The Negative Allosteric Modulator EU1794-4 Reduces Single-Channel Conductance and Ca²⁺ Permeability of GluN1/GluN2A N-Methyl-d-Aspartate Receptors

Abstract

NMDA receptors are ligand-gated ion channels that mediate a slow, Ca 2+-permeable component of excitatory synaptic currents. These receptors are involved in several important brain functions, including learning and memory, and have also been implicated in neuropathological conditions and acute CNS injury, which has driven therapeutic interest in their modulation. The EU1794 series of positive and negative allosteric modulators of NMDA receptors has structural determinants of action near the pre-M1 helix that is involved in channel gating. Here we describe the effects of the negative allosteric modulator EU1794-4 on GluN1/GluN2A channels studied in excised outside-out patches. Co-application of EU1794-4 with a maximally effective concentration of glutamate and glycine increases the fraction of time the channel is open by nearly 1.5-fold, yet reduces single channel conductance by increasing access of the channel to several subconductance levels, which has the net overall effect of reducing the macroscopic current. The lack of voltagedependence of negative modulation suggests this is unrelated to a channel block mechanism. As seen with other NMDA receptor modulators that reduce channel conductance, EU1794-4 also reduces the Ca 2+ permeability relative to monovalent cations of GluN1/GluN2A receptors. We conclude that EU1794-4 is a prototype for a new class of NMDA receptor negative allosteric modulators that reduce both the overall current that flows following receptor activation and the flux of Ca 2+ ion relative to monovalent cations. Significance Statement NMDA receptors are implicated in many neurological conditions but are challenging to target given their ubiquitous expression. Several newly identified properties of the negative allosteric modulator EU1794-4, including reducing Ca 2+ flux through NMDA receptors and attenuating channel conductance, explain why this modulator reduces but does not eliminate This article has not been copyedited and formatted. The final version may differ from this version.

Structure, Function, and Pharmacology of Glutamate Receptor Ion Channels

“…the healthy population, with the observed/expected ratio referred to as the missense tolerance ratio, can identify regions of a gene that are under purifying selection, also known as negative selection or the selective removal of alleles that are deleterious (Traynelis et al, 2017;Perszyk et al, 2021). Moreover, there are also distinct intolerant regions for each gene product, consistent with the fact that the different iGluR subunits play unique roles in brain function despite high homology and similar functional properties within a family.…”

“…The EU1794 series of nonselective NAMs of NMDA receptors produce incomplete inhibition at saturating NAM concentrations (Katzman et al, 2015) (Table 12). This incomplete inhibition reflects a reduction in single-channel amplitude for the analog EU1794-4, which is accompanied by a change in relative ionic permeability (Perszyk et al, 2021). The property of incomplete inhibition did not remove the ability of EU1794-2 [compound 4 in Katzman et al (2015)] to act as a neuroprotective agent in suppressing excitotoxicity.…”

“…47). # EU1794-4 increased open probability but decreased single channel conductance, and the overall net effect was a reduction in macroscopic current, hence the designation of NAM (Perszyk et al, 2018;Perszyk et al, 2021).…”

Development of a Dihydroquinoline–Pyrazoline GluN2C/2D-Selective Negative Allosteric Modulator of the N-Methyl-d-aspartate Receptor

“…Structures for GluN2C/D-selective NAMs: DQP-1105 ( 1 ) (Acker et al), QNZ-46 (Mosley et al; Hansen and Traynelis), UBP-1700 (Wang et al), EU1794 - 4 (Katzman et al; Perszyk et al, ), and NAB-14 (Swanger et al).…”

“…These molecules can either amplify (positive allosteric modulators, PAMs) or inhibit (negative allosteric modulators, NAMs) NMDAR activity through a variety of mechanisms. ,− In terms of subunit-selective NAMs, compounds that inhibit GluN2A- (prototype TCN-201) − and GluN2B-containing NMDARs (prototype ifenprodil) ,, have been well-described. However, while the number of reported scaffolds with GluN2C/D-selective NAM activity has steadily increased over the years − (Figure ), drug development and pharmacological characterization efforts on such compounds are lacking. Furthermore, the mechanism of action of existing GluN2C/D-selective NAMs is not well understood due to lack of structural data for their binding sites, although recent description of GluN1/GluN2C and GluN1/GluN2D receptor structures may provide a means to explore NAM binding .…”