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

Background and PurposeAllosteric modulation of the mGlu(2) receptor is a potential strategy for treatment of various neurological and psychiatric disorders. Here, we describe the in vitro characterization of the mGlu(2) positive allosteric modulator (PAM) JNJ-46281222 and its radiolabelled counterpart [H-3]-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 ApproachWe have used radioligand binding studies, functional assays, site-directed mutagenesis, homology modelling and molecular dynamics to study the binding of JNJ-46281222. Key ResultsJNJ-46281222 is an mGlu(2)-selective, highly potent PAM with nanomolar affinity (K-D = 1.7 nM). Binding of [H-3]-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 [H-3]-JNJ-46281222 binding experiments on mutant receptors. Conclusion and ImplicationsOur results obtained with JNJ-46281222 in unlabelled and tritiated form further contribute to our understanding of mGlu(2) 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 mGlu(2) and class C receptor drug discovery.