Alterations in adolescent brain serotonin (5HT)1A, 5HT2A, and dopamine (D)2 receptor systems in a nonhuman primate model of early life adversity.

Stress affects brain serotonin (5HT) and dopamine (DA) function, and the effectiveness of 5HT and DA to regulate stress and emotional responses. However, our understanding of the long-term impact of early life adversity (ELA) on primate brain monoaminergic systems during adolescence is scarce and inconsistent. Filling this gap in the literature is critical, given that the emergence of psychopathology during adolescence has been related to deficits in these systems. Here, we use a translational nonhuman primate (NHP) model of ELA (infant maltreatment by the mother) to examine the long-term impact of ELA on adolescent 5HT1A, 5HT2A and D2 receptor systems. These receptor systems were chosen based on their involvement in stress/emotional control, as well as reward and reinforcement. Rates of maternal abuse, rejection, and infant's vocalizations were obtained during the first three postnatal months, and hair cortisol concentrations obtained at 6 months postnatal were examined as early predictors of binding potential (BP) values obtained during adolescence using positron emission tomography (PET) imaging. Maltreated animals demonstrated significantly lower 5HT1A receptor BP in prefrontal cortical areas as well as the amygdala and hippocampus, and lower 5HT2A receptor BP in striatal and prefrontal cortical areas. Maltreated animals also demonstrated significantly lower D2 BP in the amygdala. None of the behavioral and neuroendocrine measurements obtained early in life predicted any changes in BP data. Our findings suggest that early caregiving experiences regulate the development of brain 5HT and DA systems in primates, resulting in long-term effects evident during adolescence.

Vutiglabridin Alleviates Cellular Senescence with Metabolic Regulation and Circadian Clock in Human Dermal Fibroblasts.

The process of cellular senescence, which is characterized by stable cell cycle arrest, is strongly associated with dysfunctional cellular metabolism and circadian rhythmicity, both of which are reported to result from and also be causal to cellular senescence. As a result, modifying any of them-senescence, metabolism, or the circadian clock-may affect all three simultaneously. Obesity accelerates aging by disrupting the homeostasis of reactive oxygen species (ROS) via an increased mitochondrial burden of fatty acid oxidation. As a result, if senescence, metabolism, and circadian rhythm are all linked, anti-obesity treatments may improve metabolic regulation while also alleviating senescence and circadian rhythm. Vutiglabridin is a small molecule in clinical trials that improves obesity by enhancing mitochondrial function. We found that chronic treatment of senescent primary human dermal fibroblasts (HDFs) with vutiglabridin alleviates all investigated markers of cellular senescence (SA-ß-gal, CDKN1A, CDKN2A) and dysfunctional cellular circadian rhythm (BMAL1) while remarkably preventing the alterations of mitochondrial function and structure that occur during the process of cellular senescence. Our results demonstrate the significant senescence-alleviating effects of vutiglabridin, specifically with the restoration of cellular circadian rhythmicity and metabolic regulation. These data support the potential development of vutiglabridin against aging-associated diseases and corroborate the intricate link between cellular senescence, metabolism, and the circadian clock.

Sex influences the effects of social status on socioemotional behavior and serotonin neurochemistry in rhesus monkeys.

