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Autism spectrum disorders (ASDs) are characterized by core behavioral symptoms in the domains of sociability, language/communication, and repetitive or stereotyped behaviors. Deficits in the prefrontal and hippocampal excitatory/inhibitory balance due to a functional loss of GABAergic interneurons are proposed to underlie these symptoms. Increasing the postsynaptic effects of GABA with compounds that selectively modulate GABAergic receptors could be a potential target for treating ASD symptoms. In addition, deficits in GABAergic interneurons have been linked to dopamine (DA) system dysregulation, and, despite conflicting evidence, abnormalities in the DA system activity may underly some ASD symptoms. Here, we investigated whether the positive allosteric modulator of α5‐containing GABAA receptors (α5‐GABAARs) SH‐053‐2′F‐R‐CH3 (10 mg/kg) attenuates behavioral abnormalities in rats exposed to valproic acid (VPA) in utero, an established risk factor for autism. We also evaluated if animals exposed to VPA in utero present changes in the ventral tegmental area (VTA) DA system activity using in vivo electrophysiology and if SH‐053‐2′F‐R‐CH3 could attenuate these changes. SH‐053‐2′F‐R‐CH3 was administered intraperitoneally 30 min before each behavioral test and electrophysiology. In utero VPA exposure caused male and female rats to present increased repetitive behavior (self‐grooming) in early adolescence and deficits in social interaction in adulthood. Male, but not female VPA rats, also presented deficits in recognition memory as adults. SH‐053‐2′F‐R‐CH3 attenuated the impairments in sociability and cognitive function in male VPA‐exposed rats without attenuating the decreased social interaction in females. Adult male and female VPA‐exposed rats also showed an increased VTA DA neuron population activity, which was not changed by SH‐053‐2′F‐R‐CH3. Despite sex differences, our findings indicate that α5‐GABAARs positive allosteric modulators may effectively attenuate some core ASD symptoms. Lay Summary: Autism spectrum disorders (ASDs) are a group of conditions characterized by abnormalities in social interaction and communication, and repetitive behaviors. These deficits often stem from underlying imbalances in the brain's chemical messengers. Efforts to address it have led researchers to explore substances that can improve brain function in ASD. One promising candidate is GABA, a neurotransmitter critical for brain communication. Basic research has indicated that boosting GABA activity may alleviate some of the core ASD symptoms. Furthermore, some studies have uncovered a potential link between GABA and dopamine, another neurotransmitter implicated in ASD symptoms. In this study, we investigated the effects of SH‐053‐2′F‐R‐CH3, a drug that can potentiate GABA effects through the modulation of alpha 5‐containing GABAA receptors in rats exposed to valproic acid (VPA) in utero, an established risk factor for autism. Unlike female rats, male rats exhibited improved sociability and cognitive function after the administration of SH‐053‐2′F‐R‐CH3. Additionally, we found an increased activity of the midbrain dopamine system in VPA‐exposed rats, which was not attenuated by SH‐053‐2′F‐R‐CH3. Overall, despite sex differences, our findings indicate that boosting GABA activity through alpha 5‐containing GABAA receptors may attenuate some ASD symptoms. [ABSTRACT FROM AUTHOR]

