Development and Validation of [3H]OF-NB1 for Preclinical Assessment of GluN1/2B Candidate Drugs

GluN2B-enriched N-methyl-D-aspartate receptors (NMDARs) are implicated in several neurodegenerative and psychiatric diseases, such as Alzheimer's disease. No clinically valid GluN1/2B therapeutic exists due to a lack of selective GluN2B imaging tools, and the state-of-the-art [H-3]ifenprodil shows poor selectivity in drug screening. To this end, we developed a tritium-labeled form of OF-NB1, a recently reported selective GluN1/2B positron emission tomography imaging (PET) agent, with a molar activity of 1.79 GBq/mu mol. The performance of [H-3]OF-NB1 and [H-3]ifenprodil was compared through head-to-head competitive binding experiments, using the GluN1/2B ligand CP-101,606 and the sigma-1 receptor (sigma 1R) ligand SA-4503. Contrary to [H-3]ifenprodil, the usage of [H-3]OF-NB1 differentiated between GluN1/2B and sigma 1R binding components. These results were corroborated by observations from PET imaging experiments in Wistar rats using the sigma 1R radioligand [F-18]fluspidine. To unravel the binding modes of OF-NB1 and ifenprodil in GluN1/2B and sigma 1Rs, we performed a retrospective in silico study using a molecular operating environment. OF-NB1 maintained similar interactions to GluN1/2B as ifenprodil, but only ifenprodil successfully fitted in the sigma 1R pocket, thereby explaining the high GluN1/2B selectivity of OF-NB1 compared to ifenprodil. We successfully showed in a proof-of-concept study the superiority of [H-3]OF-NB1 over the gold standard [H-3]ifenprodil in the screening of potential GluN1/2B drug candidates.

Automated summary

GluN2B-enriched NMDARs are associated with several neurodegenerative and psychiatric diseases. In this study, a tritium-labeled form of OF-NB1 was developed as a selective GluN1/2B PET imaging agent. Competitive binding experiments and molecular simulations demonstrated the superior selectivity and performance of OF-NB1, making it a promising tool for screening potential GluN1/2B drug candidates.