Role of endoplasmic reticulum (ER) stress in cocaine-induced microglial cell death.

While it has been well-documented that drugs of abuse such as cocaine can enhance progression of human immunodeficiency virus (HIV)-associated neuropathological disorders, the underlying mechanisms mediating these effects remain poorly understood. The present study was undertaken to examine the effects of cocaine on microglial viability. Herein we demonstrate that exposure of microglial cell line-BV2 or rat primary microglia to exogenous cocaine resulted in decreased cell viability as determined by MTS and TUNEL assays. Microglial toxicity of cocaine was accompanied by an increase in the expression of cleaved caspase-3 as demonstrated by western blot assays. Furthermore, increased microglial toxicity was also associated with a concomitant increase in the production of intracellular reactive oxygen species, an effect that was ameliorated in cells pretreated with NADPH oxidase inhibitor apocynin, thus emphasizing the role of oxidative stress in this process. A novel finding of this study was the involvement of endoplasmic reticulum (ER) signaling mediators such as PERK, Elf2α, and CHOP, which were up regulated in cells exposed to cocaine. Reciprocally, blocking CHOP expression using siRNA ameliorated cocaine-mediated cell death. In conclusion these findings underscore the importance of ER stress in modulating cocaine induced microglial toxicity. Understanding the link between ER stress, oxidative stress and apoptosis could lead to the development of therapeutic strategies targeting cocaine-mediated microglial death/dysfunction.

Pharmacological modulation of NMDA receptor activity and the advent of negative and positive allosteric modulators.

The NMDA receptor (NMDAR) family of l-glutamate receptors are well known to have diverse roles in CNS function as well as in various neuropathological and psychiatric conditions. Until recently, the types of agents available to pharmacologically regulate NMDAR function have been quite limited in terms of mechanism of action and subtype selectivity. This has changed significantly in the past two years. The purpose of this review is to summarize the many drug classes now available for modulating NMDAR activity. Previously, this included competitive antagonists at the l-glutamate and glycine binding sites, high and low affinity channel blockers, and GluN2B-selective N-terminal domain binding site antagonists. More recently, we and others have identified new classes of NMDAR agents that are either positive or negative allosteric modulators (PAMs and NAMs, respectively). These compounds include the pan potentiator UBP646, the GluN2A-selective potentiator/GluN2C and GluN2D inhibitor UBP512, the GluN2D-selective potentiator UBP551, the GluN2C/GluN2D-selective potentiator CIQ as well as the new NMDAR-NAMs such as the pan-inhibitor UBP618, the GluN2C/GluN2D-selective inhibitor QZN46 and the GluN2A inhibitors UBP608 and TCN201. These new agents do not bind within the l-glutamate or glycine binding sites, the ion channel pore or the N-terminal regulatory domain. Collectively, these new allosteric modulators appear to be acting at multiple novel sites on the NMDAR complex. Importantly, these agents display improved subtype-selectivity and as NMDAR PAMs and NAMs, they represent a new generation of potential NMDAR therapeutics.

Structure-activity relationships for allosteric NMDA receptor inhibitors based on 2-naphthoic acid.

Over-activation of N-methyl-d-aspartate (NMDA) receptors is critically involved in many neurological conditions, thus there has been considerable interest in developing NMDA receptor antagonists. We have recently identified a series of naphthoic and phenanthroic acid compounds that allosterically modulate NMDA receptors through a novel mechanism of action. In the present study, we have determined the structure-activity relationships of 18 naphthoic acid derivatives for the ability to inhibit the four GluN1/GluN2(A-D) NMDA receptor subtypes. 2-Naphthoic acid has low activity at GluN2A-containing receptors and yet lower activity at other NMDA receptors. 3-Amino addition, and especially 3-hydroxy addition, to 2-naphthoic acid increased inhibitory activity at GluN1/GluN2C and GluN1/GluN2D receptors. Further halogen and phenyl substitutions to 2-hydroxy-3-naphthoic acid leads to several relatively potent inhibitors, the most potent of which is UBP618 (1-bromo-2-hydroxy-6-phenylnaphthalene-3-carboxylic acid) with an IC(50) âˆ¼ 2 µM at each of the NMDA receptor subtypes. While UBP618 is non-selective, elimination of the hydroxyl group in UBP618, as in UBP628 and UBP608, leads to an increase in GluN1/GluN2A selectivity. Of the compounds evaluated, specifically those with a 6-phenyl substitution were less able to fully inhibit GluN1/GluN2A, GluN1/GluN2B and GluN1/GluN2C responses (maximal % inhibition of 60-90%). Such antagonists may potentially have reduced adverse effects by not excessively blocking NMDA receptor signaling. Together, these studies reveal discrete structure-activity relationships for the allosteric antagonism of NMDA receptors that may facilitate the development of NMDA receptor modulator agents for a variety of neuropsychiatric and neurological conditions.