BACKGROUND: Despite observed sex differences in the prevalence of stress-related psychiatric conditions, most preclinical and translational studies have only included male subjects. Therefore, it has not been possible to effectively assess how sex interacts with other psychosocial risk factors to impact the etiology and maintenance of stress-related psychopathology. One psychosocial factor that interacts with sex to impact risk for stress-related behavioral and physiological deficits is social dominance. The current study was designed to assess sex differences in the effects of social status on socioemotional behavior and serotonin neurochemistry in socially housed rhesus monkeys. We hypothesized that sex and social status interact to influence socioemotional behaviors as well as serotonin 1A receptor binding potential (5HT1AR-BP) in regions of interest (ROIs) implicated in socioemotional behavior. METHODS: Behavioral observations were conducted in gonadally intact adult female (n = 14) and male (n = 13) rhesus monkeys. 5HT1AR-BP was assessed via positron emission tomography using 4-(2'-Methoxyphenyl)-1-[2'-(N-2"-pyridinyl)-p[18F]fluorobenzamido]ethylpiperazine ([18F]MPPF). RESULTS: Aggression emitted was greater in dominant compared to subordinate animals, regardless of sex. Submission emitted was significantly greater in subordinate versus dominant animals and greater in females than males. Affiliative behaviors emitted were not impacted by sex, status, or their interaction. Anxiety-like behavior emitted was significantly greater in females than in males regardless of social status. Hypothalamic 5HT1AR-BP was significantly greater in females than in males, regardless of social status. 5HT1AR-BP in the dentate gyrus of the hippocampus was significantly impacted by a sex by status interaction whereby 5HT1AR-BP in the dentate gyrus was greater in dominant compared to subordinate females but was not different between dominant and subordinate males. There were no effects of sex, status, or their interaction on 5HT1AR-BP in the DRN and in the regions of the PFC studied. CONCLUSIONS: These data have important implications for the treatment of stress-related behavioral health outcomes, as they suggest that sex and social status are important factors to consider in the context of serotonergic drug efficacy.

Females are more likely to suffer from stress-related conditions that impact socioemotional behavior compared to males. One thing that influences how sex impacts stress-related health problems is social dominance. We examined whether there are sex differences in the effects of social dominance on socioemotional behavior in socially housed rhesus monkeys. Because the neurotransmitter serotonin is important for socioemotional behavior, we also looked at the levels of the 5HT1AR receptor using neuroimaging. Aggression was greater in dominant compared to subordinate animals, and submission was significantly greater in subordinate versus dominant animals and greater in females than males. Anxiety and levels of 5HT1AR in the hypothalamus were significantly greater in females than in males. 5HT1AR in the hippocampus was greater in dominant compared to subordinate females but was not different between dominant and subordinate males. Overall, these data are important for the treatment of stress-related behavioral health outcomes because suggest that sex and social dominance are important factors to consider in the context of how effective drugs targeting the serotonin system are for treating stress-related behavioral health conditions.

Genome-wide kinase-MAM interactome screening reveals the role of CK2A1 in MAM Ca2+ dynamics linked to DEE66.

The endoplasmic reticulum (ER) and mitochondria form a unique subcellular compartment called mitochondria-associated ER membranes (MAMs). Disruption of MAMs impairs Ca2+ homeostasis, triggering pleiotropic effects in the neuronal system. Genome-wide kinase-MAM interactome screening identifies casein kinase 2 alpha 1 (CK2A1) as a regulator of composition and Ca2+ transport of MAMs. CK2A1-mediated phosphorylation of PACS2 at Ser207/208/213 facilitates MAM localization of the CK2A1-PACS2-PKD2 complex, regulating PKD2-dependent mitochondrial Ca2+ influx. We further reveal that mutations of PACS2 (E209K and E211K) associated with developmental and epileptic encephalopathy-66 (DEE66) impair MAM integrity through the disturbance of PACS2 phosphorylation at Ser207/208/213. This, in turn, causes the reduction of mitochondrial Ca2+ uptake and the dramatic increase of the cytosolic Ca2+ level, thereby, inducing neurotransmitter release at the axon boutons of glutamatergic neurons. In conclusion, our findings suggest a molecular mechanism that MAM alterations induced by pathological PACS2 mutations modulate Ca2+-dependent neurotransmitter release.

Sex and social status modify the effects of fluoxetine on socioemotional behaviors in Syrian hamsters and rhesus macaques.

