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.

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.

Ethanol Stimulates Endoplasmic Reticulum Inositol Triphosphate and Sigma Receptors to Promote Withdrawal-Associated Loss of Neuron-Specific Nuclear Protein/Fox-3.

BACKGROUND: Prior studies demonstrate that ethanol (EtOH) exposure induces the release of intracellular calcium (CA(2+) ) in modulation of γ-aminobutyric acid-ergic tone and produces concomitant alterations in sigma (σ)-1 protein expression that may contribute to the development EtOH dependence. However, the influence of CA(2+) released from endoplasmic reticulum (ER)-bound inositol triphosphate (IP3) and σ-1 receptors in regulating hippocampal function has yet to be delineated. METHODS: Rat hippocampal explants were subjected to chronic intermittent EtOH (CIE) exposure with or without the addition of IP3 inhibitor xestospongin C (0 to 0.5 µM) or σ-1 receptor antagonist BD-1047 (0 to 80 µM). Hippocampal viability was assessed via immunohistochemical labeling of neuron-specific nuclear protein (NeuN)/Fox-3 in CA1, CA3, and dentate gyrus (DG) subregions. RESULTS: Exposure to CIE produced consistent and significant decreases of NeuN/Fox-3 in each primary cell layer of the hippocampal formation. Co-exposure to xestospongin reversed these effects in the CA1 subregion and significantly attenuated these effects in the CA3 and DG regions. Xestospongin application also significantly increased NeuN/Fox-3 immunofluorescence in EtOH-naïve hippocampi. Co-exposure to 20 µM BD-1047 also reversed the loss of NeuN/Fox-3 during CIE exposure in each hippocampal cell layer, whereas exposure to 80 µM BD-1047 did not alter NeuN/Fox-3 in EtOH-treated hippocampi. By contrast, 80 µM BD-1047 application significantly increased NeuN/Fox-3 immunofluorescence in EtOH-naïve hippocampi in each subregion. CONCLUSIONS: These data suggest that EtOH stimulates ER IP3 and σ-1 receptors to promote hippocampal loss of NeuN/Fox-3 during CIE.

Repeated subcutaneous administration of PT150 has dose-dependent effects on sign tracking in male Japanese quail.

A devastating feature of drug dependence is the susceptibility of relapse (40-60%) after stretches of abstinence. In both animal and human research, it has been demonstrated that cues (e.g., levers, paraphernalia) associated with drug reward can instigate renewed drug taking. Research has shown animals that attend to a cue that predicts reward more than the location of reward delivery when the cue is present (sign trackers) have an increase in corticosterone (CORT), a primary stress hormone when compared with animals that do not sign track. This interaction of sign tracking and CORT implicate CORT's effects as a possible pharmacological target for cue-induced relapse behaviors. PT150 is a novel glucocorticoid receptor antagonist that reduces the effects of CORT. Previous research has shown that oral administration of 40 mg/kg PT150 reduced sign tracking. To better understand dose-dependent effects and to control for more accurate doses, the current experiment hypothesized that PT150 (20/40/60 mg/kg) given by subcutaneous (SC) injection to male quail would reduce sign tracking to a keylight conditional stimulus that predicts a grain unconditioned stimulus dose dependently. Results showed that SC injection of 20 mg/kg PT150 reduced sign tracking, but 40 or 60 mg/kg did not. The main findings from the current study are that the glucocorticoid receptor antagonist PT150 reduces sign tracking behavior dose dependently, and SC administration may provide better bioavailability compared with our previous study that used an oral route of administration. The current findings support previous literature by suggesting that the glucocorticoid receptor may be a potential pharmacological target for reducing relapse-like behaviors. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

An improved model of ethanol and nicotine co-use in female P rats: Effects of naltrexone, varenicline, and the selective nicotinic α6ß2* antagonist r-bPiDI.

Background Although pharmacotherapies are available for alcohol (EtOH) or tobacco use disorders individually, it may be possible to develop a single pharmacotherapy to treat heavy drinking tobacco smokers by capitalizing on the commonalities in their mechanisms of action. Methods Female alcohol-preferring (P) rats were trained for EtOH drinking and nicotine self-administration in two phases: (1) EtOH alone (0 vs. 15% EtOH, 2-bottle choice) and (2) concomitant access, during which EtOH access continued with access to nicotine (0.03 mg/kg/infusion, i.v.) using a 2-lever choice procedure (active vs. inactive lever) in which the fixed ratio (FR) requirement was gradually increased to FR30. When stable co-use was obtained, rats were pretreated with varying doses of naltrexone, varenicline, or r-bPiDI, an α6ß2* subtype-selective nicotinic acetylcholine receptor antagonist shown previously to reduce nicotine self-administration. Results While EtOH intake was initially suppressed in phase 2 (co-use), pharmacologically relevant intake for both substances was achieved by raising the "price" of nicotine to FR30. In phase 2, naltrexone decreased EtOH and water consumption but not nicotine intake; in contrast, naltrexone in phase 1 (EtOH only) did not significantly alter EtOH intake. Varenicline and r-bPiDI in phase 2 both decreased nicotine self-administration and inactive lever pressing, but neither altered EtOH or water consumption. Conclusions These results indicate that increasing the "price" of nicotine increases EtOH intake during co-use. Additionally, the efficacy of naltrexone, varenicline, and r-bPiDI was specific to either EtOH or nicotine, with no efficacy for co-use. Nevertheless, future studies on combining these treatments may reveal synergistic efficacy.

