There is great interest in developing pharmacological compounds that function through metabotropic glutamate receptors (mGluRs) as modulators of synaptic function and that could consequently become effective therapeutic agents for several developmental and degenerative neuropsychiatric disorders. mGluRs are located perisynaptically, acting as sensors and modulators of glutamate transmission.1 This functional specificity makes them attractive pharmacological targets. In particular, subtype 5 receptors (mGluR5) regulate local protein synthesis and messenger RNA (mRNA) translation at synapses, and are thus ideally positioned to control synaptic plasticity.2–5 A number of studies have explored the therapeutic potential of drugs acting at the mGluR5 for a variety of conditions, including Parkinson’s disease (PD), addiction, chronic pain, mood disorders, epilepsy, and fragile X, among others.6,7 Despite their diversity, these disorders may share common mechanisms involving aberrant brain plasticity.
Regarding PD, the loss of dopamine (DA)-mediated inhibition in the striatum results in excess excitatory transmission. It is therefore possible that mGluR5 modulation can improve PD symptoms directly8–10 or through modulation of cholinergic interneurons,11,12 although in vivo off-target effects of mGluR5 antagonists, particularly on N-methyl-D-aspartate (NMDA) and mGluR1s, have to be taken into account.13 Furthermore, mGluR5 antagonists appear particularly promising for the treatment of levodopa-induced dyskinesia,14–16 a frequent, invalidating complication of DA replacement therapy that represents an abnormal form of synaptic plasticity.17 Finally, pharmacological manipulation of mGluR5s could open new modalities of neuroprotection for PD and other degenerative diseases of the nervous system, by decreasing excitotoxicity,18 modulating signaling pathways, or, perhaps, through local translation of trophic factors. We have explored the anatomical basis and functional role of mGluR5 antagonists in models of PD, taking advantage of high-sensitivity positron emission tomography (PET) and the recent development of novel specific radiopharmaceuticals in a series of studies briefly reviewed here. Metabotropic Glutamate Receptors
Glutamate is the most prevalent excitatory neurotransmitter in the brain, and a balanced glutamatergic transmission is required for normal brain function. Glutamate plays a role in a variety of physiological processes, such as long-term potentiation (learning and memory), the development of synaptic plasticity, motor control, respiration, cardiovascular regulation, emotional states, and sensory perception.19 At the synaptic level glutamateis regulated by glutamate transporters and acts through two types of receptor: the fast-ligand-binding receptors, which are directly coupled to the opening of cation channels in the cell membranes of the neurons and are termed ‘ionotropic’ (iGluR); and the second type, which are the slow G-protein-coupled ‘metabotropic’ receptors (mGluR),20 and are associated with second messenger systems and lead to enhanced phosphoinositol hydrolysis (IP3), activation of phospholipase D, increases or decreases in adenyl cyclase and cyclic adenosine monophosphatase (cAMP) formation, and changes in ion channel function.21 In the past several years, molecular cloning studies have revealed the existence of eight different subtypes of mGluRs.6 mGluR subtypes can be divided into three groups according to their sequence similarities, signal transduction mechanisms, and pharmacological profiles.22 The first group, comprising mGluR1 and mGluR5, is coupled to stimulation of IP3/Ca2+ signal transduction.23 Group I mGluRs are especially effective at reducing GABA-inhibitory responses.The second group, consisting of mGluR2 and mGluR3, is negatively coupled through adenylate cyclase to cAMP formation.24 The third group, including mGluR4, mGluR6, mGluR7, and mGluR8, is also negatively linked to adenylate cyclase activity but shows different agonist preference.25
mGluR5 was cloned in 199226 and, like the other seven subtypes, is a G-protein-coupled receptor with a large N-amino terminal domain. mGluR5 is expressed in limbic cortex, hippocampus, amygdala, basal ganglia, thalamus, and motor and premotor cortices. The receptor is localized mostly post-synaptically and co-localized with adenosine A2a,27 DA, and NMDA receptors. Brain areas expressing mGluR5s are related to nociception, emotion, motivation, and motor control, leading to the assumption that mGluRs may have a critical role in pain, anxiety, depression, and neurodegenerative disorders, including PD. Interestingly, mGluR5 is enriched in cortical and subcortical areas and prominently activated in response to DA release (see Figure 1).28
The diversity and heterogeneous distribution of mGluR subtypes through the central nervous system (CNS) provides an opportunity for developing compounds (or drugs) to selectively target a specific subsystem, aiming to ameliorate symptoms of distinct neurological disorders while limiting the disruptive effects of altered glutamate transmission on brain function. Such compounds represent both pharmacological tools to further investigate mGluR5 physiology and potential therapeutic agents for the treatment of several conditions that have been associated with abnormal activation of mGluR5 function.