Social subordination increases risk for psychiatric disorders, while dominance increases resilience to these disorders. Fluoxetine, a selective serotonin (5HT) reuptake inhibitor whose actions are mediated in part by the 5HT1A receptor (5HT1AR), has sex- and social status-specific effects on socioemotional behavior and aggressive behavior. However, the impact of social status on these sex-specific effects remains unclear. The current study evaluated the impact of acute fluoxetine treatment and social status on dominance-related behaviors in female and male hamsters, and the impact of chronic fluoxetine treatment on socioemotional behavior and 5HT1AR binding potential (5HT1ARBP) in female rhesus macaques. We hypothesized that sex differences in the effects of fluoxetine on aggression in hamsters would be diminished in dominant and enhanced in subordinate males and that aggression in female hamsters would be enhanced in dominants and diminished in subordinates. In female rhesus macaques, we hypothesized that chronic fluoxetine would alter socioemotional behaviors and site-specific 5HT1ARBP in a status-dependent manner. Male (n = 46) and female (n = 56) hamsters were paired with conspecifics for three days to establish social rank. Hamsters received a single dose of 20 mg/kg fluoxetine or vehicle two-hours prior to a test with a non-aggressive intruder. Female rhesus monkeys (n = 14) housed were administered fluoxetine (2.8 mg/kg/day) or vehicle injections chronically for 14-days, separated by a three-week washout period. On Day 15, positron emission tomography neuroimaging for 5HT1ARBP was conducted. Fluoxetine treatment decreased aggression in subordinate female monkeys and subordinate female hamsters but not in dominant females of either species. Fluoxetine decreased aggression in dominant but not in subordinate male hamsters. Fluoxetine also reduced and increased prefrontal 5HT1ARBP in dominant and subordinate females, respectively. Taken together, these results provide cross-species evidence that social status and sex impact how increased 5HT modulates agonistic behavior.

SHISA5/SCOTIN restrains spontaneous autophagy induction by blocking contact between the ERES and phagophores.

ABBREVIATIONS: ATG2: autophagy related 2; BECN1: beclin 1; COPII: coat protein II; DMSO: dimethyl sulfoxide; EBSS: Earle's balanced salt solution; EGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; ERES: ER exit site(s); GFP: green fluorescent protein; H89: H-89 dihydrochloride hydrate; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; NS5A: nonstructural protein 5A; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PLA: proximity ligation assay; PtdIns3P: phosphatidylionositol-3-phosphate; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RFP: red fluorescent protein; RPS6KB1/S6K: ribosomal protein S6 kinase B1; SBP: streptavidin binding protein; SEC16A: SEC16 homolog A, endoplasmic reticulum export factor; SEC31A: SEC31 homolog A, COPII coat complex component; siRNA: small interfering RNA; Str: streptavidin; ULK1: unc-51-like autophagy activating kinase 1; VSVG: vesicular stomatitis virus glycoprotein; WIPI2: WD repeat domain, phosphoinositide interacting 2; WT: wild type.

Synthesis and Evaluation of [11C]7-Halogen-2-Phenyl Isoindolone Derivatives: Potential PET Radioligands for in vivo Imaging of 5-HT2 C Receptors.

The serotonin 5-HT2 C receptor (5-HT2 C R) is abundantly expressed throughout the central nervous system, and involved in a variety of neuroendocrine and neurobehavioral processes. The development of a selective radioligand that will enable in vivo imaging and quantification of 5-HT2 C R densities represents a significant technological advancement in understanding both the normal function and pathophysiology of the 5-HT2 C R. Four 7-halogen-2-phenyl isoindolones (7-F, Cl, Br, I) were synthesized and displayed high affinities for 5-HT2 C R and high selectivity over 5-HT2 A and 5-HT2 B . [11C]7-Chloro-2-[4-methoxy-3-[2-(4-methylpiperidin-1-yl)ethoxy]phenyl]isoindolin-1-one (6) and [11C]7-iodo-2-[4-methoxy-3-[2-(4-methylpiperidin-1-yl)ethoxy]phenyl]isoindolin-1-one (9) were synthesized in high radiochemical yield of 37-44% [n = 10, decay corrected from end of (11C)CH3I synthesis] with high radiochemical purity via O-methylation with [11C]CH3I, respectively. MicroPET imaging studies in male rats with or without 5-HT2 C antagonist SB-242084 showed that [11C]6 and [11C]9 display specific bindings to 5-HT2 C R in the choroid plexus and hippocampus. In vivo microPET brain imaging studies in rhesus monkeys demonstrated that [11C]6 and [11C]9 exhibit excellent blood-brain barrier penetration. The contrast of bindings to the choroid plexus and hippocampus compared to the cerebellum peaked at 2.7 and 1.6, respectively, for [11C]6, and 3.7 and 2.7, respectively, for [11C]9, which were reduced by administration of a dose of SB-242084. Our results support the candidacy of [11C]6 and [11C]9 for further study as radioligands for in vivo quantitation of 5-HT2 C sites by PET.