Previous studies have identified an association between the gene glyoxalase 1 (Glo1) and anxiety-like behavior in mice and have shown that the substrate of GLO1, methylglyoxal, is a competitive partial agonist at GABAA receptors. Given the well-established role of GABAA receptors in the behavioral effects of ethanol (EtOH), we investigated the role of Glo1 in voluntary EtOH consumption in mice using the drinking in the dark (DID) paradigm. Transgenic mice overexpressing Glo1 on both FVB/NJ (FVB) or C57BL/6J (B6) backgrounds showed increased voluntary EtOH consumption compared to their wild-type littermates in DID. Furthermore, transgenic Glo1 knockdown mice on a B6 background showed decreased voluntary EtOH consumption in DID. These genetic manipulations of Glo1 had no effect on sucrose, saccharin or water consumption. Finally, we found that a small molecule GLO1 inhibitor (S-bromobenzylglutathione cyclopentyl diester (pBBG; 6.25, 12.5 mg/kg)) reduced EtOH consumption compared to vehicle treated B6 mice without altering saccharin or water consumption. Sucrose consumption was only reduced by the higher (12.5 mg/kg) dose of pBBG. We did not observe differences in the loss of righting reflex (LORR) or EtOH-induced foot slips on the balance beam in response to acute EtOH administration (LORR: 4 g/kg, Balance Beam: 1.25 g/kg) in B6 or FVB mice overexpressing Glo1, nor in B6 mice treated with pBBG. These data are the first to implicate Glo1 in EtOH-related behaviors and suggest that GLO1 inhibitors may have therapeutic potential for the treatment of alcohol use disorders.

A systematic study with phase 1 and phase 2 metabolites of cholesterol and Vitamin D was conducted to determine whether their biological activity is mediated by the Vitamin D receptor (VDR). The investigation necessitated the development of novel synthetic routes for lithocholic acid (LCA) glucuronides (Gluc). Biochemical and cell-based assays were used to demonstrate that hydroxylated LCA analogs were not able to bind VDR. This excludes VDR from mediating their biological and pharmacological activities. Among the synthesized LCA conjugates a novel VDR agonist was identified. LCA Gluc II increased the expression of CYP24A1 in DU145 cancer cells especially in the presence of the endogenous VDR ligand 1,25(OH)2D3.Furthermore, the methyl ester of LCA was identified as novel VDR antagonist. For the first time, we showed that calcitroic acid, the assumed inactive final metabolite of Vitamin D, was able to activate VDR-mediated transcription to a higher magnitude than bile acid LCA. Due to a higher metabolic stability in comparison to Vitamin D, a very low toxicity, and high concentration in bile and intestine, calcitroic acid is likely to be an important mediator of the protective Vitamin D properties against colon cancer.

AimTo investigate the in vivo effects of 3-indolylmethanamines 31B and PS121912 in treating ovarian cancer and leukemia, respectively.Materials and methodsTerminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and western blotting were applied to demonstrate the induction of apoptosis. Xenografted mice were investigated to show the antitumor effects of 3-indolylmethanamines. (13)C-Nuclear magnetic resource (NMR) and western blotting were used to demonstrate inhibition of glucose metabolism.Results31B inhibited ovarian cancer cell proliferation and activated caspase-3, cleaved poly (ADP-ribose) polymerase 1 (PARP1), and phosphorylated mitogen-activated protein kinases (MAPK), JUN N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38. 31B reduced ovarian cancer xenograft tumor growth and PS121912 inhibited the growth of HL-60-derived xenografts without any sign of toxicity. Compound 31B inhibited de novo glycolysis and lipogenesis mediated by the reduction of fatty acid synthase and lactate dehydrogenase-A expression.Conclusion3-Indolylmethanamines represent a new class of antitumor agents. We have shown for the first time the in vivo anticancer effects of 3-indolylmethanamines 31B and PS121912.

The development of targeted therapeutics for neuroblastoma, the third most common tumor in children, has been limited by a poor understanding of growth signaling mechanisms unique to the peripheral nerve precursors from which tumors arise. In this study, we combined genetics with gene-expression analysis in the peripheral sympathetic nervous system to implicate arginase 1 and GABA signaling in tumor formation in vivo. In human neuroblastoma cells, either blockade of ARG1 or benzodiazepine-mediated activation of GABA-A receptors induced apoptosis and inhibited mitogenic signaling through AKT and MAPK. These results suggest that ARG1 and GABA influence both neural development and neuroblastoma and that benzodiazepines in clinical use may have potential applications for neuroblastoma therapy.

