Jeffery N. Talbot

Identification of small-molecule inhibitors of RGS4 using a high-throughput flow cytometry protein interaction assay.

Regulators of G-protein signaling (RGS) proteins are important components of signal transduction pathways initiated through G-protein-coupled receptors (GPCRs). RGS proteins accelerate the intrinsic GTPase activity of G-protein α-subunits (Gα) and thus shorten the time course and reduce the magnitude of G-protein α- and βγ-subunit signaling. Inhibiting RGS action has been proposed as a means to enhance the activity and specificity of GPCR agonist drugs, but pharmacological targeting of protein-protein interactions has typically been difficult. The aim of this project was to identify inhibitors of RGS4. Using a Luminex 96-well plate bead analyzer and a novel flow-cytometric protein interaction assay to assess Gα-RGS interactions in a high-throughput screen, we identified the first small-molecule inhibitor of an RGS protein. Of 3028 compounds screened, 1, methyl N-[(4-chlorophenyl)sulfonyl]-4-nitrobenzenesulfinimidoate (CCG-4986), inhibited RGS4/Gαo binding with 3 to 5 μM potency. It binds to RGS4, inhibits RGS4 stimulation of Gαo GTPase activity in vitro, and prevents RGS4 regulation of μ-opioid-inhibited adenylyl cyclase activity in permeabilized cells. Furthermore, CCG-4986 is selective for RGS4 and does not inhibit RGS8. Thus, we demonstrate the feasibility of targeting RGS/Gα protein-protein interactions with small molecules as a novel means to modulate GPCR-mediated signaling processes.

RanBPM, a Scaffolding Protein in the Immune and Nervous Systems

We review the literature for Ran Binding Protein in the Microtubule-Organizing Center (RanBPM; RanBP9), a 90-kDa protein that possesses many characteristics of a scaffolding protein, including protein-interaction motifs, a cytoskeletal-binding domain, and multiple canonical docking sites for signaling intermediates. We focus on studies that have examined functional interactions between RanBPM and other proteins. These studies suggest that RanBPM provides a platform for the interaction of a variety of signaling proteins, including cell surface receptors, nuclear receptors, nuclear transcription factors, and cytosolic kinases. These studies indicate that RanBPM acts as a scaffolding protein and is important in regulating cellular function in both the immune system and the nervous system.

α1A- and α1B-Adrenergic Receptors Differentially Modulate Antidepressant-Like Behavior in the Mouse

Tricyclic antidepressant (TCA) drugs are used for the treatment of chronic depression, obsessive-compulsive disorder (OCD), and anxiety-related disorders. Chronic use of TCA drugs increases the expression of alpha(1)-adrenergic receptors (alpha(1)-ARs). Yet, it is unclear whether increased alpha(1)-AR expression contributes to the antidepressant effects of these drugs or if this effect is unrelated to their therapeutic benefit. In this study, mice expressing constitutively active mutant alpha(1A)-ARs (CAM alpha(1A)-AR) or CAM alpha(1B)-ARs were used to examine the effects of alpha(1A)- and alpha(1B)-AR signaling on rodent behavioral models of depression, OCD, and anxiety. CAM alpha(1A)-AR mice, but not CAM alpha(1B)-AR mice, exhibited antidepressant-like behavior in the tail suspension test and forced swim test. This behavior was reversed by prazosin, a selective alpha(1)-AR inverse agonist, and mimicked by chronically treating wild type mice with cirazoline, an alpha(1A)-AR agonist. Marble burying behavior, commonly used to model OCD in rodents, was significantly decreased in CAM alpha(1A)-AR mice but not in CAM alpha(1B)-AR mice. In contrast, no significant differences in anxiety-related behavior were observed between wild type, CAM alpha(1A)-AR, and CAM alpha(1B)-AR animals in the elevated plus maze and light/dark box. This is the first study to demonstrate that alpha(1A)- and alpha(1B)-ARs differentially modulate antidepressant-like behavior in the mouse. These data suggest that alpha(1A)-ARs may be a useful therapeutic target for the treatment of depression.

Sex-specific impairment of spatial memory in rats following a reminder of predator stress

Abstract It has been suggested that cognitive impairments exhibited by people with post-traumatic stress disorder (PTSD) result from intrusive, flashback memories transiently interfering with ongoing cognitive processing. Researchers have further speculated that females are more susceptible to developing PTSD because they form stronger traumatic memories than males, hence females may be more sensitive to the negative effects of intrusive memories on cognition. We have examined how the reminder of a naturalistic stress experience would affect rat spatial memory and if sex was a contributing factor to such effects. Male and female Sprague–Dawley rats were exposed, without contact, to an adult female cat for 30 min. Five weeks later, the rats were trained to locate a hidden platform in the radial-arm water maze and given a single long-term memory test trial 24 h later. Before long-term memory testing, the rats were given a 30-min reminder of the cat exposure experienced 5 weeks earlier. The results indicated that the stress reminder impaired spatial memory in the female rats only. Control manipulations revealed that this effect was not attributable to the original cat exposure adversely impacting learning that occurred 5 weeks later, or to merely exposing rats to a novel environment or predator-related cues immediately before testing. These findings provide evidence that the reminder of a naturalistic stressful experience can impair cognitive processing in rats; moreover, since female rats were more susceptible to the memory-impairing effects of the stress reminder, the findings could lend insight into the existing sex differences in susceptibility to PTSD.

Blunted Corticosterone Response to Acute Predator Stress Results in Long-Term Spatial Memory Impairment

Clinical research suggests that a blunted corticosteroid response to trauma may be associated with increased risk of developing post-traumatic stress disorder (PTSD). Therefore, we examined the influence of a blunted corticosterone response to stress on the development of PTSD-like behaviors, such as increased anxiety and impaired learning and memory, in rats. Male Sprague-Dawley rats were injected with metyrapone, an inhibitor of corticosterone synthesis, or vehicle prior to being exposed to an adult female cat for one hour. One week later, the rats were tested for anxiety-like behaviors (e.g., reduced open arm exploration, decreased locomotor activity) on an elevated plus maze and for spatial learning and memory in the radial-arm water maze. Analyses of post-stress serum samples verified that metyrapone blocked the stressinduced increase of corticosterone levels. Behaviorally, the blunted corticosterone response to stress had no effect on the development of anxiety-like behaviors. On the other hand, we found that metyrapone administration prior to stress significantly impaired long-term spatial memory. These findings suggest that reduced corticosteroid levels could interact with stress to produce deleterious effects on cognitive performance. Moreover, because anxiety-like behaviors were not intensified by the blunted corticosteroid response to stress, our findings also suggest that specific physiological responses to an acute trauma may intensify some, but not all, PTSD-like symptoms.

Peptide III-BTD

Peptide III-BTD is a relatively non-selective antagonist at the opioid receptor-like receptor ( …