A novel family of negative and positive allosteric modulators of NMDA receptors.

The N-methyl-D-aspartate (NMDA) receptor family regulates various central nervous system functions, such as synaptic plasticity. However, hypo- or hyperactivation of NMDA receptors is critically involved in many neurological and psychiatric conditions, such as pain, stroke, epilepsy, neurodegeneration, schizophrenia, and depression. Consequently, subtype-selective positive and negative modulators of NMDA receptor function have many potential therapeutic applications not addressed by currently available compounds. We have identified allosteric modulators with several novel patterns of NMDA receptor subtype selectivity that have a novel mechanism of action. In a series of carboxylated naphthalene and phenanthrene derivatives, compounds were identified that selectively potentiate responses at GluN1/GluN2A [e.g., 9-iodophenanthrene-3-carboxylic acid (UBP512)]; GluN1/GluN2A and GluN1/GluN2B [9-cyclopropylphenanthrene-3-carboxylic acid (UBP710)]; GluN1/GluN2D [3,5-dihydroxynaphthalene-2-carboxylic acid (UBP551)]; or GluN1/GluN2C and GluN1/GluN2D receptors [6-, 7-, 8-, and 9-nitro isomers of naphth[1,2-c][1,2,5]oxadiazole-5-sulfonic acid (NSC339614)] and have no effect or inhibit responses at the other NMDA receptors. Selective inhibition was also observed; UBP512 inhibits only GluN1/GluN2C and GluN1/GluN2D receptors, whereas 6-bromo-2-oxo-2H-chromene-3-carboxylic acid (UBP608) inhibits GluN1/GluN2A receptors with a 23-fold selectivity compared with GluN1/GluN2D receptors. The actions of these compounds were not competitive with the agonists L-glutamate or glycine and were not voltage-dependent. Whereas the N-terminal regulatory domain was not necessary for activity of either potentiators or inhibitors, segment 2 of the agonist ligand-binding domain was important for potentiating activity, whereas subtype-specific inhibitory activity was dependent upon segment 1. In terms of chemical structure, activity profile, and mechanism of action, these modulators represent a new class of pharmacological agents for the study of NMDA receptor subtype function and provide novel lead compounds for a variety of neurological disorders.

N-methyl-D-aspartate (NMDA) receptor NR2 subunit selectivity of a series of novel piperazine-2,3-dicarboxylate derivatives: preferential blockade of extrasynaptic NMDA receptors in the rat hippocampal CA3-CA1 synapse.

N-Methyl-d-aspartate (NMDA) receptor antagonists that are highly selective for specific NMDA receptor 2 (NR2) subunits have several potential therapeutic applications; however, to date, only NR2B-selective antagonists have been described. Whereas most glutamate binding site antagonists display a common pattern of NR2 selectivity, NR2A > NR2B > NR2C > NR2D (high to low affinity), (2S*,3R*)-1-(phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid (PPDA) has a low selectivity for NR2C- and NR2D-containing NMDA receptors. A series of PPDA derivatives were synthesized and then tested at recombinant NMDA receptors expressed in Xenopus laevis oocytes. In addition, the optical isomers of PPDA were resolved; the (-) isomer displayed a 50- to 80-fold greater potency than the (+) isomer. Replacement of the phenanthrene moiety of PPDA with naphthalene or anthracene did not improve selectivity. However, phenylazobenzoyl (UBP125) or phenylethynylbenzoyl (UBP128) substitution significantly improved selectivity for NR2B-, NR2C-, and NR2D-containing receptors over NR2A-containing NMDA receptors. Phenanthrene attachment at the 3 position [(2R*,3S*)-1-(phenanthrene-3-carbonyl)piperazine-2,3-dicarboxylic acid (UBP141); (2R*,3S*)-1-(9-bromophenanthrene-3-carbonyl)piperazine-2,3-dicarboxylic acid (UBP145); (2R*,3S*)-1-(9-chlorophenanthrene-3-carbonyl)piperazine-2,3-dicarboxylic acid (UBP160); and (2R*,3S*)-1-(9-iodophenanthrene-3-carbonyl)piperazine-2,3-dicarboxylic acid (UBP161)] displayed improved NR2D selectivity. UBP141 and its 9-brominated homolog (UBP145) both display a 7- to 10- fold selectivity for NR2D-containing receptors over NR2B- or NR2A-containing receptors. Schild analysis indicates that these two compounds are competitive glutamate binding site antagonists. Consistent with a physiological role for NR2D-containing receptors in the hippocampus, UBP141 (5 muM) displayed greater selectivity than PPDA for inhibiting the slow-decaying component of the NMDA receptor-mediated CA3-CA1 synaptic response in rat hippocampal slices. UBP125, UBP128, UBP141, and UBP145 may be useful tools for determining the function of NMDA receptor subtypes.