PurposePS121912 has been developed as selective vitamin D receptor (VDR)-coregulator inhibitor starting from a high throughput screening campaign to identify new agents that modulate VDR without causing hypercalcemia. Initial antiproliferative effects of PS121912 were observed that are characterized herein to enable future in vivo investigation with this molecule.MethodsAntiproliferation and apoptosis were determined using four different cancer cell lines (DU145, Caco2, HL-60 and SKOV3) in the presence of PS121912, 1,25-(OH)₂D₃, or a combination of 1,25-(OH)₂D₃ and PS121912. VDR si-RNA was used to identify the role of VDR during this process. The application of ChIP enabled us to determine the involvement of coregulator recruitment during transcription, which was investigated by RT-PCR with VDR target genes and those affiliated with cell cycle progression. Translational changes of apoptotic proteins were determined with an antibody array. The preclinical characterization of PS121912 includes the determination of metabolic stability and CYP3A4 inhibition.ResultsPS121912 induced apoptosis in all four cancer cells, with HL-60 cells being the most sensitive. At sub-micromolar concentrations, PS121912 amplified the growth inhibition of cancer cells caused by 1,25-(OH)₂D₃ without being antiproliferative by itself. A knockout study with VDR si-RNA confirmed the mediating role of VDR. VDR target genes induced by 1,25-(OH)₂D₃ were down-regulated with the co-treatment of PS121912. This process was highly dependent on the recruitment of coregulators that in case of CYP24A1 was SRC2. The combination of PS121912 and 1,25-(OH)₂D₃ reduced the presence of SRC2 and enriched the occupancy of corepressor NCoR at the promoter site. E2F transcription factors 1 and 4 were down-regulated in the presence of PS121912 and 1,25-(OH)₂D₃ that in turn reduced the transcription levels of cyclin A and D, thus arresting HL-60 cells in the S or G2/M phase. In addition, proteins with hematopoietic functions such as cyclin-dependent kinase 6, histone deacetylase 9 and transforming growth factor beta 2 and 3 were down-regulated as well. Elevated levels of P21 and GADD45, in concert with cyclin D1, also mediated the antiproliferative response of HL-60 in the presence of 1,25-(OH)₂D₃ and PS121912. Studies at higher concentration of P121912 identified a VDR-independent pathway of antiproliferation that included the enzymatic and transcriptional activation of caspase 3/7.ConclusionOverall, we conclude that PS121912 behaves like a VDR antagonist at low concentrations but interacts with more targets at higher concentrations leading to apoptosis mediated by caspase 3/7 activation. In addition, PS121912 showed an acceptable metabolic stability to enable in vivo cancer studies.

Nuclear receptor coregulators are master regulators of transcription and selectively interact with the vitamin D receptor (VDR) to modulate cell differentiation, cell proliferation and calcium homeostasis. Herein, we report the syntheses and evaluation of highly potent and selective VDR-coactivator inhibitors based on a recently identified 3-indolylmethanamine scaffold. The most active compound, PS121912, selectively inhibited VDR-mediated transcription among eight other nuclear receptors tested. PS121912 is also selectively disrupting the binding between VDR and the third nuclear receptor interaction domain of the coactivator SRC2. Genetic studies revealed that PS121912 behaves like a VDR antagonist by repressing 1,25-(OH)2D3 activated gene transcription. In addition, PS121912 induced apoptosis in HL-60.

