Unique Signaling Profiles of Positive Allosteric Modulators of Metabotropic Glutamate Receptor Subtype 5 Determine Differences in In Vivo Activity

doi:10.1016/j.biopsych.2012.09.012

Biological Psychiatry

Volume 73, Issue 6, 15 March 2013, Pages 501-509

https://doi.org/10.1016/j.biopsych.2012.09.012 Get rights and content

Background

Metabotropic glutamate receptor subtype 5 (mGlu₅) activators have emerged as a novel approach to the treatment of schizophrenia. Positive allosteric modulators (PAMs) of mGlu₅ have generated tremendous excitement and fueled major drug discovery efforts. Although mGlu₅ PAMs have robust efficacy in preclinical models of schizophrenia, preliminary reports suggest that these compounds may induce seizure activity. Prototypical mGlu₅ PAMs do not activate mGlu₅ directly but selectively potentiate activation of mGlu₅ by glutamate. This mechanism may be critical to maintaining normal activity-dependence of mGlu₅ activation and achieving optimal in vivo effects.

Methods

Using specially engineered mGlu₅ cell lines incorporating point mutations within the allosteric and orthosteric binding sites, as well as brain slice electrophysiology and in vivo electroencephalography and behavioral pharmacology, we found that some mGlu₅ PAMs have intrinsic allosteric agonist activity in the absence of glutamate.

Results

Both in vitro mutagenesis and in vivo pharmacology studies demonstrate that VU0422465 is an agonist PAM that induces epileptiform activity and behavioral convulsions in rodents. In contrast, VU0361747, an mGlu₅ PAMs optimized to eliminate allosteric agonist activity, has robust in vivo efficacy and does not induce adverse effects at doses that yield high brain concentrations.

Conclusions

Loss of the absolute dependence of mGlu₅ PAMs on glutamate release for their activity can lead to severe adverse effects. The finding that closely related mGlu₅ PAMs can differ in their intrinsic agonist activity provides critical new insights that is essential for advancing these molecules through clinical development for treatment of schizophrenia.

Section snippets

Materials

Dulbecco’s Modified Eagle’s Medium, fetal bovine serum, and antibiotics were purchased from Invitrogen (Carlsbad, California). Dihydroxyphenylglycine (DHPG) and LY341495 were purchased from Ascent Scientific (Bristol, United Kingdom) and Tocris (Ellisville, Missouri), respectively. VU0422465 (18), VU0361747 (10), 5-methyl-6-(phenylethynyl)-pyridine (5MPEP) (19), and 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine (MTEP) (20) were synthesized as described previously. Unless otherwise stated, all other

Optimization of mGlu₅ PAMs with Allosteric Agonist Activity

We previously reported discovery of a novel mGlu₅ PAM series, finding that some compounds behaved as pure PAMs, whereas others exhibited allosteric agonist activity 9, 10. Interestingly, this allosteric agonist activity was only observed in cell lines expressing high levels of mGlu₅ and not in lower-expressing cell lines or in native systems, suggesting that agonist activity can be an artifact of overexpression of the receptor and not indicative of physiologic effects or in vivo efficacy (9).

Discussion

Positive allosteric modulation of mGlus has emerged as an exciting new therapeutic strategy for major brain disorders, including schizophrenia, cognitive impairments, and some genetic childhood developmental disorders, and they are being rapidly advanced in drug development efforts 1, 2, 3, 4. It has been postulated that targeting allosteric sites to avoid direct activation of the receptor could lower the risk of adverse effects that may occur than with agonist-induced overstimulation. This may

