A putative model of bradykinin bound to the rat B2 receptor was generated using a combination of homology modeling (from the known transmembrane structure of bacteriorhodopsin), energy minimization, molecular dynamics, and a two-stage conformational search as a docking simulation. Overall, the proposed bound ligand adopts a twisted "S" shape, wherein a C-terminal beta-turn is buried in the receptor just below the extracellular boundary of the cell membrane and the N-terminus is interacting with negatively charged residues in extracellular loop 3 of the receptor (most notably Asp268 and Asp286). Mutagenesis experiments describing mutations which result in both a loss of bradykinin affinity as well as those which have no effect on bradykinin affinity are in good agreement with the proposed structure. In short, the mutagenesis results and the computational simulations each point to the same region of the receptor as likely to bind bradykinin. A double mutation, predicted as being likely to have a dramatic effect on bradykinin binding affinity, was confirmed experimentally, adding some validation to the proposed complex. Moreover, a new pseudopeptide bradykinin receptor antagonist (D-Arg0-Arg1-[12-aminododecanoyl]2- Ser3-D-Tic4-Oic5-Arg6) was designed on the basis of the model, and found to have good receptor affinity. Speculation regarding other possible sites for mutagenesis are also described.

A glutamate-binding protein from rat brain synaptic plasma membranes has been purified to apparent homogeneity. This protein has a Mr of 14,300 based on amino acid and carbohydrate analyses. The protein is enriched with tryptophan residues, which contribute substantially to its hydrophobic nature. It also has a relatively high content of acidic amino acids, which determine is low isoelectric point (4.82). The protein exhibits either a single, high-affinity class of sites for L-[3H]glutamate binding (KD = 0.13 microM) when binding is measured at low protein concentrations, or two classes of sites with high (KD = 0.17 microM) and low affinities (KD = 0.8 microM) when binding is measured at high protein concentrations. These observations suggest preferential binding of L-glutamate to a self-associating form of the protein. The displacement of protein-bound L-[3H]glutamic acid by other neuroactive amino acids has characteristics similar to those observed for displacement of L-glutamate from membrane binding sites. Chemical modification of the cysteine and arginine residues results in an inhibition of glutamate binding activity. The possible function of this protein in the physiologic glutamate receptor complex of neuronal membranes is discussed.

Agonist stimulation of group III metabotropic glutamate receptors (mGluRs) induces an inhibition of neurotransmitter release from neurons. The group III mGluRs are pharmacologically defined by activation with the glutamate analog L-amino-4-phosphonobutyric acid (L-AP4). The affinities of these receptors for L-AP4 and glutamate vary over approximately a 1500-fold concentration range. The goal of this study was to elucidate the molecular basis for this dispersion of agonist affinities for the group III receptors mGluR4, mGluR6, and mGluR7. were used to predict amino acids that may affect ligand affinity. Mutations were made in mGluR7 to convert selected residues into the equivalent amino acids present in the high-affinity mGluR4 receptor. The mGluR7 N74K mutation caused a 12-fold increase in affinity in a functional assay, whereas the N74K mutation in combination with mutations in residues 258 to 262, which lie outside the binding pocket, caused a 112-fold increase in affinity compared with unmutated mGluR7. Our results demonstrate that the binding site residues at position lysine 74 in mGluR4, glutamine 58 in mGluR6, and asparagine 74 in mGluR7 are key determinants of agonist affinity and that additional residues situated outside of the binding pocket, including those present in the extreme amino terminus, also contribute to agonist affinity and the pharmacological profiles of the group III mGluRs.The group III metabotropic glutamate receptors (mGluRs; mGluR4, -6, -7, and -8) share 70 to 74% amino acid identity and are selectively activated by the synthetic agonist L-amino-4-phosphonobutyric acid (L-AP4) and the endogenous amino acid L-serine-O-phosphate (L-SOP) (Fig.

Extensive research into the functions of glutamate and glutamate receptors in the central nervous system (CNS) has shown an essential role of metabotropic glutamate (mGlu) receptors in normal brain functions, but also in neurological and psychiatric disorders. The precise functions of these receptors remain undefined, and progress toward understanding their functions has been hampered by the lack of selective ligands with appropriate pharmacokinetic properties. The Group I mGlu receptor, mGlu5, is well positioned to regulate and fine-tune neuronal excitability and synaptic transmission through its modulation of various signal transduction pathways and interactions with other transmitter systems. Therefore, the mGlu5 receptor may be an important therapeutic target for the treatment of disorders of the central nervous system. The discovery of MPEP 3, a non-competitive mGlu5 receptor antagonist, provided a potent, selective, systemically active tool compound for proof of concept studies in animal models of various disease states. These studies have led to greater understanding of possible therapeutic applications of mGlu5 receptor antagonists in recent years, suggesting their use in a number of disease states, including chronic pain, various psychiatric and neurological disorders, substance abuse and withdrawal, obesity and gastroesophageal reflux disease (GERD). Together, these findings have intensified efforts to find other non-competitive mGlu5 receptor antagonists and have led to the discovery of several second-generation compounds, a few of which are in preclinical evaluations. There have been several recent reviews on mGlu receptor. This article highlights recent efforts on the design, synthesis and development of novel, non-competitive mGlu5 receptor antagonists and studies to understand their in vitro mechanisms of action and in vivo pharmacological profiles. Emphasis is also given to recent advances in the potential therapeutic applications of non-competitive mGlu5 receptor antagonists.

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