A novel metabotropic glutamate receptor 5 positive allosteric modulator acts at a unique site and confers stimulus bias to mGlu5 signaling

Abstract

Metabotropic glutamate receptor 5 (mGlu5) is a target for the treatment of central nervous system (CNS) disorders, such as schizophrenia and Alzheimer's disease. Furthermore, mGlu5 has been shown to play an important role in hippocampal synaptic plasticity, specifically in long-term depression (LTD) and long-term potentiation (LTP), which is thought to be involved in cognition. Multiple mGlu5-positive allosteric modulators (PAMs) have been developed from a variety of different scaffolds. Previous work has extensively characterized a common allosteric site on mGlu5, termed the MPEP (2-Methyl-6-(phenylethynyl)pyridine) binding site. However, one mGlu5 PAM, CPPHA (N-(4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl)-2-hydroxybenzamide), interacts with a separate allosteric site on mGlu5. Using cell-based assays and brain slice preparations, we characterized the interaction of a potent and efficacious mGlu5 PAM from the CPPHA series termed NCFP (N-(4-chloro-2-((4-fluoro-1,3-dioxoisoindolin-2-yl)methyl)phenyl)picolinamide). NCFP binds to the CPPHA site on mGlu5 and potentiates mGlu5-mediated responses in both recombinant and native systems. However, NCFP provides greater mGlu5 subtype selectivity than does CPPHA, making it more suitable for studies of effects on mGlu5 in CNS preparations. Of interest, NCFP does not potentiate responses involved in hippocampal synaptic plasticity (LTD/LTP), setting it apart from other previously characterized MPEP site PAMs. This suggests that although mGlu5 PAMs may have similar responses in some systems, they can induce differential effects on mGlu5-mediated physiologic responses in the CNS. Such stimulus bias by mGlu5 PAMs may complicate drug discovery efforts but would also allow for specifically tailored therapies, if pharmacological biases can be attributed to different therapeutic outcomes.

Fig. 1.

NCFP potentiates the response to glutamate in a manner similar to that of CPPHA. (A) Structures of CPPHA and NCFP. (B) The potencies of CPPHA (black squares) and NCPF (open triangles) were determined by adding increasing concentrations of each PAM to HEK293 mGlu₅ cells 60 seconds before the addition of a concentration of glutamate eliciting a 20% maximal response (EC₂₀, 40–60 nM). The calcium response was normalized to the response induced by a maximally effective concentration of glutamate (10 µM). (C) Potencies of the compounds were determined as in B, except rat cortical astrocytes were used. (EC₂₀, 600–650 nM). (D and E) Progressive fold shift values were determined by treating HEK293 mGlu₅ cells with fixed concentrations (300 nM, black triangles, 1 µM black circle, 3 µM open square or 10 µM open triangle) of CPPHA (D) or NCFP (E), followed by the addition of a concentration response curve to glutamate. Calcium responses were normalized to the response induced by a maximally effective concentration of glutamate (10 µM). (F) The level of ERK1/2 phosphorylation (fold/basal) was determined by treating HEK293 mGlu₅ cells with a fixed concentration of mGlu₅ compounds (3 µM; control black square, NCFP open triangle, VU0092273 black circle) or glutamate (1 mM; black diamond) for the times indicated. Data represent the mean ± S.E.M. of 3–4 independent experiments performed in duplicate.

Fig. 2.

NCFP does not inhibit [³H]methoxyPEPy binding in cells expressing high levels of mGlu₅. Cells expressing high levels of mGlu₅ were treated with increasing concentrations of NCFP (open triangles), CPPHA (black squares), or MPEP (black circles) and 2 nM [³H]methoxyPEPy. Reactions were allowed to incubate for 1 hour before termination. Nonspecific binding was determined using 10 µM MPEP. Data represent the mean ± S.E.M. of three individual experiments conducted in duplicate.

Fig. 3.

NCFP activity is blocked by a mutation in the CPPHA site, but not affected by an MPEP site ligand. (A) Cells expressing the F585I mutant mGlu₅ receptor were treated with a fixed concentration (3 µM) of NCFP (open triangles) or CPPHA (black triangles), followed by the addition of increasing concentrations of glutamate. (B) mGlu₅ cells were first treated with fixed concentrations of 5MPEP (3 µM black triangles, 10 µM black circles or 30 µM black diamonds); then, increasing concentrations of NCFP were added, and finally an EC₂₀ concentration of glutamate was applied. In all cases, the calcium response was normalized to the response induced by a maximally effective concentration of glutamate (10µM). Data represent the mean ± S.E.M. of four independent experiments performed in duplicate.