Effects of the nicotinic agonist varenicline, nicotinic antagonist r-bPiDI, and DAT inhibitor (R)-modafinil on co-use of ethanol and nicotine in female P rats.

RATIONALE: Co-users of alcohol and nicotine are the largest group of polysubstance users worldwide. Commonalities in mechanisms of action for ethanol (EtOH) and nicotine proposes the possibility of developing a single pharmacotherapeutic to treat co-use. OBJECTIVES: Toward developing a preclinical model of co-use, female alcohol-preferring (P) rats were trained for voluntary EtOH drinking and i.v. nicotine self-administration in three phases: (1) EtOH alone (0 vs. 15%, two-bottle choice), (2) nicotine alone (0.03 mg/kg/infusion, active vs. inactive lever), and (3) concurrent access to both EtOH and nicotine. Using this model, we examined the effects of (1) varenicline, a nicotinic acetylcholine receptor (nAChR) partial agonist with high affinity for the α4ß2* subtype; (2) r-bPiDI, a subtype-selective antagonist at α6ß2* nAChRs; and (3) (R)-modafinil, an atypical inhibitor of the dopamine transporter (DAT). RESULTS: In phases 1 and 2, pharmacologically relevant intake of EtOH and nicotine was achieved. In the concurrent access phase (phase 3), EtOH consumption decreased while nicotine intake increased relative to phases 1 and 2. For drug pretreatments, in the EtOH access phase (phase 1), (R)-modafinil (100 mg/kg) decreased EtOH consumption, with no effect on water consumption. In the concurrent access phase, varenicline (3 mg/kg), r-bPiDI (20 mg/kg), and (R)-modafinil (100 mg/kg) decreased nicotine self-administration but did not alter EtOH consumption, water consumption, or inactive lever pressing. CONCLUSIONS: These results indicate that therapeutics which may be useful for smoking cessation via selective inhibition of α4ß2* or α6ß2* nAChRs, or DAT inhibition, may not be sufficient to treat EtOH and nicotine co-use.

Broad-spectrum protein kinase inhibition by the staurosporine analog KT-5720 reverses ethanol withdrawal-associated loss of NeuN/Fox-3.

Chronic, intermittent ethanol (CIE) exposure is known to produce neuroadaptive alterations in excitatory neurotransmission that contribute to the development of dependence. Although activation of protein kinases (e.g., cyclic AMP [cAMP]-dependent protein kinase) is implicated in the synaptic trafficking of these receptors following CIE exposure, the functional consequences of these effects are yet to be fully understood. The present study sought to delineate the influence of protein kinase in regulating cytotoxicity following CIE exposure, as well as to examine the relative roles of ethanol exposure and ethanol withdrawal (EWD) in promoting these effects. Rat hippocampal explants were exposed to a developmental model of CIE with or without co-application of broad-spectrum protein kinase inhibitor KT-5720 (1 µM) either during ethanol exposure or EWD. Hippocampal cytotoxicity was assessed via immunofluorescence (IF) of neuron-specific nuclear protein (NeuN) with thionine staining of Nissl bodies to confirm IF findings. Concomitant application of ethanol and KT-5720 restored the loss of NeuN/Fox-3 IF in pyramidal CA1 and granule DG cell layers produced by CIE, but there was no restoration in CA3. Application of KT-5720 during EWD failed to significantly alter levels of NeuN IF, implying that ethanol exposure activates protein kinases that, in part, mediate the effects of EWD. KT-5720 application during EWD also restored thionine staining in CA1, suggesting kinase regulation of both neurons and non-neuronal cells. These data demonstrate that CIE exposure alters protein kinase activity to promote ethanol withdrawal-associated loss of NeuN/Fox-3 and highlight the influence of kinase signaling on distinct cell types in the developing hippocampus.

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.

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.

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.

Terfestatins B and C, New p-Terphenyl Glycosides Produced by Streptomyces sp. RM-5-8.

Terfestatins B (1) and C (2), new p-terphenyls bearing a novel unsaturated hexuronic acid (4-deoxy-α-L-threo-hex-4-enopyranuronate), a unique ß-D-glycosyl ester of 5-isoprenylindole-3-carboxylate (3) and the same rare sugar, and two new hygromycin precursors, were characterized as metabolites of the coal mine fire isolate Streptomyces sp. RM-5-8. EtOH damage neuroprotection assays using rat hippocampal-derived primary cell cultures with 1, 2, 3 and echoside B (a terfestatin C-3'-ß-D-glucuronide from Streptomyces sp. RM-5-8) revealed 1 as potently neuroprotective, highlighting a new potential application of the terfestatin scaffold.