Biased mGlu5-Positive Allosteric Modulators Provide In Vivo Efficacy without Potentiating mGlu5 Modulation of NMDAR Currents

Abstract

Schizophrenia is associated with disruptions in N-methyl-D-aspartate glutamate receptor subtype (NMDAR)-mediated excitatory synaptic signaling. The metabotropic glutamate receptor subtype 5 (mGlu5) is a closely associated signaling partner with NMDARs and regulates NMDAR function in forebrain regions implicated in the pathology of schizophrenia. Efficacy of mGlu5 positive allosteric modulators (PAMs) in animal models of psychosis and cognition was previously attributed to potentiation of NMDAR function. To directly test this hypothesis, we identified VU0409551 as a novel mGlu5 PAM that exhibits distinct stimulus bias and selectively potentiates mGlu5 coupling to Gαq-mediated signaling but not mGlu5 modulation of NMDAR currents or NMDAR-dependent synaptic plasticity in the rat hippocampus. Interestingly, VU0409551 produced robust antipsychotic-like and cognition-enhancing activity in animal models. These data provide surprising new mechanistic insights into the actions of mGlu5 PAMs and suggest that modulation of NMDAR currents is not critical for in vivo efficacy. VIDEO ABSTRACT.

Figure 1. VU0409551 is a potent mGlu₅ PAM in HEK293A-mGlu₅ rat cells and native systems

(A) Representative raw calcium traces following the addition of 30 μM VU0409551 and the subsequent additions of EC₂₀ and EC₈₀ concentrations of glutamate. (B) VU0409551 potentiates an EC₂₀ concentration of glutamate with a potency of 235 nM in mGlu₅-expressing R10A rat cells (C) VU0409551 shifts the glutamate concentration response curve with a maximum fold shift of 11 at 30 μM (D) VU0409551 did not shift the agonist concentration response curve in mGlu_{1,2,3,4,6,7,8}-expressing cells, displaying its high selectivity for mGlu₅. (E) VU0409551 potentiates glutamate-stimulated ERK1/2 phosphorylation and exhibits robust agonist activity in this assay. (F) VU0409551 potentiates glutamate-induced calcium mobilization in rat cortical astrocytes with a potency of 317 nM and maximum glutamate response of 79.5%, which is blocked by addition of 10 μM of the mGlu₅ antagonist MPEP. Data represent mean ± S.E.M. from 2-4 independent experiments performed in duplicate or triplicate.

Figure 2. VU0409551 potentiates NMDAR-independent synaptic plasticity in the hippocampus

(A-B) VU0409551 potentiates DHPG-induced long term depression (LTD). (A) Bath application of 75 μM DHPG for 10 min (open circle, solid line) resulted in LTD of the fEPSP slope. In contrast, bath application of 25 μM DHPG for 10 min (gray circles, solid line) resulted in a slight decrease in fEPSP slope 55 min after compound washout. Application of 10 μM VU0409551 for 10 min (dashed line), first alone and then in combination with 25 μM DHPG (solid line) for 10 min (black circles), resulted in a significant LTD in fEPSP slope (p = 0.0005, n = 6 - 7). Inset shows representative fEPSP traces for each condition for baseline (black trace) and 55 min after compound washout (gray trace). (B) Quantification of the change in fEPSP slope measured 55 min after compound washout. Data represent mean ± S.E.M. ** p < 0.01, *** p < 0.001 when compared with 25 μM DHPG.

Figure 3. Differential effects of mGlu₅ PAMs on potentiation of NMDAR currents in CA1 pyramidal cells of the hippocampus

(A-C) DHPG induces a concentration-dependent increase in NMDAR currents. Top: representative traces of NMDA-evoked currents in hippocampal CA1 pyramidal cells and Bottom: time courses of normalized NMDAR current amplitude before, during and after application of DHPG at different concentrations. (D-E) Top: representative traces of NMDA-evoked currents and Bottom: time courses of normalized NMDAR current amplitude in control, during applications of mGlu₅ PAM alone and a combination of mGlu₅ PAM and DHPG (3 μM), and after washout of the compounds. (D) The mGlu₅ PAM 5PAM523 (20 μM) potentiates the effect of DHPG on NMDAR currents. (E) VU0409551 (10 μM), a highly selective mGlu₅ PAM, does not significantly potentiate NMDAR currents. (F) Summary of DHPG and mGlu₅ PAMs (p < 0.05 n = 5 - 8) alone and in the presence of 3 μM DHPG on NMDAR currents. (G-J) VU0409551 does not have a significant effect on the EPSC_NMDA/EPSC_AMPA ratio at SC-CA1 synapses. (G-H) Representative traces of EPSCs_AMPA and EPSCs_NMDA at holding potentials of −60 mV and +40 mV in controls, during application of 3 μM DHPG, 10 μM VU0409551, or co-application of VU0409551 and DHPG, respectively. Traces in right panels in G and H are scaled to the peak of AMPAR-EPSCs and superimposed, demonstrating that VU0409551 did not potentiate the EPSC_NMDA/EPSC_AMPA ratio when applied alone or co-applied with DHPG. (I-J) Summary of the effects of 3 μM DHPG, 10 μM VU0409551, and 10 μM VU0409551 along with 3 μM DHPG on EPSC_AMPA amplitude and the EPSC_NMDA/EPSC_AMPA ratio, respectively (n = 4 - 6). Data represent mean ± S.E.M., * p < 0.05, ** p < 0.005.

