Meredith A. Saunders

Acute oral administration of the novel, competitive and selective glucocorticoid receptor antagonist ORG 34517 reduces the severity of ethanol withdrawal and related hypothalamic–pituitary–adrenal axis activation

BACKGROUND The development of ethanol dependence is associated with alterations in hypothalamic-pituitary-adrenal (HPA) axis and activation of type II glucocorticoid receptors (GR). These effects may contribute to withdrawal-associated anxiety, craving and relapse to drinking. The present studies examined acute and oral administration of the novel, selective and competitive GR antagonist ORG 34517 on the severity of ethanol withdrawal. METHODS Adult, male Sprague-Dawley rats were administered ethanol (4g/kg/i.g.) twice daily for 5 days followed by 2 days of withdrawal for 1, 2 or 3 consecutive cycles. Blood ethanol levels (BELs) were determined at 0930 on Day 4 of each week, while blood corticosterone levels (BCLs) were obtained at 11:00hours on the first day of each ethanol withdrawal. During early withdrawal, subjects received oral administration of ORG 345617 (60mg/kg/i.g.) or a placebo and withdrawal was monitored. RESULTS Peak BELs of 225.52mg/dl were observed during the third week. Withdrawal from three cycles of the regimen produced marked behavioral abnormalities (e.g., aggression, rigidity, and hypoactivity) and significant increases in BCLs of ethanol-dependent subjects. Acute, oral administration of ORG 34517 during early withdrawal significantly reduced both the severity of ethanol withdrawal, as reflected in reduced rigidity, aggression, and hypoactivity, and elevations in BCL without producing any sedative-like effects. CONCLUSIONS The present findings demonstrate that repeated ethanol exposure and withdrawal is associated with significant behavioral abnormalities and dysregulation of HPA axis activation. Further these data suggest that selective GR antagonists should be further considered as putative pharmacotherapies for treatment of ethanol dependence.

Spoxazomicin D and Oxachelin C, Potent Neuroprotective Carboxamides from the Appalachian Coal Fire-Associated Isolate Streptomyces sp. RM-14-6

The isolation and structure elucidation of six new bacterial metabolites [spoxazomicin D (2), oxachelins B and C (4, 5), and carboxamides 6-8] and 11 previously reported bacterial metabolites (1, 3, 9-12a, and 14-18) from Streptomyces sp. RM-14-6 is reported. Structures were elucidated on the basis of comprehensive 1D and 2D NMR and mass spectrometry data analysis, along with direct comparison to synthetic standards for 2, 11, and 12a,b. Complete 2D NMR assignments for the known metabolites lenoremycin (9) and lenoremycin sodium salt (10) were also provided for the first time. Comparative analysis also provided the basis for structural revision of several previously reported putative aziridine-containing compounds [exemplified by madurastatins A1, B1, C1 (also known as MBJ-0034), and MBJ-0035] as phenol-dihydrooxazoles. Bioactivity analysis [including antibacterial, antifungal, cancer cell line cytotoxicity, unfolded protein response (UPR) modulation, and EtOH damage neuroprotection] revealed 2 and 5 as potent neuroprotectives and lenoremycin (9) and its sodium salt (10) as potent UPR modulators, highlighting new functions for phenol-oxazolines/salicylates and polyether pharmacophores.

Group 1 mGlu-family proteins promote neuroadaptation to ethanol and withdrawal-associated hippocampal damage

BACKGROUND Group 1 mGlu-family proteins (i.e., mGlu) consist of mGlu1 and mGlu5 and their activity may influence voluntary ethanol intake. The present studies sought to examine the influence of these receptors on the development of ethanol dependence using in vitro and in vivo models of chronic, intermittent ethanol (CIE). METHODS Rat hippocampal explants were exposed to CIE with or without the addition of mGlu1 antagonist (7-hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt; 0.5, 1, and 3μM) or mGlu5 antagonist (E)-2-methyl-6-styryl-pyridine (SIB-1893; 20, 100, and 200μM) to assess sparing of withdrawal-induced cytotoxicity. In a separate study, adult male rats were administered CIE with or without the addition of oral administration of group 1 mGlu antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP; 3mg/kg). Blood ethanol levels (BELs) were determined at 0930h on Day 2 of Weeks 1, 2, and 3. Withdrawal behavior was monitored during Day 6 of the third consecutive withdrawal. RESULTS CIE produced significant hippocampal cytotoxicity. These effects were attenuated by co-exposure to CPCCOEt (3μM) with ethanol in the CA3. By contrast, these effects were blocked by SIB-1893 (20μM) in each primary cell layer. Oral administration of MPEP with ethanol significantly attenuated behavioral effects of subsequent withdrawal and reduced BELs. CONCLUSIONS These data demonstrate that ethanol activates group 1 mGlu-family proteins to promote withdrawal-associated cytotoxicity in vitro and physical dependence in vivo. These findings suggest that group 1 mGlu-family proteins may be therapeutic targets for treatment of alcohol use disorders.