Hypotensive influences of benzodiazepines and other GABAA receptor ligands, recognized in clinical practice, seem to stem from the existence of "vascular" GABAA receptors in peripheral blood vessels, besides any mechanisms in the central and peripheral nervous systems. We aimed to further elucidate the vasodilatatory effects of ligands acting through GABAA receptors. Using immunohistochemistry, the rat aortic smooth muscle layer was found to express GABAA γ2 and α1-5 subunit proteins. To confirm the role of "vascular" GABAA receptors, we investigated the vascular effects of standard benzodiazepines, midazolam, and flumazenil, as well as the novel compound MP-III-058. Using two-electrode voltage clamp electrophysiology and radioligand binding assays, MP-III-058 was found to have modest binding but substantial functional selectivity for α5β3γ2 over other αxβ3γ2 GABAA receptors. Tissue bath assays revealed comparable vasodilatory effects of MP-III-058 and midazolam, both of which at 100 µmol/L concentrations had efficacy similar to prazosin. Flumazenil exhibited weak vasoactivity per se, but significantly prevented the relaxant effects of midazolam and MP-III-058. These studies indicate the existence of functional GABAA receptors in the rat aorta, where ligands exert vasodilatory effects by positive modulation of the benzodiazepine binding site, suggesting the potential for further quest for leads with optimized pharmacokinetic properties as prospective adjuvant vasodilators. [ABSTRACT FROM AUTHOR]

Insect‐specific viruses (ISVs) can affect insect health and fitness, but can also interact with other insect‐associated microorganisms. Despite this, ISVs are often studied in isolation from each other, in laboratory populations. Consequently, their diversity, prevalence and associations with other viruses in field populations are less known, yet these parameters are important to understanding virus epidemiology. To help address this knowledge gap, we assessed the diversity, prevalence and coinfections of three ISVs (horizontally transmitted cripavirus, biparentally transmitted sigmavirus and maternally transmitted iflavirus) in 29 field populations of Queensland fruit fly, Australia's most significant horticultural pest, in the context of their different transmission modes. We detected new virus variant diversity. In contrast to the very high virus prevalence in laboratory populations, 46.8% of 293 field flies carried one virus and 4.8% had two viruses. Cripavirus and sigmavirus occurred in all regions, while iflavirus was restricted to subtropical and tropical regions. Cripavirus was most prevalent (37.5%), followed by sigmavirus (13.7%) and iflavirus (4.4%). Cripavirus coinfected some flies with either one of the two vertically transmitted viruses. However, sigmavirus did not coinfect individuals with iflavirus. Three different modelling approaches detected negative association patterns between sigmavirus and iflavirus, consistent with the absence of such coinfections in laboratory populations. This may be linked with their maternal transmission and the ineffective paternal transmission of sigmavirus. Furthermore, we found that, unlike sigmavirus and iflavirus, cripavirus load was higher in laboratory than field flies. Laboratory and mass‐rearing conditions may increase ISV prevalence and load due to increased transmission opportunities. We conclude that a combination of field and laboratory studies is needed to uncover ISV interactions and further our understanding of ISV epidemiology. [ABSTRACT FROM AUTHOR]

Despite aggressive treatment approaches, the overall survival of glioblastoma (GBM) patients remained poor with a strong need for more effective chemotherapeutic agents. A previous study has shown that ARN14988 is more cytotoxic to GBM cells compared to US Food and Drug Administration‐approved temozolomide. This finding makes ARN14988 a desirable candidate for further pharmacological assessment. Therefore, an efficient analytical method is needed to quantify ARN14988. Herein, we have developed and validated sample preparation and LC–MS/MS triple quadrupole (QQQ) method for quantification of ARN14988 in mouse plasma. In this method, the liquid–liquid extraction of ARN14988 from mouse plasma was performed using 5% ethyl acetate in hexane. The chromatographic separation was achieved using a C18‐column with mobile phases of 10 mm ammonium acetate (pH 5) and 0.1% formic acid in methanol, within a runtime of 10 min. The monitored transitions were m/z 391.20 → m/z 147.00 for ARN14988, and m/z 455.30 → m/z 165.00 for verapamil (internal standard) in positive electrospray ionization. The developed method for ARN14988 showed linearity over the range of 10–5,000 ng/ml (r2 > 0.99). The selectivity, sensitivity, matrix effect, recovery, stability, inter‐day and intraday accuracy and precision were determined using four quality control samples. This validated method was successfully applied to the pharmacokinetic study of ARN14988 in mice. [ABSTRACT FROM AUTHOR]