Figure 4. In contrast to other mGlu₅ PAMs, VU0409551 does not potentiate NMDAR-mediated hippocampal synaptic plasticity

(A) VU0409551 does not potentiate NMDAR-mediated, theta burst stimulation-induced long term potentiation (LTP). Saturation TBS resulted in a significant increase in fEPSP slope 35 min after stimulation. Threshold TBS resulted in a small increase in fEPSP slope measured 35 min after stimulation. Bath application of 10 μM VU0409551 for 20 min, followed by threshold TBS, resulted in no change in fEPSP slope from threshold TBS alone. In contrast, bath application of 1 μM VU0092273 for 20 min, followed by threshold TBS, resulted in a significant enhancement of fEPSP slope measured 35 min after stimulation. Inset shows representative fEPSP traces for each condition for baseline (black trace) and 35 min after TBS (gray trace). (B) Quantification of the change in fEPSP slope measured 35 min after TBS stimulation. In contrast to VU0092273, bath application of VU0409551 for 20 min followed by threshold TBS did not result in significant LTP 35 min after stimulation (p = 0.0002, n = 6 - 11). Data represent mean ± S.E.M. ** p < 0.01, when compared with threshold TBS.

Figure 5. VU0409551 displays wake-promoting effects in vivo

Sleep-wake electroencephalography (EEG) studies demonstrate that VU0409551 (10 or 60 mg/kg), administered at the 2^nd hr of the light cycle, dose-dependently decreases the percent time spent asleep (p < 0.0001, n=8). Sleep polygraphic variables were measured in rats for 24 hr beginning at the start of the light cycle. Data represent mean ± S.E.M.. * p < 0.05.

Figure 6. VU0409551 demonstrates antipsychotic-like activity and cognition enhancement in rodent behavioral models

(A) VU0409551 (10 – 100 mg/kg) reverses MK-801-induced hyperlocomotion in rats with a minimum effective dose of 10 mg/kg and maximum reversal of 49.7% at 100 mg/kg. Data are expressed as the mean total number of beam breaks per 5-min intervals ± S.E.M.. Area under the curve from time interval t = 60 to 120 min was quantified for each treatment group and expressed as total ambulations. Percent reversal was calculated after normalizing to the vehicle + MK-801 treatment group. (p < 0.0001, n = 10 - 12). (B) VU0409551 dose-dependently enhances acquisition of contextual fear conditioning in rats. Pretreatment with 1, 3, 5.6 and 10 mg/kg VU0409551 prior to conditioning significantly enhanced acquisition of fear conditioning assessed 24 hr later (p = 0.0005, n = 10-13). Data are expressed as mean ± S.E.M. *** p < 0.001, ** p < 0.01 and * p < 0.05. (C) VU0409551 dose-dependently enhances recognition memory in rats. Pretreatment with 3 and 10 mg/kg VU0409551 prior to exposure to identical objects significantly enhanced recognition memory assessed 24 hr later (p = 0.0014, n = 19). Data are expressed as mean ± S.E.M.. *** p < 0.001 and * p < 0.05. (D) VU0409551 demonstrates pro-cognitive effects in the delayed non-matching to position task in rats. Pretreatment with VU0409551 (10 – 60 mg/kg) prior to testing significantly increased the proportion of correct responses at 10 sec and 20 sec delays, respectively (p = 0.0236, n = 15 – 35). Data are expressed as mean ± S.E.M. * p < 0.05.

Figure 7. Repeat administration of VU0409551 does not result in positive Fluoro-Jade C staining in the rat brain

(A-B) mGlu₅ ago-PAM VU0424465 (3 mg/kg) induces positive Fluoro-Jade C (FJC) staining in the auditory cortex 24 hr following acute dosing. Unlike VU0424465, VU0409551 does not induce neurotoxicity in rats at doses above those required for robust in vivo efficacy in behavioral assays as demonstrated by negative FJC staining following repeat dosing of 120 mg/kg once daily for four consecutive days (p < 0.0001, n = 3). Data represent mean ± S.E.M. *** p < 0.0001, when compared with VU0409551. (C) mGlu₅ PAMs VU0424465 (3 mg/kg) and PAM 5PAM523 (56.6 mg/kg) induce robust behavioral convulsions as measured by the modified Racine scale (0-5). VU0409551 is void of seizure activity following acute or repeat dosing (120 mg/kg) once daily for four consecutive days. Data represent mean ± S.E.M.