Use of Bioinformatics and Genetics to Identify a New Class of Drugs for Neurological Disease

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NMDA receptors (NMDARs) can trigger synaptic plasticity due to their ability to act as coincident detectors of simultaneous neuronal firing and excitatory synaptic input, which can strengthen synapses by a cellular process known as long term potentiation (LTP). NMDARs play a critical role in cognition and memory formation, which is compromised in several neurological diseases. NMDARs are assemblies of two GluN1 subunits and two GluN2A-D subunits. Increased expression of GluN2B-containing NMDARs in vivo can enhance synaptic plasticity and memory, suggesting that GluN2B-selective positive allosteric modulators may facilitate learning. Finding ways to improve synaptic plasticity and memory formation could improve quality of life for patients with Alzheimer’s disease or intellectual disability. GluN2B-selective negative allosteric modulators (i.e. inhibitors) such as ifenprodil and analogues bind to the GluN1/GluN2B heterodimer amino terminal domain (ATD) interface. However, no drug-like GluN2B- selective positive allosteric modulator (i.e. potentiator) that binds to this site has been discovered despite the pharmaceutical industry screening many millions of compounds. Thus, if such modulators exist, they are absent from large screening libraries, which provide the starting point for virtually all medicinal chemistry efforts. In this proposal we exploit two advances in our understanding of NMDARs that could enable us to find new scaffolds for positive modulators. First, a crystallographic study of GluN2B-selective NMDAR inhibitors demonstrated a unique binding mode for the 93-series of Emory compounds, which occupy three branches of the triangular pocket located at the interface between the GluN1 and GluN2B ATDs. No other GluN2B- selective modulator binds in this fashion. We also discovered two GluN1 mutations near the modulator binding site that convert the action of Emory-synthesized GluN2B inhibitors, but not other classes of GluN2B inhibitors, into potentiators. This is the first evidence that a GluN2B-selective potentiator of NMDARs acting at the ifenprodil site can exist, and provides insight into what that molecule does to protein structure to make it a potentiator. We believe this insight together with structure-based design methods will support discovery of GluN2B potentiators of wild type NMDARs. Second, genetic variation in the healthy population has revealed that GluN1 and GluN2B residues in contact with ifenprodil have significantly fewer variants than expected, revealing that the ifenprodil binding pocket is under selective pressure. The only possible reasons for this are that these residues are critically important for receptor function, or an undiscovered endogenous modulator binds within this pocket and is important for health. We propose to test these two possibilities, including a screen of CSF for actions of a small molecule that binds within this pocket. We will carry out two sets of experiments: (1) design and synthesize GluN2B-selective positive allosteric modulators that act at the ifenprodil site, and (2) screen CSF for an endogenous modulator acting at the ifenprodil binding site.