Virginia Research Day 2021

Pharmacology of a Novel Biased Allosteric Modulator for NMDA receptors Lina Cortes Kwapisz 2 , Brittney Mehrkens 3 , Blaise M. Costa 1,2&3* 1 Edward Via College of Osteopathic Medicine, Blacksburg, VA, USA, 2 Virginia–Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, 3 Center for One Health Research, Virginia Tech, Blacksburg, VA, USA

Figure 2. CNS4 Exhibits NMDAR Subunit Selectivity with different glutamate concentration

Figure 5. CNS4 Increases Ca 2+ influx in cortical and striatal neurons with induced excitotoxicity

INTRODUCTION

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o The N-methyl-D-aspartate (NMDA) receptor plays a crucial role in brain physiology and pathogenesis of disorders. o Functional NMDA receptors are heteromers composed of four subunits: two obligatory glycine-binding GluN1 subunits and two other identical or different glutamate (GluN2) or glycine (GluN3) subunits. o Recent studies report that synaptic glutamate levels are highly fluctuant. There is a need to develop compounds that can modulate distinct GLuN2 expression without global NMDAR overactivation.

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Figure 5. Ca2+ assay in cortical neurons with NMDAR-induced excitotoxicity with increasing doses of NMDA and constant 100µM CNS4. Statistical significance was determined through One-Way ANOVA Multiple Comparisons P (<0.05 ). Similar results were found in striatal neurons Background NMDA Mem+NMDA CNS4 (100uM) +0.3uM +1uM +3uM +10uM +30uM +100uM +300uM NMDA

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o From our ongoing efforts to discover small molecules to dissect the function of NMDA receptor based on their subunit composition, CNS4 was computationally identified as one of the top hit compounds. This compound was synthesized at Syngene International and the pharmacological characterization of CNS4 work is currently in progress.

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Figure 1. A. GluN2D relative response with increasing CNS4 concentration (1 μ M, 3 μ M, 10 μ M, 30 μ M and 100 μ M) in agonist max (100 μ M Glu/ 100 μ M Gly). Statistical significance was determined at the alpha level p < 0.0 5(*) using one-way ANOVA test . (ii) Traces from GluN2D DRC recordings (B) Relative response of all NMDAR with agonist super max solution

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Figure 6. CNS4 does not alter neuronal viability

METHODS

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Figure 3. CNS4 does not replace either glutamate nor glycine

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o Two-electrode voltage clamp (TEVC) and patch clamp electrophysiology techniques were used to study the CNS4 induced changes in NMDA receptor function. o Appropriate combination of cRNA encoding different NMDA receptor subunits were injected into xenopus oocytes and incubated in 18 ° C for 2-4 days before TEVC assays with a -60-mV holding potential. CNS4 effect on endogenous agonist (glutamate) and co-agonist (glycine) activated NMDA receptors was studied as described in the results section. o Neuron culture on cortical and striatal neurons with induced excitotoxicity was performed to identify CNS4 Ca 2+ influx and neuronal viability . o Female rat embryos (E19) were retrieved according to IACUC guideless. Cortical and striatal neurons were dissected and grown for two weeks before being treated. Imaging assays were performed based on manufacture instructions

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(A) MTS Assay in cortical neurons with NMDAR-induced excitotoxicity with increasing doses of CNS4. Statistical significance was determined through One-Way ANOVA Multiple Comparisons P (<0.05) (B) MTS assay in cortical and striatal untreated neurons versus 100µM CNS4. Statistical difference was determined with unpaired T-Test P (<0.001)

Figure 4. CNS4 does not block channel pore of NMDAR at the I-V plot Figure 3 (A) GluN2A and (B) GluN2B using agonist max solution with glutamate and glycine alone plus 100 μ M CNS4. Statistical significance was determined at the alpha level p<0.05 ( ∗ ) and p < 0.0005 ( ∗∗∗∗ ) using one-way ANOVA test. .

RESULTS

Figure 1. CNS4 Exhibits NMDA Receptor Subunit Selectivity in TEVC Assay

CONCLUSIONS

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o CNS4 modulates NMDA receptor currents based on various factors including agonist concentration and co-expressing GluN2 subunit in xenopus oocytes as identified by TEVC electrophysiology assay. o CNS4 potentiates NMDA receptors based on glutamate concentration. o I-V plot TEVC electrophysiological recordings suggested the non-voltage dependent activity of CNS4 that alters ion inward current without blocking NMDA receptors o In both cortical and striatal neurons with induced excitotoxicity CNS4 increased Ca 2+ uptake o At the MTS assay, CNS4 did not increased further cell death and when used by itself it did not alter neuronal viability Acknowledgement: Authors thank Dr.Pierre Paoletti, CNRS France, for providing triheteromeric receptor subunit constructs (GluN16A, GluN2Ar1 & 2Br2) published in Stroebel et al, 2014. Also thank Dr. Daniel Monaghan (UNMC) for critical input with data interpretation. References : Costa BM, Irvine MW, Fang G, Eaves RJ, Mayo-Martin MB, Skifter DA, Jane DE, Monaghan DT. A novel family of negative and positive allosteric modulators of NMDA receptors. J Pharmacol Exp Ther. 2010 Dec;335(3):614-21. Stroebel D, Carvalho S, Grand T, Zhu S, Paoletti P. Controlling NMDA receptor subunit composition using ectopic retention signals. J Neurosci. 2014 Dec 10;34(50):16630-6.

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Figure 1. A. GluN2A, GluN2B, GluN2C and GluN2D relative response with increasing CNS4 concentration (1 μ M, 3 μ M, 10 μ M, 30 μ M and 100 μ M) in agonist (0.3 μ M Glu/ 100 μ M Gly). Statistical significance determined at alpha p < 0.0 5(*) using one-way ANOVA test. B Traces from GluN2C DRC recordings with increasing CNS4 (1μM, 3μM, 10μM, 30μM and 100μM) with agonist (0.3μM Glu/ 100μM Gly)

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Figure 4: I-V curve of the (A) GluN2C and (B) GLuN2D subunit using agonist solution (0.3µM Glu/100µGly), agonist plus 100µM CNS4, agonist plus 40µM MgCl2 and agonist plus 40µM MgCl2 plus 100µM CNS4. Significant level of the reversal potential was determined at alpha level p < 0.05( ∗ ) and p < 0.0005( ∗∗∗∗ ) using one way ANOVA Tukey’s multiple comparisons test.

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This work was funded by the American Heart Association Scientist Development Grant (AHA-SDG) awarded to BC.

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