Metabotropic glutamate receptors: a structural view point

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Introduction

Most neurons and glia in the mammalian central nervous system express multiple receptor subtypes activated by l-Glutamic acid (l-Glu, 1). These receptors have been classified into two main families, termed ionotropic glutamate (iGlu) receptors and metabotropic glutamate (mGlu) receptors (Fig. 1).

The ionotropic glutamate receptors are integral membrane-spanning ion channels, formed by different stoichiometric arrangements of hetero-subunits which determine the cation selectivity (Fig. 2a). They are pharmacologically classified as NMDA, AMPA and KA receptors, and are involved in the control of the fast (AMPA) and slow (NMDA) component of excitatory postsynaptic currents.

Metabotropic glutamate receptors, first discovered in 1985 (Sladeczek et al., 1985; Nicoletti et al., 1986), constitute a heterogeneous family of GTP-binding proteins (Fig. 2b). So far, eight mGlu receptor subtypes (and several splice variants) have been identified and classified intro three groups according to sequence homology, transduction mechanisms and agonist pharmacology. Group I includes mGluR1 and mGluR5 which are coupled, when expressed in heterologous systems to the activity of phospholipase C. Group II (mGluR2 and mGluR3) and group III (mGluR4, mGluR6-mGluR8) are negatively coupled to adenylyl cyclase (AC) but are endowed with a completely different localization and pharmacology (Conn and Pin, 1997; Pellicciari et al., 1999). In addition to the cloned mGlu receptor subtypes, a metabotropic glutamate receptors directly coupled to the activity of phospholipase D (PLD) has been pharmacologically identified (Albani Torregrossa et al., 1999).

Over the last decade, mGlu receptors have become the object of an intense research activity, aimed at the discovery of new therapeutic agents that may help to control malfunctioning of glutamatergic pathways in a clinically useful way. Indeed, the molecular diversity, the modulatory properties and the peculiar synaptic localization of metabotropic glutamate receptors make them an especially attractive targets for the developing of neuroprotective agents devoid of the severe side effects associated with ionotropic glutamate receptor modulators. In particular, it is currently accepted that antagonists of the group I receptor subtypes and agonists of the group II/III have neuroprotective properties following ischemic insult in several models of brain ischemia (Strasser et al., 1998; Bruno et al., 1999). There are, however, indications that this paradigm is a relatively simplistic one, and that the understanding of the precise involvement of individual subtypes requires that selective and potent ligands become available.

Central to the design and synthesis of selective ligands is the comprehension of the structural factors that regulate the interaction between ligands and the receptor sites of individual subtypes. The increasing knowledge on the molecular and structural biology of metabotropic glutamate receptors has recently provided the rational basis upon which functional profile, selectivity and potency of available ligands can be understood.

In this chapter, we will present our recent results on the molecular modeling and synthesis of selective mGlu receptor ligands, with a major emphasis on group I mGlu receptors.

Section snippets

Ionotropic receptors

In 1990, Seeburg et al. reported the first cloning of a gene expresing a functional ionotropic glutamate receptor (Keinanen et al., 1990). Since then, the family of ionotropic glutamate receptors has continuously grown up, and now at least 14 several spliced variant subunits have been molecularly characterized and shown to assemble into functionally active NMDA, AMPA or KA receptors. Ionotropic glutamate receptors are oligomeric assembly of four or five subunits which form the channel pore and

Role of the amino terminal domain in mGluR1. A medicinal chemistry perspective

Starting from the initial findings of O'Hara et al. on the sequence homology of mGluR1 with PBPs, we engaged ourselves in a research program aimed at the construction of heuristic models of the ATD of mGluR1 that may help in disclosing those structural features which account for the observed ligand potency and selectivity. By taking advantage of the known 3D-structure of the open form of LIVBP, we reported in 1996 a three-dimensional model of the ATD of mGluR1 (Costantino and Pellicciari, 1996

Agonist binding modes

According to the site-directed mutagenesis experiments, the Ser165 and Thr188 residues must be directly implicated in the binding of l-Glu (1) and other agonists. Once having in our hands the 3D-structure of the ATD of mGluR1, we performed a manual docking experiment and we fitted l-Glu (1) in the putative binding site. The amino acidic moiety was accommodated in the close proximity of Ser165 and Thr188 in such a way to form hydrogen bonds with the side chain hydroxy groups. The distal

Antagonist binding mode: the interdomain binding hypothesis

According to the classical definition, mGlu receptor antagonists are chemical entities that cause the receptor to be in a functionally inactive state. Given the operational model of domain closure for the ATD of mGlu receptors, it can be hypothesized that antagonists may either block the ATD in the open form or prevents the transduction of the signal after the closed form is achieved.

Antagonists of mGluR1 generally belong to the class of carboxyphenylglycine (Chart II) (Eaton et al., 1993).

CPGs

Comparison between mGluR1 and mGluR5. Structural origin of antagonist selectivity

The two mGlu receptor subtypes belonging to group I, namely mGluR1 and mGluR5, share more than 90% of sequence homology. It is therefore expectable that they will also share a common 3D-folding and that the methodological protocol used to build up the homology model of the ATD of mGluR1 can also be applied to the ATD of mGluR5. Thus, we have engaged ourselves in the task of constructing the 3D-model of the ATD of mGluR5 with the aim of obtaining structural information upon which selectivity

Agonist-mediated domain closure: a mechanistic hypothesis

According to the molecular mechanism domain closure of PBPs, the ATD of mGlu1 receptors should exist in equilibrium between an open and a closed form, both in their ligand-bound or unbound state. The closed, ligand-bound, state is the functionally active one. The shift of the equilibrium towards the closed, ligand-bound, state that takes place upon agonist binding to the open form of the ATD must be driven by some modification of the chemical environment of the two globular domains. In order to

Conclusions

Advances in the molecular biology of metabotropic glutamate receptors, coupled with the application of homology modeling techniques, allow to gain insights into the molecular mechanism responsible for ligand interaction and signal transduction. The availability of homology models of the ATD of mGluR1, in particular, has permitted us to postulate a molecular mechanism for the action of agonists and antagonists. The information thereby obtained can be usefully instrumental in the design of new,

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