptors.

The design and synthesis of conformationally restricted analogues of alpha-amino acids is an often used strategy in medicinal chemistry research. Here we present the rational design, synthesis, and pharmacological evaluation of 2-azanorbornane-3-exo,5-endo-dicarboxylic acid (1), a novel conformationally restricted (S)-glutamic acid (Glu) analogue intended as a mimic of the folded Glu conformation. The synthesis of 1 was completed in its racemic form in eight steps from commercially available starting materials. As a key step, the first facially selective hydroboration of a 5-methylidene[2.2.1]bicyclic intermediate was investigated. In this transformation, the catalytic methodology of Wilkinson's/catechol borane proved superior to stoichiometric borane or dialkyl borane reagents, in terms of higher diastereomeric excess and chemical yield. To our surprise (+/-)-1 did not show affinity in binding studies on native 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) (IC(50)300 microM, [(3)H]AMPA) or kainic acid (IC(50)160 microM, [(3)H]kainic acid) receptors nor in binding studies on the cloned iGluR5,6 subtypes (IC(50)300 microM, [(3)H]kainic acid).

A number of 1-hydroxyazole derivatives were synthesized as bioisosteres of (S)-glutamic acid (Glu) and as analogues of the AMPA receptor agonist (R,S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA, 3b). All compounds were subjected to in vitro pharmacological studies, including a series of Glu receptor binding assays, uptake studies on native as well as cloned Glu uptake systems, and the electrophysiological rat cortical slice model. Compounds 7a,b, analogues of AMPA bearing a 1-hydroxy-5-pyrazolyl moiety as the distal carboxylic functionality, showed only moderate affinity for [3H]AMPA receptor binding sites (IC(50) = 2.7 +/- 0.4 microM and IC(50) = 2.6 +/- 0.6 microM, respectively), correlating with electrophysiological data from the rat cortical wedge model (EC(50) = 280 +/- 48 microM and EC(50) = 586 +/- 41 microM, respectively). 1-Hydroxy-1,2,3-triazol-5-yl analogues of AMPA, compounds 8a,b, showed high affinity for [3H]AMPA receptor binding sites (IC(50) = 0.15 +/- 0.03 microM and IC(50) = 0.13 +/- 0.02 microM, respectively). Electrophysiological data showed that compound 8a was devoid of activity in the rat cortical wedge model (EC(50) > 1000 microM), whereas the corresponding 4-methyl analogue 8b was a potent AMPA receptor agonist (EC(50) = 15 +/- 2 microM). In accordance with this disparity, compound 8a was found to inhibit synaptosomal [3H]D-aspartic acid uptake (IC(50) = 93 +/- 25 microM), as well as excitatory amino acid transporters (EAATs) EAAT1 (IC(50) = 100 +/- 30 microM) and EAAT2 (IC(50) = 300 +/- 80 microM). By contrast, compound 8b showed no appreciable affinity for Glu uptake sites, neither synaptosomal nor cloned. Compounds 9a-c and 10a,b, possessing 1-hydroxyimidazole as the terminal acidic function, were devoid of activity in all of the systems tested. Protolytic properties of compounds 7a,b, 8b, and 9b were determined by titration, and a correlation between the pK(a) values and the activity at AMPA receptors was apparent. Optimized structures of all the synthesized ligands were fitted to the known crystal structure of an AMPA-GluR2 construct. Where substantial reduction or abolition of affinity at AMPA receptors was observed, this could be rationalized on the basis of the ability of the ligand to fit the construct. The results presented in this article point to the utility of 1-hydroxypyrazole and 1,2,3-hydroxytriazole as bioisosteres of carboxylic acids at Glu receptors and transporters. None of the compounds showed significant activity at metabotropic Glu receptors.

Our previous publication (J. Med. Chem. 1996, 39, 3188-3194) described (RS)-2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid (Homo-AMPA) as a highly selective agonist at the mGlu6 subtype of metabotropic excitatory amino acid (EAA) receptors. Homo-AMPA has already become a standard agonist for the pharmacological characterization of mGlu6 (Trends Pharmacol. Sci. Suppl. 1997, 37-39), and we here report the resolution, configurational assignment, and pharmacology of (S)- (6) and (R)- (7) Homo-AMPA. Using the "Ugi four-component condensation", 3-(3-ethoxy-5-methylisoxazol-4-yl)propanal (10) was converted into the separable diastereomeric derivatives of 6 and 7, compounds 12 and 11, respectively. Deprotection of 12, in one or two steps, gave extensively racemized 6, which was converted in low yield into 6 (99.0% ee) through several crystallizations. 6 (99.7% ee) and 7 (99.9% ee) were finally obtained by preparative chiral HPLC. The configurational assignments of 6 and 7 were based on 1H NMR spectroscopic studies on 12 and 11, respectively, and circular dichroism studies on 6 and 7. Values of optical rotations using different solvents and the chiral HPLC elution order of 6 and 7 supported the results of the spectroscopic configurational assignments. The activities of 6 and 7 at ionotropic EAA (iGlu) receptors and at mGlu1-7 were studied. (S)-Homo-AMPA (6) was shown to be a specific agonist at mGlu6 (EC50 = 58 +/- 11 microM) comparable in potency with the endogenous mGlu agonist (S)-glutamic acid (EC50 = 20 +/- 3 microM). Although Homo-AMPA did not show significant effects at iGlu receptors, (R)-Homo-AMPA (7), which was inactive at mGlu1-7, turned out to be a weak N-methyl-D-aspartic acid (NMDA) receptor antagonist (IC50 = 131 +/- 18 microM).

The homologous series of acidic amino acids, ranging from aspartic acid (1) to 2-aminosuberic acid (5), and the corresponding series of 3-isoxazolol bioisosteres of these amino acids, ranging from (RS)-2-amino-2-(3-hydroxy-5-methylisoxazol-4-yl)acetic acid (AMAA, 6) to (RS)-2-amino-6-(3-hydroxy-5-methylisoxazol-4-yl)hexanoic acid (10), were tested as ligands for metabotropic excitatory amino acid receptors (mGlu1 alpha, mGlu2, mGlu4a, and mGlu6). Whereas AMAA (6) and (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propinoic acid (AMPA, 7) are potent and highly selective agonists at N-methyl-D-aspartic acid (NMDA) and AMPA receptors, respectively, the higher homologue of AMPA (7), (RS)-2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid (homo-AMPA, 8), is inactive at ionotropic excitatory amino acid receptors. Homo-AMPA (8), which is a 3-isoxazolol bioisostere of 2-aminoadipic acid (3), was, however, shown to be a specific and rather potent agonist at mGlu6, approximately 4 times weaker than the nonselective excitatory amino acid receptor agonist (S)-glutamic acid. 2-Aminoadipic acid (3), which shows a complex excitatory amino acid synaptic pharmacology, was an agonist at mGlu6 as well as mGlu2. AMPA (7) and the higher homologue of homo-AMPA (8), (RS)-2-amino-5-(3-hydroxy-5-methylisoxazol-4-yl)pentanoic acid (9), showed relatively weak agonist effects at mGlu6. It is concluded that homo-AMPA (8) is likely to be a useful tool for studies of the pharmacology and physiological role of mGlu6. We describe a new versatile synthesis of this homologue of AMPA and the synthesis of compound 10.