A study of N-methyl-D-aspartate receptor allosteric modulators with diverse mechanisms of action Restricted; Files Only

Perszyk, Riley (Spring 2018)

Permanent URL: https://etd.library.emory.edu/concern/etds/m900nt40f?locale=en


N-methyl-d-aspartate receptors (NMDARs) are obligatory heterotetrameric ionotropic cell-surface receptors. Throughout the brain, NMDARs are expressed in synaptic and extrasynaptic spaces. Glutamate release at excitatory synapses leads to NMDAR activation, subsequent depolarization, and calcium influx. There are many endogenous molecular modulatory factors that influence NMDAR activity, including the co-agonist glycine, magnesium ions, zinc ions, neurosteroids, and extracellular protons. Additionally, NMDAR activity is controlled by the membrane potential due to extracellular block by magnesium ions. NMDARs play a role in initiating several forms of plasticity, integrating synaptic signals, and brain development. Modulation of NMDARs has been proposed as beneficial intervention in numerous neurological disorders. Previous attempts to target NMDARs have not been fruitful due to on-target side effects. A more detailed understanding of various NMDAR subtypes or subpopulations and how these factions contribute to the overall NMDAR function is required. This knowledge along with the development of novel pharmacological agents, with selective capabilities, will enhance future endeavors of producing safe and tolerated NMDAR-targeting drugs.

In this dissertation, three new compound series are evaluated for their actions on NMDARs and their utility in neural tissues. One series contains analogs with opposing actions and particular properties that should allow these compounds to selectively act based on the pattern of synaptic stimulation. The subunit selectivity of a series of positive allosteric modulators allows for selective targeting of a subpopulation of neurons and enhanced excitability of those cells. A non-selective NMDAR positive allosteric modulator series may have cell-type preferring actions in neuronal tissue derived from to differences in potentiation across the NMDAR subunits. Study of these compound series has aided in a greater understanding of NMDAR allosteric modulation and how their use might alter physiological NMDAR-dependent processes. A discussion of various theoretical models of NMDAR modulation includes their potential use and limitations. The potential utility of these novel modulators will also be discussed. This work has utility in furthering our understanding of NMDARs and in the development of new pharmacological compounds that possess diverse modes of action.

Table of Contents

Chapter 1: Introduction                                                                                                     1

Pathological roles of NMDA receptors                                                                       5

Neurological roles of NMDA receptors                                                                       7

Molecular Composition and Function of NMDA Receptors                                  10

      Architecture of NMDA Receptors                                                                           11

      NMDA Receptor Function                                                                                      13

      Intercellular Consequences of NMDA Receptor Activity                                        18

Mechanisms of NMDA receptor allosteric modulation                                           21

      Regulation of NMDA receptor function by allosteric ligands of the ATD               23

Regulation of NMDA receptor function by allosteric ligands of the agonist binding domain         29

      Regulation of NMDA receptor function by allosteric ligands of the TMD              35

      Competitive antagonists of NMDA receptor                                                           43

      Channel blockers of NMDA receptors                                                                    44

      Regulation of NMDA receptor function by the carboxyl-terminal domain              47

      Summary of modulation mechanisms                                                                     48

Models of NMDA receptor behavior                                                                        48

Macroscopic models of NMDA receptors                                                                 51

Microscopic models of NMDA receptors                                                                  52

Uses of NMDA receptor models                                                                                58

Conclusion                                                                                                                   60


Chapter 2: Materials and Methods                                                                                 62

Molecular biology                                                                                                        63

Two-electrode voltage-clamp recordings from Xenopus laevis                               63

Whole cell patch-clamp recordings of heterologous cells                                         64

Electrophysiological recordings of rodent hippocampus                                         65

Miniature and spontaneous post synaptic current detection and analysis             69

Hippocampal field tissue preparation and western blotting for GluN2D              71

Modelling receptor function                                                                                       72

Statistical analysis                                                                                                        73


Chapter 3: An NMDAR positive and negative allosteric modulator series share a binding site and are interconverted by methyl groups.                                                                                    75

Abstract                                                                                                                       76

Introduction                                                                                                                 77

Results                                                                                                                          80

Identification of a new class of positive allosteric modulators of NMDAR function 80

Allosteric modulation of agonist potency by NAMs and PAMs                               84

PAM and NAM display both glutamate and glycine dependence                           96

Evaluation of interactions with known modulatory sites                                       110

Mutagenesis suggests shared structural determinants of action for PAMs and NAMs            115

PAMs and NAMs exert their opposing effects via a shared binding site on NMDARs            121

Discussion                                                                                                                  135

The site and mechanism of action for the EU1794 series                                     135

EU1794 series links positive and negative allosteric modulators that act at the TMD.           137

Differential actions on synaptic and extrasynaptic receptors by submaximal EU1794 analogues     138


Chapter 4: GluN2D-containing NMDA receptors mediate synaptic transmission in hippocampal interneurons and regulate interneuron activity                                                            141

Abstract                                                                                                                     142

Introduction                                                                                                               144

Results                                                                                                                        147

Enantiomeric preference of a series of GluN2C/D-selective NMDAR positive allosteric modulators                                                                                                                              147

GRIN2D mRNA and the GluN2D protein are expressed in hippocampal interneurons         151

The NMDAR-component of mEPSCs in hippocampal interneurons is potentiated by (+)-CIQ         155

(+)-CIQ does not alter the NMDAR EPSC time course                                       163

(+)-CIQ increases spontaneous interneuron activity in mouse hippocampal brain slices.     165

Discussion                                                                                                                  168


Chapter 5: 1622-14 is a highly efficacious NMDA-receptor positive allosteric modulator that acts on hippocampal pyramidal cells and interneurons                                                           174

Abstract                                                                                                                     175

Introduction                                                                                                               176

Results                                                                                                                        179

Identification of a new class of positive allosteric modulators of NMDAR function. 179

1622-14 is a more potent and efficacious analog of 1622.                                    185

1622-14 potentiates the NMDAR-component of synaptic hippocampal excitatory transmission in both pyramidal cells and interneurons.                                                                         193

1622-14 enhances theta-burst potentials but not low frequency EPSPs in the CA1. 198

Discussion                                                                                                                  203

Mechanism and binding site                                                                                  203

Actions at neuronal receptors                                                                               205

Differences between pyramidal neurons and interneurons                                   208

Speculation about the in vivo actions of 1622-14                                                  208


Chapter 6: Discussion                                                                                                     211

Biophysical constraints on potentiation                                                                   213

The allosteric two-state receptor model                                                                217

Coupling of positive allosteric modulator enhancement and apparent agonist potency         229

The actions of a positive allosteric modulator on a model with multiple gating steps 234

The actions of a positive allosteric modulator on NMDA receptor hidden Markov gating models suggest activity is based at which step modulation occurs                                    245

Properties of several modulator series align with receptor modeling predictions 251

Which model is best?                                                                                                258

Moving forward in pursuit of novel NMDAR modulators with utility                261

The 1180 series                                                                                                     261

The 1622 series                                                                                                     262

The 1794 series                                                                                                     263

The future of pharmacological agent development                                               267

Conclusions                                                                                                                268


Chapter 7: References                                                                                                    270

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