Unique signaling profiles of positive allosteric modulators of metabotropic glutamate receptor subtype 5 determine differences in in vivo activity

Abstract

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.

Figure 1

VU0424465, an mGlu₅ positive allosteric modulator optimized for direct agonist activity, induces calcium mobilization in the absence of glutamate. (A) Representative raw calcium traces following the addition of 30 μM VU0424465 (compound; 3–143 seconds; agonist) and followed by an EC₂₀ of glutamate (143–240 s); PAM) and EC₈₀ of glutamate (240–350 seconds); control trace represents the response to an EC₂₀ and EC₈₀ of glutamate following the addition of vehicle alone. Static ratio represents relative fluorescence units normalized to initial values. VU0424465, but not VU0361747, clearly induces a concentration-dependent release of calcium when added alone. Potencies were determined by adding a concentration-response curve of VU0424465 (B) followed by an EC₂₀ of glutamate (PAM) or (C) in the absence of glutamate (agonist). VU0424465 potentiated the calcium mobilization evoked by glutamate in a concentration-dependence manner. The PAM EC₅₀ of VU0424465 is 1.5 nM, while the agonist potency is 171 nM with a 65% maximum glutamate response. Data represent the mean ± S.E.M. of 5 independent experiments performed in duplicate. For these studies, rat mGlu₅-expressing cell lines were utilized for full in vitro characterization of novel compounds to correlate in vitro data to in vivo studies in rats; similar potency/efficacy profiles were observed in human mGlu₅-expressing cell lines.

Figure 2

VU0424465-induced agonist activity is mediated via the mGlu₅ allosteric MPEP site. (A) VU0424465-induced calcium mobilization was not inhibited by 1 mM LY341495, an mGlu orthosteric antagonist (mGlu₅ IC₅₀ 8.2 μM). Increasing concentrations of 5MPEP, a neutral MPEP site ligand, induced a parallel rightward shift in the VU0424465 agonist concentration-response curve with no effect on the maximum response, suggesting a competitive interaction of VU0424465 with the allosteric mGlu₅ MPEP site. (B) Multiple single point mutations that eliminate calcium mobilization by the orthosteric agonists, glutamate and DHPG, did not abolish VU0424465-induced agonist activity. (C) Introduction of the single point mutation A809V, which abolishes activity at the mGlu₅ allosteric MPEP-site, eliminated agonist activity evoked by VU0424465. This mutation had no effect on orthosteric agonist activity of glutamate or DHPG (data not shown). The single point mutation, F585I, known to inhibit activity of the allosteric PAM CPPHA, had no effect on VU0424465 agonist activity. Data represent the mean ± S.E.M. of 3 independent experiments performed in duplicate.

Figure 3

VU0424465 demonstrates true agonist activity in native systems. (A) Bath application of 10 μM VU0424465 (solid line) for 10 minutes resulted in the induction of LTD measured 55 minutes after compound washout (n=6). (B) Maximum suppression of the fEPSP response measured as percent baseline. DHPG and VU0361747 were quantified from Noetzel et. al., 2012 in the absence of glutamate (agonist) (9). Data represent mean ± S.E.M.

Figure 4

The mGlu₅ agonist-PAM VU0424465, but not the pure PAM VU0361747, induces epileptiform activity in CA3 in the hippocampal formation. Inter-event interval and amplitude of spontaneous firing were measured using field recordings in CA3 of the hippocampal formation. (A) Application of 50 μM DHPG or 10 μM VU0424465 for 10 minutes induced a robust decrease in the inter-event interval of spontaneous firing while having no significant effect on the amplitude (n=8). In contrast, application of 10 μM VU0361747 had no effect on either the inter-event interval or amplitude of spontaneous firing (n=8). In the presence of 10 μM MTEP, 10 μM VU0424465 had no effect on the inter-event interval or amplitude of spontaneous firing (n=7). Error bars represent S.E.M. (B) Sample traces taken pre-drug (control) and after administration of compound. (C) Cumulative probability plots for the compounds of interest.

Figure 5

The mGlu₅ agonist-PAM VU0424465, but not the pure PAM VU0361747, induces dose-dependent increases in behavioral convulsions. Rats treated with increasing doses of VU0424465 (0.1 – 10 mg/kg) exhibited dose-dependent seizure activity as measured by the modified Racine scale (0–5). Rats receiving a high dose of VU0361747 (56.6 mg/kg) did not display observable behavioral seizure activity. Compounds were diluted in 10% Tween 80 (pH 7.0, 1 mg/3 ml) and injected intraperitoneally. Data represent mean ± S.E.M (n=5).

Figure 6

Representative cortical EEG recordings from (A) vehicle, (B) VU0424465 and (C) VU0361747-treated rats demonstrate that only the agonist-PAM VU0424465 induces epileptiform activity. (1–3) Compounds were diluted in 10% Tween 80 (pH 7.0, 1 mg/3 ml) and injected intraperitoneally. Traces below are temporally expanded portions of the EEG recording located within the boxes. Both behavioral and electrographic seizures were observed in VU0424465-treated animals (n=4). Seizure activity was quantified by the analysis of the rate of EEG spiking. The * denotes the time of drug administration. The arrow indicates the occurrence of a behavioral seizure with no EEG correlate. The # designates changes in the EEG due to movement artifact. (D) Comparison of spike rate (mean + 95% CI) between vehicle and VU0424452-treated animals revealed that after 4 h, EEG spiking greatly increased in the VU0424465-treated animals. (E) Comparison of spike rate (mean + 95% CI) showed that no difference in EEG spiking between vehicle and VU00361747-treated animals was observed (n=4). ^ p < 0.05, two-tailed t-test.