Identification of the corticotropin-releasing factor receptor 1 antagonists as inhibitors of Chikungunya virus replication using a Gaussia luciferase-expressing subgenomic replicon.

The Chikungunya virus (CHIKV), an enveloped RNA virus that has been identified in over 40 countries and is considered a growing threat to public health worldwide. However, there is no preventive vaccine or specific therapeutic drug for CHIKV infection. To identify a new inhibitor against CHIKV infection, this study constructed a subgenomic RNA replicon expressing the secretory Gaussia luciferase (Gluc) based on the CHIKV SL11131 strain. Transfection of in vitro-transcribed replicon RNA to BHK-21 cells revealed that Gluc activity in culture supernatants was correlated with the intracellular replication of the replicon genome. Through a chemical compound library screen using the Gluc reporter CHIKV replicon, we identified several compounds that suppressed CHIKV infection in Vero cells. Among the hits identified, CP-154,526, a non-peptide antagonist of the corticotropin-releasing factor receptor type-1 (CRF-R1), showed the strongest anti-CHIKV activity and inhibited CHIKV infection in Huh-7 cells. Interestingly, other CRF-R1 antagonists, R121919 and NGD 98-2, also exhibited inhibitory effects on CHIKV infection. Time-of-drug addition and virus entry assays indicated that CP-154,526 suppressed a post-entry step of infection, suggesting that CRF-R1 antagonists acted on a target in the intracellular replication process of CHIKV. Therefore, the Gluc reporter replicon system established in this study would greatly facilitate the development of antiviral drugs against CHIKV infection.

Sex-specific plasticity in CRF regulation of inhibitory control in central amygdala CRF1 neurons after chronic voluntary alcohol drinking.

Despite strong preclinical evidence for the ability of corticotropin releasing factor 1 (CRF1) antagonists to regulate alcohol consumption, clinical trials have not yet demonstrated therapeutic effects of these compounds in alcohol use disorder (AUD) patients. Several confounding factors may limit the translation of preclinical CRF1 research to patients, including reliance on experimenter-administered alcohol instead of voluntary consumption, a preponderance of evidence collected in male subjects only and an inability to assess the effects of alcohol on specific brain circuits. A population of particular interest is the CRF1-containing neurons of the central amygdala (CeA). CRF1 CeA neurons are sensitive to ethanol, but the effects of alcohol drinking on CRF signalling within this population are unknown. In the present study, we assessed the effects of voluntary alcohol drinking on inhibitory control of CRF1+ CeA neurons from male and female CRF1:GFP mice using ex vivo electrophysiology and determined the contributions of CRF1 signalling to inhibitory control and voluntary alcohol drinking. Chronic alcohol drinking produced neuroadaptations in CRF1+ neurons that increased the sensitivity of GABAA receptor-mediated sIPSCs to the acute effects of alcohol, CRF and the CRF1 antagonist R121919, but these adaptations were more pronounced in male versus female mice. The CRF1 antagonist CP-154,526 reduced voluntary alcohol drinking in both sexes and abolished sex differences in alcohol drinking. The lack of alcohol-induced adaptation in the female CRF1 system may be related to the elevated alcohol intake exhibited by female mice and could contribute to the ineffectiveness of CRF1 antagonists in female AUD patients.

Blockade of corticotropin-releasing factor-1 receptors in the infralimbic cortex prevents stress-induced reinstatement of alcohol seeking in male Wistar rats: Evidence of interaction between CRF1 and orexin receptor signaling.

