An Integrated Approach for Screening and Identification of Positive Allosteric Modulators of N-Methyl-D-Aspartate Receptors

Abstract

N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that play an important role in synaptic plasticity and learning and memory formation. Malfunctioning of NMDARs, in particular the reduction in NMDAR activity, is thought to be implicated in major neurological disorders. NMDAR positive allosteric modulators (PAMs) represent potential therapeutic interventions for restoring normal NMDAR function. We report a novel screening approach for identification and characterization of NMDAR-PAMs. The approach combines high-throughput fluorescence imaging with automated electrophysiological recording of glutamate-evoked responses in HEK-293 cells expressing NR1/NR2A NMDAR subunits. Initial high-throughput screening (HTS) of a chemical library containing >810,000 compounds using a calcium flux assay in 1536-well plate format identified a total of 864 NMDAR-PAMs. Concentration response determination in both calcium flux and automated electrophysiological assays found several novel chemical series with EC50 values between 0.49 and 10 µM. A small subset (six series) was selected and analyzed for pharmacological properties, subtype selectivity, mode of action, and activity at native NMDARs. Our approach demonstrates the successful application of HTS functional assays that led to identification of NMDAR-PAMs providing the foundation for further medicinal chemistry work that may lead to novel therapies for treatment of cognitive impairment associated with Alzheimer's disease and schizophrenia.

Epilepsy-associated GRIN2A mutations reduce NMDA receptor trafficking and agonist potency – molecular profiling and functional rescue

“…The search for new potentiators for clinical enhancement of NMDAR function has been sporadic. However, with NMDAR hypofunction now shown here to be a disease mechanism in epilepsy, as well as schizophrenia 40 , cognitive decline and Alzheimer’s disease 41 , this may be changing 42 . Rescue of mutant receptors that are trapped within the ER may also be possible using cell-and-ER permeable competitive antagonists, as has been achieved for D732A-GluN1 using the competitive glycine site antagonist DCKA 32 , however cell permeability may be an issue for GluN2A-specific antagonists.…”

The dual‐functional memantine nitrate MN‐08 alleviates cerebral vasospasm and brain injury in experimental subarachnoid haemorrhage models

“…Stock solutions of MN‐08 (100 μM) and memantine (150 μM) were prepared and diluted into working solutions: MN‐08 (0.8, 1.6, 3.2, 6.5, 12.5, 25, 50, and 100 μM) and memantine (1.17, 2.34, 4.69, 9.38, 8.8, 37.5, 75, and 150 μM). The patch procedure has been described in previous reports (Jambrina et al, ; Maki, Cummings, Paganelli, Murthy, & Popescu, ). In short, electrodes were filled with an intracellular solution of 50‐mM CsCl, 90‐mM CsF, 2‐mM MgCl 2 , 5‐mm EGTA, and 10‐mM HEPES, which was then adjusted to pH 7.2 with CsOH.…”

“…Stock solutions of MN-08 (100 μM) and memantine (150 μM) were prepared and diluted into working solutions: 1.6,3.2,6.5,12.5,25,50, and 100 μM) and memantine (1.17, 2.34, 4.69, 9.38, 8.8, 37.5, 75, and 150 μM). The patch procedure has been described in previous reports (Jambrina et al, 2016;Maki, Cummings, Paganelli, Murthy, & Popescu, 2014).…”

Identification of Potential Pharmacoperones Capable of Rescuing the Functionality of Misfolded Vasopressin 2 Receptor Involved in Nephrogenic Diabetes Insipidus

“…7 The SDDL has been curated from over 20 commercial and academic sources and contains more than 40,000 compounds unique to Scripps. SDDL compounds are selected based on scaffold novelty, physical properties and spatial connectivity.…”