N-{4-Chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl}-2-hydroxybenzamide (CPPHA) Acts through a Novel Site as a Positive Allosteric Modulator of Group 1 Metabotropic Glutamate Receptors

Abstract

Recent studies suggest that a novel positive allosteric modulator (PAM) of the metabotropic glutamate receptor (mGluRs), mGluR5, termed 4-nitro-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (VU-29), potentiates mGluR5 responses by actions at a site that is overlapping with the binding site of 2-methyl-6-(phenylethynyl)pyridine (MPEP), a previously identified negative allosteric modulator of this receptor. It is interesting that a structurally distinct PAM, N-{4-Chloro-2- [(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl}-2-hydroxybenzamide (CPPHA), does not to bind to the MPEP site. We now report that CPPHA potentiates mGluR5 responses by a mechanism that is distinct from that of VU-29. VU-29-and CPPHA-induced potentiation of mGluR5 responses are blocked by a neutral ligand at the MPEP allosteric site termed 5-methyl-2-(phenylethynyl)pyridine (5MPEP). However, increasing concentrations of 5MPEP induce parallel rightward shifts in the VU-29 concentration-response curve, whereas 5MPEP inhibits CPPHA potentiation in a noncompetitive manner. Consistent with this, a mutation (A809V/mGluR5) that reduces binding of ligands to the MPEP site eliminates the effect of VU-29 but has no effect on the response to CPPHA. On the other hand, a mutation (F585I/mGluRs) that eliminates the effect of CPPHA does not alter the response to VU-29. CPPHA is also a PAM at mGluR1. It is interesting that the corresponding mutation of F585I/mGluR5 in mGluR1 (F599I/ mGluR1) eliminates CPPHA's effect without altering the potentiation of a known PAM of mGluR1, (S)-2-(4-fluorophenyl)-1-(toluene-4-sulfonyl)pyrrolidine (Ro 67-7476). Likewise, another mutation (V757L/mGluR1) that abolishes potentiation of Ro 67-7476 has no effect on CPPHA. Finally, CPPHA does not displace binding of a radioligand for the mGluR1 allosteric antagonist characterized previously. Together, these data suggest that CPPHA acts at a novel allosteric site on both mGluR1 and -5 to potentiate responses to activation of these receptors.Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. In addition to eliciting fast excitatory synaptic responses, glutamate has neuromodulatory effects by activation of G protein-coupled receptors (GPCRs) termed metabotropic glutamate receptors (mGluRs). There are eight subtypes of mGluRs divided into three groups. Group I mGluRs (mGluR1 and mGluR5) couple to G␣ q/11 and activate phospholipase C. Group II (mGluR2 and mGluR3) and group III (mGluR4, mGluR6, mGluR7, and mGluR8) mGluRs couple to the inhibition of adenylyl cyclase and other effectors through G␣ i/o (Conn and Pin,

Overlapping binding sites drive allosteric agonism and positive cooperativity in type 4 metabotropic glutamate receptors

“…The existence of multiple allosteric sites in the 7TM of mGluRs has been proposed, notably in mGlu 5 (12,52). For example, the 2 mGlu 5 PAMs, CPPHA and VU0357121, do not displace MPEP, contrary to other mGlu 5 PAMs such as VU29, for example (53,54). Interestingly, the pocket in which VU0415374 is binding in mGlu 4 7TM seems to be homologous to the binding pocket of DFB, an mGlu 5 PAM that has been described to occupy an overlapping but distinct binding site to MPEP in mGlu 5 (55).…”

A Novel Metabotropic Glutamate Receptor 5 Positive Allosteric Modulator Acts at a Unique Site and Confers Stimulus Bias to mGlu5 Signaling

“…However, at least two mGlu 5 PAMs, CPPHA (N-(4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl] phenyl)-2-hydroxybenzamide) (O'Brien et al, 2004;Zhao et al, 2007;Chen et al, 2008) and VU0357121 (Hammond et al, 2010), have been identified that interact noncompetitively with the MPEP site. Of interest, Zhang et al (2005) reported that CPPHA and an MPEP site mGlu 5 PAM, DFB (difluorobenzaldazine), induce similar potentiation of mGlu 5 -mediated calcium mobilization in cortical astrocytes.…”

Structure and ligand recognition of class C GPCRs

“…The PAMs stabilize the active conformation of this group by facilitating the movement of a conserved Trp in TM6, whereas the NAMs prevent the relative movement between TM6 and TM3 [66] . For the mGlu5 receptor, most PAMs and NAMs share an overlapping binding pocket that is composed of TM3, 5, 6, and 7 [73] , except for a small number of distinct sites [74] . For the mGlu1 receptor, however, the PAMs and NAMs bind to distinct sites in the 7TM [64,65,[75][76][77] , except for a shared site that consists of Val757 in TM3 [65,77] .…”

“…An unique PAM for both the mGlu1 and mGlu5 receptors, CPPHA, was shown to bind to this site. Phe585 in TM1 of mGlu5 (Phe599 at the corresponding position in mGlu1) is essential for the recognition of CPPHA [74] .…”