The principal site of action of phencyclidine (PCP) in the brain is the N- methyl-D-aspartate (NMDA) subtype of glutamate receptor. PCP binding to the NMDA receptor is modulated by Mg2+ and polyamines. However, although polyamines and Mg2+ have substantial modulatory effects on the binding of PCP, the sites and mechanisms by which these modulators alter receptor function is poorly understood. The long term goal of the present study is to understand the mechanism of action of these modulators one the binding of PCP to the NMDA receptor, and to use these modulator sites as targets for drugs that will alter or block the pharmacological effects of PCP. We will take three convergent approaches, using synthetic chemistry, molecular biology and pharmacology to achieve this goal.
The specific aims of this project are: 1. To pursue rational design and synthesis of polyamine antagonists. Using arcaine as a lead compound and systematically modifying size, conformation and hydrophobicity of novel bisguanidines we will develop polyamine antagonists that are more potent and/or more selective than the currently available drugs. 2. To pharmacologically characterize novel polyamine ligands. We will use ligand binding assays and intracellular Ca2+ assays to establish the activity and specificity of newly synthesized polyamine site drugs. We will also determine the extent to which PCP action can be modified by polyamine antagonists. 3. To determine the effect of polyamine agonists and antagonists on ligand binding to recombinant NMDA receptor subunits expressed in mammalian cells. We will assess polyamine action on defined NMDA receptor subunit combinations, which offers a more specific approach to investigating the mechanism of action of this modulator on the NMDA receptor complex. 4. To determine the effects of elevated intracellular free Mg2+ on inhibition of NMDA receptors by PCP site ligands. We will use Ca2+- dependent and Ca2+-independent methods to elevate intracellular [Mg2+] to test the hypothesis that elevated [Mg2+] will decrease PCP binding to the NMDA receptor. Understanding the molecular mechanisms by which Mg2+ and polyamines influence the actions of PCP will provide important new information about the molecular mechanisms of action of this important substance of abuse. This project will also focus on strategies that will prevent the effects of PCP on the NMDA receptor, and thereby offer a therapeutic target for the treatment of PCP abuse.