Identification of Neuroprotective Spoxazomicin and Oxachelin Glycosides via Chemoenzymatic Glycosyl-Scanning

The assessment of glycosyl-scanning to expand the molecular and functional diversity of metabolites from the underground coal mine fire-associated Streptomyces sp. RM-14-6 is reported. Using the engineered glycosyltransferase OleD Loki and a 2-chloro-4-nitrophenylglycoside-based screen, six metabolites were identified as substrates of OleD Loki, from which 12 corresponding metabolite glycosides were produced and characterized. This study highlights the first application of the 2-chloro-4-nitrophenylglycoside-based screen toward an unbiased set of unique microbial natural products and the first reported application of the 2-chloro-4-nitrophenylglycoside-based transglycosylation reaction for the corresponding preparative synthesis of target glycosides. Bioactivity analysis (including antibacterial, antifungal, anticancer, and EtOH damage neuroprotection assays) revealed glycosylation to attenuate the neuroprotective potency of 4, while glycosylation of the structurally related inactive spoxazomicin C (3) remarkably invoked neuroprotective activity.

Corticosterone enhances N-methyl-D-aspartate receptor signaling to promote isolated ventral tegmental area activity in a reconstituted mesolimbic dopamine pathway.

Elevations in circulating corticosteroids during periods of stress may influence activity of the mesolimbic dopamine reward pathway by increasing glutamatergic N-methyl-D-aspartate (NMDA) receptor expression and/or function in a glucocorticoid receptor-dependent manner. The current study employed organotypic co-cultures of the ventral tegmental area (VTA) and nucleus accumbens (NAcc) to examine the effects of corticosterone exposure on NMDA receptor-mediated neuronal viability. Co-cultures were pre-exposed to vehicle or corticosterone (CORT; 1 μM) for 5 days prior to a 24 h co-exposure to NMDA (200 μM). Co-cultures pre-exposed to a non-toxic concentration of corticosterone and subsequently NMDA showed significant neurotoxicity in the VTA only. This was evidenced by increases in propidium iodide uptake as well as decreases in immunoreactivity of the neuronal nuclear protein (NeuN). Co-exposure to the NMDA receptor antagonist 2-amino-7-phosphonovaleric acid (APV; 50 μM) or the glucocorticoid receptor (GR) antagonist mifepristone (10 μM) attenuated neurotoxicity. In contrast, the combination of corticosterone and NMDA did not produce any significant effects on either measure within the NAcc. Cultures of the VTA and NAcc maintained without synaptic contact showed no response to CORT or NMDA. These results demonstrate the ability to functionally reconstitute key regions of the mesolimbic reward pathway ex vivo and to reveal a GR-dependent enhancement of NMDA receptor-dependent signaling in the VTA.

NOVEL SPOXAZOMICINS DERIVED FROM STREPTOMYCES SP. RM-14−6 ATTENUATE ETHANOL INDUCED CYTOTOXICITY IN VITRO

OF THESIS NOVEL SPOXAZOMICINS DERIVED FROM STREPTOMYCES SP. RM-14−6 ATTENUATE ETHANOL INDUCED CYTOTOXITY IN VITRO An estimated 13.9% of Americans currently meet criteria for an alcohol use disorder. Ultimately, chronic alcohol use may result in neurological deficits, with up to 85% of alcoholics exhibiting signs of cognitive decline. However, biochemical and behavioral factors contributing to this decline have remained elusive. Our ongoing research program encompasses a multi-tiered screening of a natural product library and validation process to provide novel information about mechanisms underlying these deficits and to identify novel chemical scaffolds to be exploited in the development of pharmacological treatments for alcohol use disorders in a rodent organotypic hippocampal slice culture mode. Experiment 1 sought to establish a 48 h high throughput model for testing novel scaffolds against ethanol (EtOH) toxicity. Experiment 2 tested multiple natural product compounds for their ability to attenuate ethanol-induced cytotoxicity. Results from Experiment 1 revealed EtOH (100 mM) induced significant cytotoxicity at 48 h. Trolox (100 μM), a potent antioxidant, was found to reduce ethanolinduced cytotoxicity in this assay. Experiment 2 revealed two spoxazomicins (1, 1-1) demonstrated potent cytoprotective effects against ethanol toxicity. These findings highlight the potential applications of these novel scaffolds for use in the treatment of alcohol use disorder.

Correction to “Terfestatins B and C, New p-Terphenyl Glycosides Produced by Streptomyces sp. RM-5–8”

In the Abstract, second paragraph on p 2796, and last paragraph on p 2798, “5-isoprenylindole-3-carboxylate” should be revised as “6-isoprenylindole-3-carboxylate”. Page 2798, first paragraph, lines 11−15 should be revised as “Support for the C-6 isoprenyl assignment derived from a key COSY correlation between H-1′/H-2′ and HMBC correlations from H2-1′ (δH 3.40, 2H) to C-7 (δC 111.3), C-6 (δC 135.9), C5 (δC 122.5), C-2′ (δC 123.8), and C-3′ (δC 131.3)...”. In the Supporting Information, the chemical structure and name of compound 3 and the corresponding H/C chemical shifts relating to compound 3, C-4, C-5, C-6, and C-7 in Table S2, have been revised accordingly.