Der f 38 Is a Novel TLR4-Binding Allergen Related to Allergy Pathogenesis from Dermatophagoides farinae.

It is difficult to treat allergic diseases including asthma completely because its pathogenesis remains unclear. House dust mite (HDM) is a critical allergen and Toll-like receptor (TLR) 4 is a member of the toll-like receptor family, which plays an important role in allergic diseases. The purpose of this study was to characterize a novel allergen, Der f 38 binding to TLR4, and unveil its role as an inducer of allergy. Der f 38 expression was detected in the body and feces of Dermatophagoides farinae (DF). Electron microscopy revealed that it was located in the granule layer, the epithelium layer, and microvilli of the posterior midgut. The skin prick test showed that 60% of allergic subjects were Der f 38-positive. Der f 38 enhanced surface 203c expression in basophils of Der f 38-positive allergic subjects. By analysis of the model structure of Der p 38, the expected epitope sites are exposed on the exterior side. In animal experiments, Der f 38 triggered an infiltration of inflammatory cells. Intranasal (IN) administration of Der f 38 increased neutrophils in the lung. Intraperitoneal (IP) and IN injections of Der f 38 induced both eosinophils and neutrophils. Increased total IgE level and histopathological features were found in BALB/c mice treated with Der f 38 by IP and IN injections. TLR4 knockout (KO) BALB/c mice exhibited less inflammation and IgE level in the sera compared to wild type (WT) mice. Der f 38 directly binds to TLR4 using biolayer interferometry. Der f 38 suppressed the apoptosis of neutrophils and eosinophils by downregulating proteins in the proapoptotic pathway including caspase 9, caspase 3, and BAX and upregulating proteins in the anti-apoptotic pathway including BCL-2 and MCL-1. These findings might shed light on the pathogenic mechanisms of allergy to HDM.

Targeting HIF-activated collagen prolyl 4-hydroxylase expression disrupts collagen deposition and blocks primary and metastatic uveal melanoma growth.

Uveal melanoma (UM) is the most prevalent primary intraocular malignancy in adults, and patients that develop metastases (~50%) survive <1 year, highlighting the urgent need for new therapies. TCGA has recently revealed that a hypoxia gene signature is associated with poor UM patient prognosis. Here we show that expression of hypoxia-regulated collagen prolyl-4-hydroxylase genes P4HA1 and P4HA2 is significantly upregulated in UM patients with metastatic disease and correlates with poor prognosis, suggesting these enzymes might be key tumor drivers. We targeted hypoxia-induced expression of P4HA1/2 in UM with KCN1, a hypoxia inducible factor-1 (HIF-1) pathway inhibitor and found potent inhibition of primary and metastatic disease and extension of animal survival, without overt side effects. At the molecular level, KCN1 antagonized hypoxia-induced expression of P4HA1 and P4HA2, which regulate collagen maturation and deposition in the extracellular matrix. The treatment decreased prolyl hydroxylation, induced proteolytic cleavage and rendered a disordered structure to collagen VI, the main collagen produced by UM, and reduced UM cell invasion. Together, these data demonstrate that extracellular collagen matrix formation can be targeted in UM by inhibiting hypoxia-induced P4HA1 and P4HA2 expression, warranting further development of this strategy in patients with uveal melanoma.

Heterogeneity in liver histopathology is associated with GSK-3ß activity and mitochondrial dysfunction in end-stage diabetic rats on differential diets.

