Development of Clickable Photoaffinity Ligands for Metabotropic Glutamate Receptor 2 Based on Two Positive Allosteric Modulator Chemotypes

The metabotropic glutamate receptor 2 (mGlu₂) is a transmembrane-spanning class C G protein-coupled receptor that is an attractive therapeutic target for multiple psychiatric and neurological disorders. A key challenge has been deciphering the contribution of mGlu₂ relative to other closely related mGlu receptors in mediating different physiological responses, which could be achieved through the utilization of subtype selective pharmacological tools. In this respect, allosteric modulators that recognize ligand-binding sites distinct from the endogenous neurotransmitter glutamate offer the promise of higher receptor-subtype selectivity. We hypothesized that mGlu₂-selective positive allosteric modulators could be derivatized to generate bifunctional pharmacological tools. Here we developed clickable photoaffinity probes for mGlu₂ based on two different positive allosteric modulator scaffolds that retained similar pharmacological activity to parent compounds. We demonstrate successful probe-dependent incorporation of a commercially available clickable fluorophore using bioorthogonal conjugation. Importantly, we also show the limitations of using these probes to assess in situ fluorescence of mGlu₂ in intact cells where significant nonspecific membrane binding is evident.