Fig. 4.

NCFP inhibits [³H]methoxyPEPy binding in a manner suggestive of a noncompetitive interaction. (A) Cell membranes from HEK293A cells expressing a low level of mGlu₅ were treated with increasing concentrations of CPPHA (black squares), NCFP (open triangles), or MPEP (black circles) and 2 nM [³H]methoxyPEPy. Reactions were allowed to incubate for 1 hour before termination. Nonspecific binding was determined using 10 µM MPEP. (B) F585I cell membranes were treated the same as in (A). Data represent the mean ± S.E.M. of 3–7 independent experiments performed in duplicate or triplicate.

Fig. 5.

NCFP acts differently at the N-terminal–truncated receptor, compared with MPEP- site ligands. (A) Cells expressing the N-terminal–truncated mGlu₅ receptor were treated with increasing concentrations of NCFP (open triangles) or VU0092273 (black diamonds). The calcium response was normalized to the response induced by a maximally effective concentration of ionomycin (1 µM). (B) The level of ERK1/2 phosphorylation (fold/basal) was determined by treating cells with a fixed concentration of mGlu₅ compound (3 µM; NCFP open triangles, VU0092273 black diamonds), FBS (10%, black triangles), or control (black squares) for the times indicated. (C) Cell membranes were treated with increasing concentrations of NCFP (open triangles), VU0092273 (black diamonds), or MPEP (black circles) and 2 nM [³H]methoxyPEPy. Reactions were allowed to incubate for 1 hour before termination. Nonspecific binding was determined using 10 µM MPEP. Data represent the mean ± S.E.M. of 3–6 independent experiments conducted in duplicate.

Fig. 6.

NCFP potentiates the DHPG-induced change in membrane voltage in STN neurons. Bath application of 100 µM DHPG induced a robust change in membrane voltage, whereas application of 1 µM DHPG induced a small change in membrane voltage. Application of 10 µM NCFP for 10 minutes had no effect on membrane voltage alone. Co-application of 10 µM NCFP and 1 µM DHPG resulted in a significant enhancement in the change in membrane voltage, compared with 1µM DHPG alone. Data represent the mean ± S.E.M. for five individual experiments for each treatment. * P < 0.5, when compared with 1µM DHPG. Sample traces from individual experiments are presented on the right.

Fig. 7.

NCFP does not significantly enhance DHPG-induced LTD at the Schaffer collateral-CA1 synapse in hippocampus. A stimulus intensity that produced 50–60% of the maximal fEPSP response was used as the baseline response and was determined for each individual experiment. Insets are sample fEPSP traces measured pre-drug (black) and 55 minutes after drug washout (gray). (A) Bath application of 75 µM DHPG for 10 minutes (open circle, solid line) resulted in LTD of the fEPSP slope (n = 8). In contrast, bath application of 25 µM DHPG for 10 minutes (black circles, solid line) resulted in a slight decrease in fEPSP slope 55 minutes after compound washout (n = 9). Application of 10 µM NCFP for 10 minutes (dashed line), first alone and then in combination with 25 µM DHPG (solid line) for 10 minutes (gray circles), resulted in no significant change in fEPSP slope (n = 7). (B) Quantification of the change in fEPSP slope measured 55 minutes after compound washout. Error bars represent S.E.M. * P < 0.5, when compared with control (25 µM DHPG).

Fig. 8.

NCFP has no effect on potentiation of threshold TBS LTP at the Schaffer collateral-CA1 synapse in hippocampus. A stimulus intensity that produced 40–50% of the maximal fEPSP response was used as the baseline response and was determined for each individual experiment. Insets are sample fEPSP traces measured pre-drug (black) and 35 minutes after TBS stimulation for LTP (gray). (A) Threshold TBS (black circles) resulted in a small increase in fEPSP slope measured 35 minutes after stimulation (n = 11). Bath application of 10 µM NCFP for 20 minutes (solid line), followed by threshold TBS (light gray circles), resulted in no change in fEPSP slope from threshold TBS alone (n = 8). In contrast, bath application of 1 µM VU0092273 for 20 minutes (solid line), followed by threshold TBS (dark gray circles), resulted in a significant enhancement of fEPSP slope measured 35 minutes after stimulation (n = 9). (B) Quantification of the change in fEPSP slope measured 35 minutes after TBS stimulation. Error bars represent S.E.M. * P < 0.5, when compared with control (TBS threshold).