Michael J Morton1, John Malone1, Paul Sharpe1, Graham Sproat1, Malcolm Haddrick1, Joe Mather2

1 Discovery Sciences, AstraZeneca R&D, Alderley Park, Cheshire, SK10 4TG
2 Neuroscience iMed, AstraZeneca R&D, Boston

The ligand-gated N-methyl-D-aspartate receptor (NMDAR) is a multi-subunit ion channel highly expressed throughout the CNS. The functional channel is a tetrameric or pentameric complex of essential (NR1A) and modulatory (NR2A-D and NR3A-B) subunits and requires the simultaneous co-application to 2 agonists, glycine and glutamate/NMDA, for activation. De novo mutation or deletions of NR2B have been implicated in neurodevelopmental disorders and schizophrenia and intellectual disability. Identification of selective positive allosteric modulators (PAMs) of NR1A/NR2B receptors could form the basis for treatment for these disorders.

The generation of cell reagents expressing NMDAR is challenging. Activation of the channel is highly cytotoxic to the cell, compounded by the presence of co-agonists in routine culture medium. To reduce this cytotoxicity, the NMDAR has been co-expressed with the glutamate-aspartate receptor, GLAST, which removes glutamate from culture medium that would otherwise activate the receptor. This cell reagent has been generated at large scale, cryopreserved and used to development of a medium throughput automated electrophysiology assay to detect positive allosteric modulators of NR1A/2B.

Using the MaxCyte electroporation platform, HEK cells, stably expressing the Glutamate-Aspartate Transporter (GLAST), were transfected with NR1A:NR2B DNA and allowed to recover for 16-20 hours shaking in media at 37oC in the presence of 100uM AZ57 (a non-selective blocker of NMDAR). The combination of GLAST expression and AZ57 improved the viability of NR1A/2B-transfected cells to >90%, significantly more than AZ57 alone.

Using Sophion’s QPatch HTX automated patch clamp system as a screening platform; we have developed a medium throughput protocol for the identification of PAM activity for the NR2B subunit. Using 10uM glutamate (in the presence of 10uM glycine as a co-agonist), we are able to show that a concentration-dependent, subunit specific, positive allosteric modulatory effect can be generated in a NR1A/NR2B transiently expressing cell system.