Molecular mechanism of positive allosteric modulation of the metabotropic glutamate receptor 2 by JNJ-46281222

Abstract

Background and purpose: Allosteric modulation of the mGlu2 receptor is a potential strategy for treatment of various neurological and psychiatric disorders. Here, we describe the in vitro characterization of the mGlu2 positive allosteric modulator (PAM) JNJ-46281222 and its radiolabelled counterpart [(3) H]-JNJ-46281222. Using this novel tool, we also describe the allosteric effect of orthosteric glutamate binding and the presence of a bound G protein on PAM binding and use computational approaches to further investigate the binding mode.

Experimental approach: We have used radioligand binding studies, functional assays, site-directed mutagenesis, homology modelling and molecular dynamics to study the binding of JNJ-46281222.

Key results: JNJ-46281222 is an mGlu2 -selective, highly potent PAM with nanomolar affinity (KD = 1.7 nM). Binding of [(3) H]-JNJ-46281222 was increased by the presence of glutamate and greatly reduced by the presence of GTP, indicating the preference for a G protein bound state of the receptor for PAM binding. Its allosteric binding site was visualized and analysed by a computational docking and molecular dynamics study. The simulations revealed amino acid movements in regions expected to be important for activation. The binding mode was supported by [(3) H]-JNJ-46281222 binding experiments on mutant receptors.

Conclusion and implications: Our results obtained with JNJ-46281222 in unlabelled and tritiated form further contribute to our understanding of mGlu2 allosteric modulation. The computational simulations and mutagenesis provide a plausible binding mode with indications of how the ligand permits allosteric activation. This study is therefore of interest for mGlu2 and class C receptor drug discovery.

Figure 1

Chemical structure of JNJ‐46281222. The position of the tritium label in [³H]‐JNJ‐46281222 is denoted by *.

Figure 2

Characterization of [³H]‐JNJ‐46281222 binding to mGlu₂ receptors stably expressed at CHO‐K1 membranes. (A) Saturation analysis of [³H]‐JNJ‐46281222 binding. A representative experiment is shown with similar data being obtained in two additional experiments. (B) Association and dissociation kinetics of 6 nM [³H]‐JNJ‐46281222 at the mGlu₂ receptor at 15°C. Association data were best fitted using a one‐phase exponential association model, whereas data for dissociation curves were best fitted using a two‐phase exponential decay model. Data are expressed as percentage of specific [³H]‐JNJ‐46281222 binding and are shown as mean ± SEM of at least four individual experiments. Where bars are not shown, SEM values are within the symbol.

Figure 3

The effects of glutamate and GTP on [³H]‐JNJ‐46281222 binding. (A) Increasing concentrations of glutamate enhance the specific binding of 6 nM [³H]‐JNJ‐46281222 to mGlu₂ receptors expressed at CHO‐K1 cell membranes, whereas GTP inhibits specific [³H]‐JNJ‐46281222 binding. (B) Effects of glutamate and GTP on homologous displacement of [³H]‐JNJ‐46281222 from mGlu₂ receptors expressed at CHO‐K1 membranes. Data are normalized to specific binding in the absence of glutamate or GTP (set at 100%). (C, D) The effect of glutamate on mGlu₂ receptor association and dissociation of [³H]‐JNJ‐46281222. Data are expressed as specific binding of the respective curve, association plateaus are fixed at 100% specific [³H]‐JNJ‐46281222 binding and dissociation plateau is fixed at 0% specific [³H]‐JNJ‐46281222 binding. (E) Specific [³H]‐JNJ‐46281222 binding after 9 min of dissociation in the absence or presence of GTP. *P <0.05 versus control, determined using Student's two‐tailed unpaired t‐test. All graphs are shown as mean ± SEM of three to six individual experiments performed in duplicate. Where bars are not shown, SEM values are within the symbol.

Figure 4

Saturation binding of radioligands to mGlu₂ receptors expressed at CHO‐K1 membranes. (A) Saturation binding of [³H]‐JNJ‐46281222 to the mGlu₂ allosteric binding site in the absence or presence of glutamate. (B) Saturation binding of [³H]‐DCG‐IV to the mGlu₂ orthosteric binding site, determined by ‘spiked’ saturation (see text). (C) Saturation binding of [³H]‐LY341495 to the orthosteric binding site. Graphs shown are from a representative experiment performed in duplicate in each case.

Figure 5

Characterization of potency of JNJ‐46281222. (A) Stimulation of [³⁵S]‐GTPγS binding to mGlu₂ receptors induced by increasing concentrations of JNJ‐46281222. A representative experiment is shown, with similar data obtained in a second experiment. (B) Dose–response curve of JNJ‐46281222 in the presence of glutamate (EC20, 4 μM) on the binding of [³⁵S]‐GTPγS. Data are expressed as the percentage of maximal response induced by 1 mM glutamate and are shown as mean ± SEM of 13 individual experiments performed in triplicate. Where bars are not shown, SEM values are within the symbol.

Figure 6

Molecular dynamics (MD) simulations of the binding of JNJ‐46281222 to the mGlu₂ receptor. (A) The complete system used for MD simulations, including mGlu₂ receptor 7TM monomer, G protein, ligand (JNJ‐46281222, magenta), lipids and solvent. (B) Close up of 7TM binding site showing interaction with a subset of important amino acids. MD starting position for ligand is coloured magenta; snapshot after 200 ns is turquoise. Movement of W7736.48a.50c from outwards orientation (magenta) at start of simulation to inwards orientation (turquoise) is highlighted. (C) RMSD of mGlu₂ receptor monomer during the simulation.

Figure 7

(A) Radioligand binding to mGlu₂ receptor mutants F6433.36a.40c and N7355.47a.47c. Specific [³H]‐JNJ‐46281222 binding to WT or mutant mGlu₂ receptors, transiently transfected in CHO‐K1 cells. Data were normalized to % specific binding compared with binding to WT mGlu₂ receptors and are expressed as mean ± SEM of three individual experiments performed in duplicate. (B) Representative immunoblot of WT (left panel) and mutant mGlu₂ receptors (middle, F643, and right, N735, panel) and actin. *P‐value <0.05 compared with WT, analysis was carried out using unnormalized data and determined using one‐way repeated‐measures ANOVA with Dunnett's post test.