While liver histopathology is heterogeneous in diabetes, the underlying mechanisms remain unclear. We investigated whether glycemic variation resulting from differential diets can induce heterogeneity in diabetic liver and the underlying molecular mechanisms. We generated end-stage non-obese diabetic model rats by subtotal-pancreatectomy in male Sprague- Dawley rats and ad libitum diet for 7 weeks (n = 33). The rats were then divided into three groups, and fed a standard- or a low-protein diet (18 or 6 kcal%, respectively), for another 7 weeks: to maintain hyperglycemia, 11 rats were fed ad libitum (18AL group); to achieve euglycemia, 11 were calorierestricted (18R group), and 11 were both calorie- and proteinrestricted with the low-protein diet (6R group). Overnightfasted liver samples were collected after the differential diets together with sham-control (18S group), and histology and molecular changes were compared. Hyperglycemic-18AL showed glycogenic hepatopathy (GH) without steatosis, with the highest GSK-3ß inactivation because of Akt activation during hyperglycemia; mitochondrial function was not impaired, compared to the 18S group. Euglycemic-18R showed neither GH nor steatosis, with intermediate GSK-3ß activation and mitochondrial dysfunction. However, euglycemic-6R showed both GH and steatosis despite the highest GSK-3ß activity and no molecular evidence of increased lipogenesis or decreased ApoB expression, where mitochondrial dysfunction was highest among the groups. In conclusion, heterogeneous liver histopathology developed in end-stage non-obese diabetic rats as the glycemic levels varied with differential diets, in which protein content in the diets as well as glycemic levels differentially influenced GSK-3ß activity and mitochondrial function in insulin-deficient state. [BMB Reports 2020; 53(2): 100-105].

Overexpression of PkINO1 improves ethanol resistance of Pichia kudriavzevii N77-4 isolated from the Korean traditional fermentation starter nuruk.

The yeast Pichia kudriavzevii N77-4 was isolated from the Korean traditional fermentation starter nuruk. In this study, fermentation performance and stress resistance ability of N77-4 was analyzed. N77-4 displayed superior thermotolerance (up to 44°C) in addition to enhanced acetic acid resistance compared to Saccharomyces cerevisiae. Moreover, N77-4 produced 7.4 g/L of ethanol with an overall production yield of 0.37 g/g glucose in 20 g/L glucose medium. However, in 250 g/L glucose medium the growth of N77-4 slowed down when the concentration of ethanol reached 14 g/L or more and ethanol production yield also decreased to 0.30 g/g glucose. An ethanol sensitivity test indicated that N77-4 was sensitive to the presence of 1% ethanol, which was not the case for S. cerevisiae. Furthermore, N77-4 displayed a severe growth defect in the presence of 6% ethanol. Because inositol biosynthesis is critical for ethanol resistance, expression levels of the PkINO1 encoding a key enzyme for inositol biosynthesis was analyzed under ethanol stress conditions. We found that ethanol stress clearly repressed PkINO1 expression in a dose-dependent manner and overexpression of PkINO1 improved the growth of N77-4 by 19% in the presence of 6% ethanol. Furthermore, inositol supplementation also enhanced the growth by 13% under 6% ethanol condition. These findings indicate that preventing downregulation in PkINO1 expression caused by ethanol stress improves ethanol resistance and enhances the utility of P. kudriavzevii N77-4 in brewing and fermentation biotechnology.

Anks1a regulates COPII-mediated anterograde transport of receptor tyrosine kinases critical for tumorigenesis.

ErbB2 signalling, which is amplified by EphA2 binding, is an important therapeutic target for breast cancer. Despite the importance of the EphA2/ErbB2 complex in promoting breast tumorigenesis, the mechanism by which these receptor tyrosine kinases (RTKs) are exported from the endoplasmic reticulum (ER) remains poorly understood. Here we report that the PTB adaptor Anks1a is specifically localized to the ER on its own serine phosphorylation. Once there, Anks1a acts as an important regulator of COPII-mediated EphA2 ER export. The Anks1a ankyrin repeat domain binds EphA2 and causes it to accumulate at sites of ER exit. Simultaneously, the Anks1a PTB domain binds Sec23. This induces internalization of EphA2 via COPII vesicles, while Anks1a remains behind on the ER membrane. EphA2 also binds ErbB2 in the ER and seems to load ErbB2 into growing COPII carriers. Together, our study reveals a novel mechanism that regulates the loading of RTKs into COPII vesicles.

Minicircle microporation-based non-viral gene delivery improved the targeting of mesenchymal stem cells to an injury site.