mGluRs belong to class C of G-protein-coupled receptors and, in addition to the characteristic seven-strand transmembrane domain for G-protein activation, possess a large extracellular domain that is responsible for ligand recognition.29 The mGluRs have a large bi-lobed extracellularN-terminus of ~560 amino acids that has been shown by mutagenesis studies to confer glutamate binding, agonist activation of the receptor, and subtype specificity for group-selective agonists.30
A large number of pharmacological agents active at mGluRs have been reported in the literature. According to the mode of binding, mGluR pharmacological agents can be classified into competitive and noncompetitive agents and, based on the mode of action, they can be classified into agonists, antagonists, and positive/negative/neutralmodulators or potentiators.31 Competitive agonists and antagonists bind to the same orthosteric binding site as endogenous glutamate, which is a cleft between the two lobes in the extracellular N-terminus. Their binding ability depends on how much they could stabilize the closed conformation.32 These ligands received the earliest research interest and they have been well developed. They are all glutamate analogs or substituted glycines, which implies that they have poor selectivity within their group. In addition, competitive agonists and antagonists have structural carboxyl and amino groups, which make them too polar to penetrate the blood–brain barrier.30 Starting from 1996,33 a number of structural types of non-competitive negative, positive, and neutral allosteric modulators have been developed as mGluR ligands.34 These ligands modulate mGlu receptor activity by binding to allosteric binding sites that are located in the seven-strand transmembrane domain. The allosteric binding sites are structurally distinct from the classic agonist, orthosteric, binding site.35 Positive and negative modulators thus offer the potential for improved selectivity for individual mGluR family members compared with competitive agonists and antagonists at the glutamate site.32 These ligands are not amino acid derivatives and are structurally diverse. They are lipophilic and have much better CNS penetrating capability. Thus, such positive and negative modulators with high binding affinity, high subtype selectivity, and appropriate lipophilicity are good candidates for mGluR radiotracer development.36–38 These tracers do not show competitive binding with endogenous glutamate, which increases their sensitivity. In summary, classic mGluR5 agonists and antagonists are derived from endogenous glutamate, thus lacking subtype selectivity and suitable lipophilicity to cross the blood–brain barrier. By contrast, allosteric modulators— negative, positive, neutral—are structurally diverse and amino-acidunrelated, therefore possessing high binding affinity, high subtype selectivity, and appropriate lipophilicity.
The first selective mGlu5 receptor antagonists were identified in 199939 through random screening and have been followed by a large number of potent, subtype-selective, and structurally diverse allosteric modulators, mostly developed by the pharmaceutical industry. Indeed, in search of alternative chemical structures for orthosteric antagonists, Novartis AG developed the first selective antagonist, SIB-1757 (6-methyl-2- (phenylazo)-3-pyridinol),39 which inhibits glutamate-induced receptor activation without affecting the affinity for glutamate, i.e. in a noncompetitive manner. Subsequent optimization and replacement of the trans-olefinic tether with a carbon triple bond led to the synthesis of 2-methyl-6-(phenylethylyn) pyridine (MPEP),40 which displays highlyimproved mGluR5 antagonist activity and has been considered a prototypical mGluR5 antagonist, although it is also active at the NMDA receptor.13,30 Structure–activity relationship studies performed on MPEP, in which chemical modifications were made to each of the three regions of the original molecule, led to identification of MTEP ((2-methyl-1,3- thiazo-4-yl)ethynyl pyridine) and other highly potent and selective diaryl (heteroaryl) acetylenes as mGluR5 non-competitive antagonists.41 With the assumption that the (2-methyl-1,3-thiazo-4-yl)ethynyl group is an optimal chemical structure to confer mGluR5 antagonist activity to a compound, further structure–activity relationship studies on MTEP have identified other ligands containing thiazole moieties as mGluR5 noncompetitive antagonists with improved pharmacological profiles. Although most of the ligands are MPEP- or MTEP-derived, some compounds lacking the acetylenic tether have been delineated as potent and selective mGluR5 non-competitive antagonists as well.
