Volkmann RA et al. (2016), MPX-004 and MPX-007: New Pharmacological Tools ...

XB-ART-51824

PLoS One 2016 Jan 01;112:e0148129. doi: 10.1371/journal.pone.0148129.

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

Volkmann RA , Fanger CM , Anderson DR , Sirivolu VR , Paschetto K , Gordon E , Virginio C , Gleyzes M , Buisson B , Steidl E , Mierau SB , Fagiolini M , Menniti FS .

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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.

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Species referenced: Xenopus
Genes referenced: cda grin2a runx2

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	Fig 1. The structures of TCN-201, MPX-004 and MPX-007.The values are IC50s for inhibition of Ca2+ responses mediated by GluN2A receptors expressed in HEK cells.
	Fig 2. Concentration-response of TCN-201, MPX-004, and MPX-007 inhibition of Ca2+ responses mediated by GluN2A expressed in HEK cells.Cells were stimulated with glutamate and glycine (3 μM each) in the presence of compounds at a range of concentrations. Curves for inhibition of the Ca2+ response in GluN2A-expressing cells were derived from fits to the Hill equation using GraphPad Prism (v6.00 for Mac, GraphPad Software, La Jolla California USA). Whereas MPX-004 and MPX-007 achieve full inhibition of the GluN2A Ca2+ response by ~ 3 μM, TCN-201 never inhibits more than ~40% of the response. Each data point is a mean (± standard deviation) of data from 20–86 experiments).
	Fig 3. Potency or efficacy of TCN-201 and selected analogs for inhibition of Ca2+ responses mediated by GluN2A, GluN2B and GluN2D expressed in HEK cells.Cells were stimulated with glutamate and glycine (3 μM each) in the presence of compounds at a range of concentrations. IC50 for inhibition of the Ca2+ response in GluN2A-expressing cells was fitted to the Hill equation using CDD Vault. For GluN2B or GluN2D no IC50 could be determined, so the effect of each compound at 10 μM is shown as the % inhibition of the Ca2+ response (note that negative % inhibition represents an increase in Ca2+ response over glutamate plus glycine alone). Values are the mean IC50 or % ± SEM with the number of replicate curves indicated in parentheses. ND- not determined.
	Fig 4. Effect of MPX-004 and MPX-007 on glutamate/glycine-induced currents in Xenopus oocytes expressing GluN1 and GluN2A, B, C, or D.Oocytes were exposed to MPX-004 or MPX-007 at concentrations from 10 nM to 10 μM as indicated. Inhibition curves were generated using GraphPad Prism and IC50 values were generated using CCD Vault. The IC50s for inhibition of GluN2A-mediated currents were 198 ± 17 and 143 ± 10 nM for MPX-004 and MPX-007, respectively. Each data point is a mean (± standard deviation) of data from 4–12 oocytes.
	Fig 5. Effects of MPX-004 and MPX-007 on NMDA/glycine-induced currents of rat cortical neurons in primary culture.Cortical neurons were maintained in primary culture for 13–15 days and then examined using whole-cell manual patch clamp for current evoked by NMDA (100 μM) + glycine (10 μM) applied for 4 seconds during 10 second pulses to +20 or +40 mV from a holding potential of -70 mV. Inhibition of NMDA-activated currents was quantified during exposure to 10 μM MPX-007, MPX-004, Ro 25–6981, or a combination of Ro 25–6981 + MPX-004. Currents were blocked ~25–30% by either MPX-007 or MPX-004 alone, ~70% by Ro 25–6981 alone, and ~85% by Ro 25–6981 plus MPX-004.
	Fig 6. Effect of MPX-004 on isolated NMDA receptor-mediated fEPSPs in rat hippocampal CA1 stratum radiatum in response to stimulation of Schaffer collateral input.Slices were exposed to MPX-004 from 100 nM to 30 μM in half log concentration increments as well as to 50 μM. Right panel- Time course for inhibition of fEPSPs after application of different concentrations of MPX-004. Left panel- Percent inhibition at 40 min after application for each MPX-004 concentration. Maximum inhibition was approximately 60% of the fEPSPs at 30 or 50 μM. The IC50 of MPX-004 for inhibition of fEPSPs was 3.4 μM. Curves in the right panel and data point in the left panel are a mean (± SEM) of 4, 5, 6, 8, 6, 5 and 2 slices obtained from 2, 2, 3, 3, 2, 2 and 1 rats exposed to 0.1, 0.3, 1, 3, 10, 30 and 50 μM MPX-004, respectively. In separate experiments, a highly selective GluN2B NAM inhibited approximately 40% of the fEPSP (data not shown).
	Fig 7. Synthetic schemes for compounds 2–10.

References [+] :

, Correction: MPX-004 and MPX-007: New Pharmacological Tools to Study the Physiology of NMDA Receptors Containing the GluN2A Subunit. 2016, Pubmed