A study of N-methyl-D-aspartate receptor allosteric modulators with diverse mechanisms of action Restricted; Files Only
Perszyk, Riley (Spring 2018)
Published
Abstract
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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