Reversible inhibition of GluN2B-containing NMDA receptors with an in situ red-shifted, photoswitchable antagonist

NMDA receptors (NMDARs) are glutamate-gated ion channels playing a central role in synaptic transmission and plasticity. Dysregulation of NMDARs is linked to various neuropsychiatric disorders, emphasizing the need to understand the functional roles of individual NMDAR subtypes in the brain. GluN2B-containing NMDARs (GluN2B-NMDARs) are particularly important due to both pro-cognitive and pro-excitotoxic roles, although these functions remain under debate. Traditional pharmacological and genetic approaches have important shortcomings in terms of specificity and spatio-temporal resolution, limiting their use in native tissues. We therefore turned to optopharmacology, a technique based on the use of photosensitive ligands, whose activity can be reversibly tuned via illumination with different wavelengths. We developed OptoNAM-3, an azobenzene-based, photoswitchable negative allosteric modulator selective for GluN2B-NMDARs. OptoNAM-3 is a potent inhibitor of GluN2B-NMDARs in its trans configuration and inactive in its cis configuration. When bound to GluN2B-NMDARs, OptoNAM-3 displays remarkable red-shifting of its photoswitching properties that we attributed to geometric constraints imposed by the binding site onto the ligand azobenzene moiety. OptoNAM-3 allowed fast and reversible photomodulation of GluN2B-NMDAR activity in vitro using either UV/green or blue/green light illumination cycles. OptoNAM-3 furthermore acted as a reversible, red-shifted in vivo photomodulator of Xenopus tadpole locomotion. By enabling fast and reversible photocontrol of endogenous GluN2B-NMDARs with in vivo compatible photochemical properties, OptoNAM-3 should advance our understanding of the role of this class of NMDARs in brain function and dysfunction. Significance statementThis article presents the development and characterization of a photoswitchable negative allosteric modulator (NAM) targeting GluN2B-containing NMDA receptors (GluN2B-NMDARs). Traditional GluN2B-selective NAMs suffer from slow kinetics and irreversible effects, limiting their use in native tissues. OptoNAM-3 emerged as a potent and selective inhibitor of GluN2B-NMDARs, exhibiting fast temporal resolution of action and reversibility both in vitro and in vivo. OptoNAM-3 furthermore exhibited different spectral properties when in solution or bound to its target, thus behaving as an in situ "red-shifted" photodependent antagonist with improved in vivo compatibility. This study therefore provides a valuable photoswitchable tool for precise control of NMDAR activity in native tissues. It furthermore reveals the importance of the protein environment on the spectral properties of photosensitive molecules.