Development of a MS binding assay to analyze the interactions of drugs with the ifenprodil binding site of NMDA receptors with GluN2B subunit

Negative allosteric modulators interacting with the ifenprodil binding site of NMDA receptors with GluN2B subunit are of particular interest for the treatment of psychiatric and neurodegenerative disorders. In order to eliminate the radioligand [3H]ifenprodil in the standard radioactive binding assay, a novel MS binding assay was established and validated making use of the quantitative determination of non-radioactive ifenprodil by triple quadrupole MS. Kinetic, saturation and competitive experiments were carried out in a stepwise and iterative manner to optimize the final assay conditions. Under optimized assay conditions, the MS binding assay provided a Kd value for the marker ifenprodil (Kd = 11 nM), which is comparable with the Kd value obtained in the radioligand binding assay for [3H]ifenprodil (Kd = 7.6 nM). Although the MS binding assay led to higher Ki values for known NMDA receptor antagonists, both assays showed the same trend of Ki values for these inhibitors.