MPX-004 and MPX-007: New Pharmacological Tools to Study the Physiology of NMDA Receptors Containing the GluN2A Subunit

Abstract

GluN2A is the most abundant of the GluN2 NMDA receptor subunits in the mammalian CNS. Physiological and genetic evidence implicate GluN2A-containing receptors in susceptibility to autism, schizophrenia, childhood epilepsy and neurodevelopmental disorders such as Rett Syndrome. However, GluN2A-selective pharmacological probes to explore the therapeutic potential of targeting these receptors have been lacking. Here we disclose a novel series of pyrazine-containing GluN2A antagonists exemplified by MPX-004 (5-(((3-chloro-4-fluorophenyl)sulfonamido)methyl)-N-((2-methylthiazol-5-yl)methyl)pyrazine-2-carboxamide) and MPX-007 (5-(((3-fluoro-4-fluorophenyl)sulfonamido)methyl)-N-((2-methylthiazol-5-yl)methyl)methylpyrazine-2-carboxamide). MPX-004 and MPX-007 inhibit GluN2A-containing NMDA receptors expressed in HEK cells with IC50s of 79 nM and 27 nM, respectively. In contrast, at concentrations that completely inhibited GluN2A activity these compounds have no inhibitory effect on GluN2B or GluN2D receptor-mediated responses in similar HEK cell-based assays. Potency and selectivity were confirmed in electrophysiology assays in Xenopus oocytes expressing GluN2A-D receptor subtypes. Maximal concentrations of MPX-004 and MPX-007 inhibited ~30% of the whole-cell current in rat pyramidal neurons in primary culture and MPX-004 inhibited ~60% of the total NMDA receptor-mediated EPSP in rat hippocampal slices. GluN2A-selectivity at native receptors was confirmed by the finding that MPX-004 had no inhibitory effect on NMDA receptor mediated synaptic currents in cortical slices from GRIN2A knock out mice. Thus, MPX-004 and MPX-007 offer highly selective pharmacological tools to probe GluN2A physiology and involvement in neuropsychiatric and developmental disorders.

Structural Basis for Negative Allosteric Modulation of GluN2A-Containing NMDA Receptors

“…Recently, the chemical scaffold of TCN-201 was modified to yield MPX-004 and MPX-007 (Volkmann et al, 2016) (Figure 1A). We evaluated GluN2A-selectivity and potency of TCN-201, MPX-004, and MPX-007 at recombinant NMDA receptors containing GluN1 and either GluN2A, GluN2B, GluN2C or GluN2D (i.e.…”

“…The selectivity and structural similarity of TCN-201, MPX-004, and MPX-007 suggest they share this mechanism of action (Volkmann et al, 2016). To compare the glycine-sensitivity of the NAMs, we determined potency and efficacy for inhibition of GluN1/2A in the presence of 1-300 μM glycine (Figure 1C).…”

“…The use of ifenprodil and its analogs as pharmacological tools has enabled precise localization of GluN2B-containing receptors in the CNS, and identified a wide range of normal processes and neurological diseases that involve GluN2B. In this regard, the enhanced solubility and increased efficacy of MPX-004 and MPX-007 suggest that these NAMs could be useful tool compounds with which to study the contributions of GluN2A-containing NMDA receptors to normal brain function and disease (Volkmann et al, 2016). In the past decades, NMDA receptors have been widely considered as important targets for pharmacological intervention in a number of neurological and psychiatric diseases, yet drug discovery efforts have provided relatively few NMDA receptor ligands approved as therapeutic agents.…”

“…Here, we take advantage of structural and pharmacological differences between TCN-201 and two recently described analogs, MPX-004 and MPX-007 (Volkmann et al, 2016), to investigate the molecular mechanism for GluN2A-selective allosteric inhibition of glycine binding. Using an engineered disulfide bond, we show that NAM binding stabilizes the glycine-lacking, apo -state of the GluN1 LBD, and that allosteric inhibition requires a transition from the glycine-bound to the apo -state of the GluN1 LBD.…”

Structural basis of subunit selectivity for competitive NMDA receptor antagonists with preference for GluN2A over GluN2B subunits

“…Considerable progress has been made in the development of subunit-selective allosteric modulators (7)(8)(9)(10)(11)(12)(13), but the development of subtype-selective competitive NMDA receptor antagonists has been less successful. The competitive glutamate-site antagonist NVP-AAM077 (hereafter NVP) was originally reported to have 100-fold preference for GluN1/2A over GluN1/2B (14).…”