Genetic engineering approaches to improve the therapeutic potential of mesenchymal stem cells (MSCs) have been made by viral and non-viral gene delivery methods. Viral methods have severe limitations in clinical application because of potential oncogenic, pathogenic, and immunogenic risks, while non-viral methods have suffered from low transfection efficiency and transient weak expression as MSCs are hard-to-transfect cells. In this study, minicircle, which is a minimal expression vector free of bacterial sequences, was employed for MSC transfection as a non-viral gene delivery method. The conventional cationic liposome method was not effective for MSC transfection as it resulted in very low transfection efficiency (less than 5%). Microporation, a new electroporation method, greatly improved the transfection efficiency of minicircles by up to 66% in MSCs without any significant loss of cell viability. Furthermore, minicircle microporation generated much stronger and prolonged transgene expression compared with plasmid microporation. When MSCs microporated with minicircle harboring firefly luciferase gene were subcutaneously injected to mice, the bioluminescence continued for more than a week, whereas the bioluminescence of the MSCs induced by plasmid microporation rapidly decreased and disappeared in mice within three days. By minicircle microporation as a non-viral gene delivery, MSCs engineered to overexpress CXCR4 showed greatly increased homing ability toward an injury site as confirmed through in vivo bioluminescence imaging in mice. In summary, the engineering of MSCs through minicircle microporation is expected to enhance the therapeutic potential of MSCs in clinical applications.

Efficient adhesion-based plasma membrane isolation for cell surface N-glycan analysis.

Glycans, which decorate cell surfaces, play crucial roles in various physiological events involving cell surface recognition. Despite the importance of surface glycans, most analyses have been performed using total cells or whole membranes rather than plasma membranes due to difficulties related to isolation. In the present study, we employed an adhesion-based method for plasma membrane isolation to analyze N-glycans on cell surfaces. Cells were attached to polylysine-coated glass plates and then ruptured by hypotonic pressure. After washing to remove intracellular organelles, only a plasma membrane fraction remained attached to the plates, as confirmed by fluorescence imaging using organelle-specific probes. The plate was directly treated with trypsin to digest and detach the glycoproteins from the plasma membrane. From the resulting glycopeptides, N-glycans were released and analyzed using MALDI-TOF mass spectrometry and HPLC. When N-glycan profiles obtained by this method were compared to those by other methods, the amount of high-mannose type glycans mainly contaminated from the endoplasmic reticulum was dramatically reduced, which enabled the efficient detection of complex type glycans present on the cell surface. Moreover, this method was successfully used to analyze the increase of high-mannose glycans on the surface as induced by a mannosidase inhibitor treatment.

Efficient myogenic differentiation of human adipose-derived stem cells by the transduction of engineered MyoD protein.

Human adipose-derived stem cells (hASCs) have great potential as cell sources for the treatment of muscle disorders. To provide a safe method for the myogenic differentiation of hASCs, we engineered the MyoD protein, a key transcription factor for myogenesis. The engineered MyoD (MyoD-IT) was designed to contain the TAT protein transduction domain for cell penetration and the membrane-disrupting INF7 peptide, which is an improved version of the HA2 peptide derived from influenza. MyoD-IT showed greatly improved nuclear targeting ability through an efficient endosomal escape induced by the pH-sensitive membrane disruption of the INF7 peptide. By applying MyoD-IT to a culture, hASCs were efficiently differentiated into long spindle-shaped myogenic cells expressing myosin heavy chains. Moreover, these cells differentiated by an application of MyoD-IT fused to myotubes with high efficiency through co-culturing with mouse C2C12 myoblasts. Because internalized proteins can be degraded in cells without altering the genome, the myogenic differentiation of hASCs using MyoD-IT would be a safe and clinically applicable method.

Chronic interferon-α decreases dopamine 2 receptor binding and striatal dopamine release in association with anhedonia-like behavior in nonhuman primates.

