Synthesis and Pharmacological Characterization of C4-(Thiotriazolyl)-substituted-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylates. Identification of (1R,2S,4R,5R,6R)-2-Amino-4-(1H-1,2,4-triazol-3-ylsulfanyl)bicyclo[3.1.0]hexane-2,6-dicarboxylic Acid (LY2812223), a Highly Potent, Functionally Selective mGlu2 Receptor Agonist.

Identification of orthosteric mGlu(2/3) receptor agonists capable of discriminating between individual mGlu2 and mGlu3 subtypes has been highly challenging owing to the glutamate-site sequence homology between these proteins. Herein we detail the preparation and characterization of a series of molecules related to (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate 1 (LY354740) bearing C4-thiotriazole substituents. On the basis of second messenger responses in cells expressing other recombinant human mGlu2/3 subtypes, a number of high potency and efficacy mGlu2 receptor agonists exhibiting low potency mGlu3 partial agonist/antagonist activity were identified. From this, (1R,2S,4R,5R,6R)-2-amino-4-(1H-1,2,4-triazol-3-ylsulfanyl)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 14a (LY2812223) was further characterized. Cocrystallization of 14a with the amino terminal domains of hmGlu2 and hmGlu3 combined with site-directed mutation studies has clarified the underlying molecular basis of this unique pharmacology. Evaluation of 14a in a rat model responsive to mGlu2 receptor activation coupled with a measure of central drug disposition provides evidence that this molecule engages and activates central mGlu2 receptors in vivo.

Synthesis and pharmacological characterization of C4-disubstituted analogs of 1S,2S,5R,6S-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate: identification of a potent, selective metabotropic glutamate receptor agonist and determination of agonist-bound human mGlu2 and mGlu3 amino terminal domain structures.

As part of our ongoing research to identify novel agents acting at metabotropic glutamate 2 (mGlu2) and 3 (mGlu3) receptors, we have previously reported the identification of the C4α-methyl analog of mGlu2/3 receptor agonist 1 (LY354740). This molecule, 1S,2S,4R,5R,6S-2-amino-4-methylbicyclo[3.1.0]hexane-2,6-dicarboxylate 2 (LY541850), exhibited an unexpected mGlu2 agonist/mGlu3 antagonist pharmacological profile, whereas the C4ß-methyl diastereomer (3) possessed dual mGlu2/3 receptor agonist activity. We have now further explored this structure-activity relationship through the preparation of cyclic and acyclic C4-disubstituted analogs of 1, leading to the identification of C4-spirocyclopropane 5 (LY2934747), a novel, potent, and systemically bioavailable mGlu2/3 receptor agonist which exhibits both antipsychotic and analgesic properties in vivo. In addition, through the combined use of protein-ligand X-ray crystallography employing recombinant human mGlu2/3 receptor amino terminal domains, molecular modeling, and site-directed mutagenesis, a molecular basis for the observed pharmacological profile of compound 2 is proposed.

Janocchio--a Java applet for viewing 3D structures and calculating NMR couplings and NOEs.

We present a Java applet, based on the open source Jmol program, which allows the calculation of coupling constants and NOEs from a three-dimensional structure. The program has all the viewing features of Jmol, but adds the capability to calculate both H-H and H-C 3-bond couplings constants. In the case of H--H couplings, the Altona equation is used to perform this. The program also calculates NOEs using the full relaxation matrix approach. All these calculations are driven from a simple point and click interface. The program can calculate values for multi-structure files, and can produce input files for the conformational fitting program NAMFIS.

Identification and pharmacological profile of a new class of selective nicotinic acetylcholine receptor potentiators.

Here we report the discovery, by high-throughput screening, of three novel (2-amino-5-keto)thiazole compounds that act as selective potentiators of nicotinic acetylcholine receptors. Compound selectivity was assessed at seven human nicotinic acetylcholine receptors (alpha1beta1gammadelta, alpha2beta4, alpha3beta2, alpha3beta4, alpha4beta2, alpha4beta4, and alpha7) expressed in mammalian cells or Xenopus oocytes. At alpha2beta4, alpha4beta2, alpha4beta4, and alpha7, but not alpha1beta1gammadelta, alpha3beta2, or alpha3beta4, submaximal responses to nicotinic agonists were potentiated in a concentration-dependent manner by all compounds. At similar concentrations, no potentiation of 5-hydroxytryptamine, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, GABA(A), and N-methyl-d-aspartate receptors or voltage-gated Na(+) and Ca(2+) channels was observed. Furthermore, these compounds did not inhibit acetylcholine esterase. Further profiling revealed that these compounds enhanced the potency and maximal efficacy of a range of nicotinic agonists at alpha4beta2 nicotinic acetylcholine receptors, a profile typical of allosteric potentiators. At concentrations required for potentiation, the compounds did not displace [(3)H]epibatidine from the agonist-binding site, and potentiation was observed at all agonist concentrations, suggesting a noncompetitive mechanism of action. Blockade of common second messenger systems did not affect potentiation. At concentrations higher then required for potentiation the compounds also displayed intrinsic agonist activity, which was blocked by competitive and noncompetitive nicotinic acetylcholine receptor (nAChR) antagonists. These novel selective nicotinic receptor potentiators should help in clarifying the potential therapeutic utility of selective nAChR modulation for the treatment of central nervous system disorders.

High affinity ligands for the alpha7 nicotinic receptor that show no cross-reactivity with the 5-HT3 receptor.

Potent and selective ligands of the alpha7 nicotinic acetylcholine receptor are required to understand the pharmacological effect of alpha7 activation. A common cross-reactivity occurs with serotonergic 5-HT3 receptors with which alpha7 receptors have a high sequence homology. We demonstrate that certain quinuclidine 3-biaryl carboxamides are high affinity alpha7 ligands with an excellent binding selectivity over 5-HT3 receptors.

PSAB-OFP, a selective alpha 7 nicotinic receptor agonist, is also a potent agonist of the 5-HT3 receptor.

5-Hydroxytryptamine 3 (5-HT(3)) and alpha 7 nicotinic receptors share high sequence homology and pharmacological cross-reactivity. An assessment of the potential role of alpha 7 receptors in many neurophysiological processes, and hence their therapeutic value, requires the development of selective alpha 7 receptor agonists. We used a recently reported selective alpha 7 receptor agonist, (R)-(-)-5'Phenylspiro[1-azabicyclo[2.2.2] octane-3,2'-(3'H)furo[2,3-b]pyridine (PSAB-OFP) and confirmed its activity on human recombinant alpha 7 receptors. However, PSAB-OFP also displayed high affinity binding to 5-HT(3) receptors. To assess the functional activity of PSAB-OFP on 5-HT(3) receptors we studied recombinant human 5-HT(3) receptors expressed in Xenopus oocytes, as well as native mouse 5-HT(3) receptors expressed in N1E-115 neuroblastoma cells, using whole-cell patch clamp and Ca(2+) imaging. Our results show that PSAB-OFP is an equipotent, partial agonist of both alpha 7 and 5-HT(3) receptors. We conclude that it will be necessary to identify the determinant of this overlapping pharmacology in order to develop more selective alpha 7 receptor ligands.