A Novel Selective Allosteric Modulator Potentiates the Activity of Native Metabotropic Glutamate Receptor Subtype 5 in Rat Forebrain

Abstract

We found that N- {4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl}-2-hydroxybenzamide (CPPHA), is a potent and selective positive allosteric modulator of the metabotropic glutamate receptor subtype 5 (mGluR5). CPPHA alone had no agonist activity and acted as a selective positive allosteric modulator of human and rat mGluR5. CPPHA potentiated threshold responses to glutamate in fluorometric Ca 2ϩ assays 7-to 8-fold with EC 50 values in the 400 to 800 nM range, and at 10 M shifted mGluR5 agonist concentration-response curves to glutamate, quisqualate, and (R,S)-3,5-dihydroxyphenylglycine (DHPG) 4-to 7-fold to the left. The only effect of CPPHA on other mGluRs was weak inhibition of mGluR4 and 8. Neither CPPHA nor the previously described 3,3Ј-difluorobenzaldazine (DFB) affected [ 3 H]quisqualate binding to mGluR5, but although DFB partially competed for [ 3 H]3-methoxy-5-(2-pyridinylethynyl)pyridine binding, CPPHA had no effect on the binding of this 2-methyl-6-(phenylethynyl)-pyridine analog to mGluR5. Although the binding sites for the two classes of allosteric modulators seem to be different, these different allosteric sites can modulate functionally and mechanistically similar allosteric effects. In electrophysiological studies of brain slice preparations, it had been previously shown that activation of mGluR5 receptors by agonists increased N-methyl-D-aspartate (NMDA) receptor currents in the CA1 region of hippocampal slices. We found that CPPHA (10 M) potentiated NMDA receptor currents in hippocampal slices induced by threshold levels of DHPG, whereas having no effect on these currents by itself. Similarly, 10 M CPPHA also potentiated mGluR5-mediated DHPG-induced depolarization of rat subthalamic nucleus neurons. These results demonstrate that allosteric potentiation of mGluR5 increases the effect of threshold agonist concentrations in native systems.Metabotropic glutamate receptors (mGluRs) are G proteincoupled receptors (GPCRs) that bind glutamate to modulate neurotransmitter release or postsynaptic excitatory neurotransmission, and hence they modulate the strength of synaptic transmission. The mGluRs are members of GPCR family C and possess a large extracellular agonist binding domain in the amino-terminal portion of the receptor. This agonist binding domain distinguishes family C from the other GPCR families in which the agonist binding sites are associated with the seven-strand transmembrane spanning region or with the extracellular loops that connect the strands of this region. Thus, in the mGluRs, interaction of the agonist with the transmembrane domains is thought to be indirect (O'Hara et al., 1993; for reviews, see Conn and Pin, 1997;Bockaert and Pin, 1999).Article, publication date, and citation information can be found at

Progress toward Positive Allosteric Modulators of the Metabotropic Glutamate Receptor Subtype 5 (mGlu5)

“…Interestingly, unlike other mGlu 5 PAMs characterized to date, CPPHA appears to elicit receptor activation through a novel allosteric site that does not appear to directly involve the MPEP site. 17,26,43,44 Radioligand tools, either as negative allosteric modulators (NAMs), PAMs, or silent allosteric modulators (SAMs), are currently unavailable for the CPPHA site, and the potential impact of an appropriate in vivo tool compound from the CPPHA series is still lacking and thus leaves an important remaining question regarding potential in vivo efficacy for PAMs which modulate their activity outside the MPEP site. Interestingly, examination of DFB and CPPHA in independent signaling mechanisms (ERK1/2 phosphorylation and calcium mobilization) demonstrated that differential PAM signaling is possible, supporting the notion that PAMs which engage distinct allosteric binding sites and presumably unique modulator-receptor conformations can differentially facilitate downstream signaling responses.…”

“…42 Following DFB, N-[5-chloro-2-[(1,3-dioxoisoindolin-2-yl)methyl]phenyl]-2-hydroxybenzamide (CPPHA, 4) was disclosed as a much more potent mGlu 5 PAM. 17 The CPPHA benzamide series of PAMs was characterized as having a shallow structureÀactivity relationship (SAR), poor physicochemical properties, and limited activity at the rat mGlu 5 receptor and was therefore not utilized in vivo. Interestingly, unlike other mGlu 5 PAMs characterized to date, CPPHA appears to elicit receptor activation through a novel allosteric site that does not appear to directly involve the MPEP site.…”

“…Group I mGlus are predominantly expressed postsynaptically in the CNS, whereas group II and III receptors are located primarily presynaptically where they control neurotransmitter release. 10À12 Group I selective orthosteric agonists 13À16 and later PAMs 17 of the Group I mGlu receptors, and in particular mGlu 5 , demonstrated a unique ability to potentiate NMDA receptor currents in vitro in a highly specific manner. These studies in conjunction with the emerging "NMDA receptor hypofunction" hypothesis as a concept to account for the major symptom clusters of schizophrenia came to light in the mid-1990s 18À22 and provided the groundwork for a highly testable and novel hypothesis for this complex disorder which led the way to multiple strategies to enhance glutamatergic transmission.…”

Metabotropic glutamate receptor 5 as a potential target for smoking cessation

“…mGluR5 receptors regulate the activation of glutamate NMDA receptors acting as an enhancer of NMDA-activated currents and increased phosphorylation of the receptor through intracellular signalling MAPK/ERK and calcium responsive element binding (O'Brien et al 2004;Liu et al 2007) via a Src-family tyrosine kinase (Kotecha et al 2003).…”

Positive Allosteric Modulation of Metabotropic Glutamate 5 (mGlu5) Receptors Reverses N-Methyl-D-Aspartate Antagonist-Induced Alteration of Neuronal Firing in Prefrontal Cortex

“…The worsening of the detrimental effects of NMDA receptor antagonists by mGlu5 receptor antagonist suggests that activation of mGlu5 receptors may represent a plausible approach for ameliorating symptoms of schizophrenia (Marino and Conn 2002;Moghaddam 2004). Recent behavioral studies using 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB), a positive allosteric modulator of the mGlu5 receptor (Kinney et al 2005b;O'Brien et al 2004), show that pretreatment with this compound reverses amphetamine-induced hyperlocomotion and prepulse inhibition deficit, two tests which serve to model some aspects of schizophrenia in rodents (Kinney et al 2005a). …”