Compared with available negative modulators, potent and selective positive allosteric modulators of mGluR5 are less well developed. Since the discovery of the first mGluR5-positive modulator,42 3,3’- difluorobenzaldazine, Merck has reported three series of positive allosteric modulators for mGlu5 receptor, which are the benzaldazine series, benzamide series, and pyrazole series.42–44 The discovery of non-competitive allosteric modulators with high binding affinity andsubtype selectivity facilitates the exploration of the physiological and pathological functions of mGluR5 in normal and diseased states.
mGluR5 Binding in the Normal and Parkinsonian Brain
DA exerts a complex regulation of glutamate neurotransmission in the basal ganglia circuitry through differential effects on striatopallidal and striatonigral neurons mediated by DA receptors D1 and D2.45 In PD, loss of DA results in an imbalance in other neurotransmitters, mostly excitatory. Taking advantage of new PET tracers, we have examined the distribution of mGluR5 in the rodent and primate brain and the effects of DA denervation in parkinsonian animals. In our recent studies,28,38,46 we have synthesized and radiolabeled five non-competitive antagonists for mGluR5—[11C]M-MPEP (2-[(3-methoxyphenyl)ethynyl]-6-methylpyridine), [11C]M-PEPy (3-methoxy-5-[(2-pyridyl)ethynyl]pyridine,), [11C]MPEP (2- methyl-6-(2-phenylethynyl) pyridine), [18F]FMTEP (2-fluoro-5-(2-(2-methylthiazol-4-yl)ethynyl)pyridine), and [18F]FPEB (3-fluoro-5-(2-pyridylethynyl) benzonitrile)—and conducted in vivo PET imaging studies in different disease models to investigate mGluR5 expression and function.All of the compounds showed prominent binding in the striatum and limbic regions of the brain. Using [11C]MPEP in rats with a unilateral lesion of the DA system, we have described a small enhancement on the side of thelesion in the striatum, cortex and hippocampus.46 Figure 2 illustrates the enhancement observed in the striatum, on the side of DA lesion, withthree mGluR5 compounds in the 6-hydroxy-DA rat model of PD.
In naïve primates, the mGluR5 tracer [11C]MPEPy rapidly accumulates in discrete cortical and subcortical regions (see Figure 3), including the premotor and cingulate cortices, superior temporal gyrusand limbic (paraentorhinal/amygdala/hippocampal) cortex, the nucleus accumbens, caudate, and putamen, the ventral thalamus, and the midbrain. This distribution matches the areas that have high mGluR5 mRNA expression in the rodent brain.47 Following systemic administration of a neurotoxin, MPTP (1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine), we found a significant enhancement of mGluR5 binding (~20%) in the motor regions of the striatum.28 In the primate brain, the distribution of mGluR5 radiotracers corresponds well with DA-responsive areas (see Figure 1). From an technical imaging point of view, we found that the accumulation of pyridine analogs into the brain mGluR5s is very fast (one to five minutes) and is followed by a fast washout, which is problematic, causing poor image quality because of statistically low counts. This makes them unfavorable for development for human studies in spite of their excellent in vitro pharmacological characteristics. Instead, benzonitrile derivatives have turned out to be promising ligands to target mGluR5 in vivo and are good candidates for human applications.38,48
mGluR5 Antagonists in Parkinson’s Disease Therapy
From our in vivo studies we can conclude that the prevalent distribution of mGluR5 in the striatum and limbic structures supports their role in modulating DA- and glutamate-dependent signaling and synaptic plasticity within the basal ganglia cortico–subcortical loops. In PD, thedeath of DA neurons in the substantia nigra pars compacta causes a loss of DA in the basal ganglia. DA modulation of neurotransmission in the striatum and other basal ganglia structures is crucial to gate cortical and thalamic excitatory input through the direct and indirect pathways. We have found an upregulation of mGluR5 following DA denervation in animal models of PD,28,46 which probably represents a local compensatory mechanism, directed to dampen an excessive excitability of striatopallidal neurons. Drugs targeting the mGluR5 might providenew approaches by selectively reducing glutamate transmission in the areas where it is abnormally enhanced. While current surgical approaches in PD aimed at reducing or interrupting transmission in the indirect pathway are quite effective, they are invasive and expensive, and mGluR5 antagonists could provide an alternative approach.
In addition, we and others have found enhanced mGluR5 expression in several brain areas related to the indirect pathway in models of L-3,4- dihydrophenylalanine (L-DOPA)-induced dyskinesias, and some studies have shown promising therapeutic results after using mGluR5 antagonists.15,49 At present is unclear whether pharmacologicalnormalization of DA levels in PD patients is capable of modifying the adaptive post-synaptic changes and enhancement of mGluR5. If changes in striatal mGluR5 are sustained or evolve independently, it may be necessary to target specifically this pathway in order to obtain full reversal of PD symptomatology and L-DOPA-induced dyskinesias.
Lastly, the possibility of modifying the progression of PD with these compounds is appealing, but more work needs to be undertaken, with more selective modulators, to validate mGluR5s in the basal ganglia circuitry as targets for neuroprotective purposes.