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Building on this, we further examined whether the individual 7TM from mGlu5 receptor dimer can induce G-protein signaling upon PAM activation, independent of VFT/CRD orientation relative to the plane of the membrane and the 7TM dimer interface. The ago-PAM VU0424465 can activate mGlu5 (Rook et al., 2013), and here we show that this response can only be partially inhibited by saturating concentrations of the competitive antagonist LY341495 that prevents VFT closure (Figure 5A; Table S8). Previously, it has been reported that the open VFTs can still move to reach the active orientation, as illustrated by the crystal structure of the mGlu1 VFT dimer with bound LY341495 (PDB: 3KS9), we cannot exclude the possibility of ago-PAM VU0424465 inducing the active (A) state of the VFT dimer.
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The metabotropic glutamate receptor 5 (mGlu₅) is a promising therapeutic target for multiple CNS disorders. Recent mGlu₅ drug discovery has focused on targeting binding sites within the mGlu₅ 7-transmembrane domain (7TM) that are topographically distinct from that of the endogenous ligand. mGlu₅ primarily couples to G_q/11 proteins leading to mobilization of intracellular Ca²⁺ (iCa²⁺), but also activates iCa²⁺ independent signaling pathways, with biased agonism/modulation operative for multiple positive allosteric modulator (PAM) and PAM-agonist chemotypes. Although several residues within the common allosteric binding pocket are key determinants of PAM activity, how these residues affect biased modulation is unknown. The current study probed the molecular basis of mGlu₅ PAM biased modulation. Modulation of mGlu₅ activity by four chemically distinct mGlu₅ PAMs (VU0424465, DPFE, VU29 and VU0409551) was assessed across two distinct receptor endpoints (iCa²⁺ mobilization and ERK1/2 phosphorylation) at mGlu₅ receptors containing single-point mutations of allosteric binding pocket residues informed by computational modeling. Many mutations had differential effects on PAM affinity and cooperativity across signaling endpoints, resulting in gain or reversal of bias at the level of both affinity and functional cooperativity. Additionally, mutants had differential effects on functional cooperativity between the orthosteric ligands, DHPG and glutamate, and the PAMs, DPFE and VU29, but not VU0409551, indicating that probe dependence is linked to orthosteric agonists conferring activation states that differentially influence allosteric ligand-receptor interactions in a chemotype dependent fashion. Collectively, these data provide crucial insight into the residues that govern different activation states adopted by mGlu₅ in order to signal via distinct intracellular pathways when co-bound by orthosteric agonists and PAMs.

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: Authors JMR and MJN contributed equally to this work.

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Priority CommunicationUnique Signaling Profiles of Positive Allosteric Modulators of Metabotropic Glutamate Receptor Subtype 5 Determine Differences in In Vivo Activity

Background

Methods

Results

Conclusions

Section snippets

Materials

Optimization of mGlu5 PAMs with Allosteric Agonist Activity

Discussion

Trends Pharmacol Sci

Bioorg Med Chem Lett

Electroencephalogr Clin Neurophysiol

J Biol Chem

Allosteric modulators of GPCRs: A novel approach for the treatment of CNS disorders

Nat Rev Drug Discov

Mutations causing syndromic autism define an axis of synaptic pathophysiology

Nature

Toward fulfilling the promise of molecular medicine in fragile X syndrome

Annu Rev Med

mGluR5 positive allosteric modulators facilitate both hippocampal LTP and LTD and enhance spatial learning

Neuropsychopharmacology

Interaction of N-methyl-D-aspartate and group 5 metabotropic glutamate receptors on behavioral flexibility using a novel operant set-shift paradigm

Behav Pharmacol

A novel selective positive allosteric modulator of metabotropic glutamate receptor subtype 5 has in vivo activity and antipsychotic-like effects in rat behavioral models

J Pharmacol Exp Ther

ADX47273 [S-(4-fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]-oxadiazol-5-yl]-piper idin-1-yl}-methanone]: A novel metabotropic glutamate receptor 5-selective positive allosteric modulator with preclinical antipsychotic-like and procognitive activities

J Pharmacol Exp Ther

Functional impact of allosteric agonist activity of selective positive allosteric modulators of metabotropic glutamate receptor subtype 5 in regulating central nervous system function

Mol Pharmacol

Discovery of novel allosteric modulators of metabotropic glutamate receptor subtype 5 reveals chemical and functional diversity and in vivo activity in rat behavioral models of anxiolytic and antipsychotic activity

Mol Pharmacol

Priority Communication
Unique Signaling Profiles of Positive Allosteric Modulators of Metabotropic Glutamate Receptor Subtype 5 Determine Differences in In Vivo Activity

Optimization of mGlu₅ PAMs with Allosteric Agonist Activity