Alcohol use dysregulates responsivity to stress, which is mediated by corticotropin-releasing factor (CRF). With repeated cycles of alcohol use, the hypothalamic-pituitary-adrenal axis becomes hyporesponsive, rendering individuals vulnerable to the reinstatement of alcohol-seeking behavior during stressful episodes. Orexin (Orx; also called hypocretin) plays a well-established role in regulating diverse physiological processes, including stress, and interacts with CRF. The infralimbic cortex (IL) is a CRF-rich region. Anatomical evidence suggests that CRF and Orx interact in this area. To test the behavioral implication of CRF and Orx transmission in the IL during the stress-induced reinstatement of alcohol-seeking behavior, male Wistar rats were trained to self-administer 10% alcohol for 3 weeks. The rats then underwent two weeks of extinction training (identical to the alcohol self-administration sessions, but alcohol was withheld). The day after the last extinction session, the rats received a bilateral intra-IL injection of the CRF1 receptor antagonist CP154,526 (0.6 µg/0.5 µl/side), the dual Orx receptor antagonist TCS1102 (15 µg/0.5 µl/side), or their combination and then were tested for the footshock stress-induced reinstatement of alcohol-seeking behavior. CP154,526 significantly prevented reinstatement, but TCS1102 did not produce such an effect. Interestingly, the co-administration of TCS1102 and CP154,526 reversed the effect of CP154,526 alone, and footshock stress induced a significant increase in Crhr1 and Hcrtr2 mRNA expression in the IL. These results demonstrate a functional interaction between Orx receptor and CRF1 receptor signaling and suggest that CRF1 receptor antagonism may ameliorate stress-induced alcohol-seeking behavior.

Antagonism of corticotropin-releasing factor CRF1 receptors blocks the enhanced response to cocaine after social stress.

Numerous studies have shown that social defeat stress induces an increase in the rewarding effects of cocaine. In this study we have investigated the role played by the main hypothalamic stress hormone, corticotropin-releasing factor (CRF), in the effects that repeated social defeat (RSD) induces in the conditioned rewarding effects and locomotor sensitization induced by cocaine. A total of 220 OF1 mice were divided into experimental groups according to the treatment received before each social defeat: saline, 5 or 10 mg/kg of the nonpeptidic corticotropin-releasing factor CRF1 receptor antagonist CP-154,526, or 15 or 30 µg/kg of the peptidic corticotropin-releasing factor CRF2 receptor antagonist Astressin2-B. Three weeks after the last defeat, conditioned place preference (CPP) induced by 1 mg/kg of cocaine was evaluated. Motor response to 10 mg/kg of cocaine was also studied after a sensitization induction. Blockade of corticotropin-releasing factor CRF1 receptor reversed the increase in cocaine CPP induced by social defeat. Conversely, peripheral corticotropin-releasing factor CRF2 receptor blockade produced similar effects to those observed in socially stressed animals. The effect of RSD on cocaine sensitization was again blocked by the corticotropin-releasing factor CRF1 receptor antagonist, while peripheral CRF2 receptor antagonist did not show effect. Acute administration of Astressin2-B induced an anxiogenic response. Our results confirm that CRF modulates the effects of social stress on reinforcement and sensitization induced by cocaine in contrasting ways. These findings highlight CRF receptors as potential therapeutic targets to be explored by research about stress-related addiction problems.

CRF1 but not glucocorticoid receptor antagonists reduce separation-induced distress vocalizations in guinea pig pups and CRF overexpressing mouse pups. A combination study with paroxetine.

RATIONALE: Given the large number of patients that does not respond sufficiently to currently available treatment for anxiety disorders, there is a need for improved treatment. OBJECTIVES: We evaluated the anxiolytic effects of corticotropin releasing factor (CRF)1 receptor antagonists and glucocorticoid receptor (GR) antagonists in the separation-induced vocalization test in guinea pigs and transgenic mice with central CRF overexpression. Furthermore, we explored effects of these drugs when given in combination with a suboptimal dose of a selective serotonin re-uptake inhibitor (SSRI). METHODS: In guinea pig pups, the CRF1 receptor antagonists CP-154,526 and DMP695, and the GR antagonists mifepristone and Org34517 (all at 2.5, 10 and 40mg/kg intraperitoneally (IP)) were tested alone or in combination with 0.63mg/kg paroxetine IP. In CRF overexpressing mouse pups and wild type littermates, effects of CP-154,526 (10, 20 and 40mg/kg subcutaneously (SC)) and mifepristone (5, 15, 45mg/kg SC) were studied alone or in combination with 0.03mg/kg paroxetine SC. RESULTS: CRF1 but not GR antagonists reduced the number of calls relative to vehicle in guinea pigs and mice, independent of genotype. Treatment of CRF1 receptor or GR antagonists with paroxetine had no combined effect in guinea pigs, wild type or CRF overexpressing mice. CONCLUSIONS: Current results indicate robust anxiolytic properties of CRF1 receptor antagonists in guinea pigs and mice overexpressing CRF, and lack thereof of GR antagonists. Although no combined treatment effects were observed, it would be interesting to study combined treatment of CRF1 receptor antagonists with SSRIs following chronic drug administration.

Stress and Withdrawal from Chronic Ethanol Induce Selective Changes in Neuroimmune mRNAs in Differing Brain Sites.

Stress is a strong risk factor in alcoholic relapse and may exert effects that mimic aspects of chronic alcohol exposure on neurobiological systems. With the neuroimmune system becoming a prominent focus in the study of the neurobiological consequences of stress, as well as chronic alcohol exposure proving to be a valuable focus in this regard, the present study sought to compare the effects of stress and chronic ethanol exposure on induction of components of the neuroimmune system. Rats were exposed to either 1 h exposure to a mild stressor (restraint) or exposure to withdrawal from 15 days of chronic alcohol exposure (i.e., withdrawal from chronic ethanol, WCE) and assessed for neuroimmune mRNAs in brain. Restraint stress alone elevated chemokine (C-C motif) ligand 2 (CCL2), interleukin-1-beta (IL-1ß), tumor necrosis factor alpha (TNFα) and toll-like receptor 4 (TLR4) mRNAs in the cerebral cortex within 4 h with a return to a control level by 24 h. These increases were not accompanied by an increase in corresponding proteins. Withdrawal from WCE also elevated cytokines, but did so to varying degrees across different cytokines and brain regions. In the cortex, stress and WCE induced CCL2, TNFα, IL-1ß, and TLR4 mRNAs. In the hypothalamus, only WCE induced cytokines (CCL2 and IL-1ß) while in the hippocampus, WCE strongly induced CCL2 while stress and WCE induced IL-1ß. In the amygdala, only WCE induced CCL2. Finally-based on the previously demonstrated role of corticotropin-releasing factor 1 (CRF1) receptor inhibition in blocking WCE-induced cytokine mRNAs-the CRF1 receptor antagonist CP154,526 was administered to a subgroup of stressed rats and found to be inactive against induction of CCL2, TNFα, or IL-1ß mRNAs. These differential results suggest that stress and WCE manifest broad neuroimmune effects in brain depending on the cytokine and brain region, and that CRF inhibition may not be a relevant mechanism in non-alcohol exposed animals. Overall, these effects are complex in terms of their neuroimmune targets and neuroanatomical specificity. Further investigation of the differential distribution of cytokine induction across neuroanatomical regions, individual cell types (e.g., neuronal phenotypes and glia), severity of chronic alcohol exposure, as well as across differing stress types may prove useful in understanding differential mechanisms of induction and for targeting select systems for pharmacotherapeutic intervention in alcoholism.

Lifelong disturbance of serotonin transporter functioning results in fear learning deficits: Reversal by blockade of CRF1 receptors.

The inability to associate aversive events with relevant cues (i.e. fear learning) may lead to maladaptive anxiety. To further study the role of the serotonin transporter (SERT) in fear learning, classical fear conditioning was studied in SERT knockout rats (SERT(-/-)) using fear potentiation of the startle reflex. Next, fear acquisition and concomitant development of contextual conditioned fear were monitored during training. To differentiate between developmental and direct effects of reduced SERT functioning, effects of acute and chronic SSRI treatment were studied in adult rats. Considering the known interactions between serotonin and corticotropin-releasing factor (CRF), we studied the effect of the CRFR1 antagonist CP154,526 on behavioral changes observed and determined CRF1 receptor levels in SERT(-/-) rats. SERT(-/-) showed blunted fear potentiation and enhanced contextual fear, which resulted from a deficit in fear acquisition. Paroxetine treatment did not affect acquisition or expression of fear-potentiated startle, suggesting that disturbed fear learning in SERT(-/-) results from developmental changes and not from reduced SERT functioning. Although CRF1 receptor levels did not differ significantly between genotypes, CP154,526 treatment normalized both cue- and contextual fear in SERT(-/-) during acquisition, but not expression of fear-potentiated startle. The disrupted fear acquisition and concomitant increase in contextual conditioned fear-potentiated startle fear in SERT(-/-) resembles the associative learning deficit seen in patients with panic disorder and suggests that normal SERT functioning is crucial for the development of an adequate fear neuro-circuitry. Moreover, the normalization of fear acquisition by CP154,526 suggests a role for central CRF signaling in the generalization of fear.