Neuroimaging studies in humans have demonstrated that inflammatory cytokines target basal ganglia function and presynaptic dopamine (DA), leading to symptoms of depression. Cytokine-treated nonhuman primates also exhibit evidence of altered DA metabolism in association with depressive-like behaviors. To further examine cytokine effects on striatal DA function, eight rhesus monkeys (four male, four female) were administered interferon (IFN)-α (20 MIU/m(2) s.c.) or saline for 4 weeks. In vivo microdialysis was used to investigate IFN-α effects on DA release in the striatum. In addition, positron emission tomography (PET) with [(11)C]raclopride was used to examine IFN-α-induced changes in DA2 receptor (D2R) binding potential before and after intravenous amphetamine administration. DA transporter binding was measured by PET using [(18)F]2ß-carbomethoxy-3ß-(4-chlorophenyl)-8-(2-fluoroethyl)nortropane. Anhedonia-like behavior (sucrose consumption) was assessed during saline and IFN-α administration. In vivo microdialysis demonstrated decreased release of DA after 4 weeks of IFN-α administration compared with saline. PET neuroimaging also revealed decreased DA release after 4 weeks of IFN-α as evidenced by reduced displacement of [(11)C]raclopride following amphetamine administration. In addition, 4 weeks of IFN-α was associated with decreased D2R binding but no change in the DA transporter. Sucrose consumption was reduced during IFN-α administration and was correlated with decreased DA release at 4 weeks as measured by in vivo microdialysis. Taken together, these findings indicate that chronic peripheral IFN-α exposure reduces striatal DA release in association with anhedonia-like behavior in nonhuman primates. Future studies examining the mechanisms of cytokine effects on DA release and potential therapeutic strategies to reverse these changes are warranted.

Radiofluorinated carbohydrates for positron emission tomography.

2-Deoxy-2-[(18)F]fluoro-D-glucose (2-(18)FDG) has represented radiofluorinated carbohydrates as the most successful tracer for positron emission tomography (PET). 2-(18)FDG uptake depends on glucose metabolism, which is related to a disease progression. 2-(18)FDG has been widely used in oncology, neurology, cardiology, infectious diseases, and inflammation, to complement anatomical modalities such as CT and MRI. Followed by the success of 2-(18)FDG, various radiofluorinated carbohydrates have been evaluated as PET tracers, which include analogs of D-ribose, D-mannose, D-galactose, D-talose, D-fructose, D-allose, lactose, L-fucose, N-acetylneuraminic acid, and L-ascorbic acid. Among those radiofluorinated carbohydrates, several have implied potential for further development. 2-Deoxy-2-[(18)F]fluoro-D-galactose has been developed to assess liver function and diagnose hepatic carcinoma. 6-Deoxy-6-[(18)F]fluoro-D-fructose showed promising characteristics for diagnosis of breast cancer. Three radiofluorinated analogs of lactose have been designed as the substrates of the overexpressed hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein in peritumoral pancreatic tissue for early diagnosis of pancreatic cancer. The metabolism of 6-[(18)F]fluoro-L-fucose suggested that it is a bioactive analog of L-fucose in the synthesis of glycoconjugate macromolecules. 6-Deoxy-6-[(18)F]fluoro-L-ascorbic acid was evaluated to assess antioxidant function of L-ascorbic acid in rodent models of transient global ischemia and glutathione deficiency.

Arylsulfonamide KCN1 inhibits in vivo glioma growth and interferes with HIF signaling by disrupting HIF-1α interaction with cofactors p300/CBP.

PURPOSE: The hypoxia-inducible factor-1 (HIF-1) plays a critical role in tumor adaptation to hypoxia, and its elevated expression correlates with poor prognosis and treatment failure in patients with cancer. In this study, we determined whether 3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-phenylbenzenesulfonamide, KCN1, the lead inhibitor in a novel class of arylsulfonamide inhibitors of the HIF-1 pathway, had antitumorigenic properties in vivo and further defined its mechanism of action. EXPERIMENTAL DESIGN: We studied the inhibitory effect of systemic KCN1 delivery on the growth of human brain tumors in mice. To define mechanisms of KCN1 anti-HIF activities, we examined its influence on the assembly of a functional HIF-1α/HIF-1ß/p300 transcription complex. RESULTS: KCN1 specifically inhibited HIF reporter gene activity in several glioma cell lines at the nanomolar level. KCN1 also downregulated transcription of endogenous HIF-1 target genes, such as VEGF, Glut-1, and carbonic anhydrase 9, in a hypoxia-responsive element (HRE)-dependent manner. KCN1 potently inhibited the growth of subcutaneous malignant glioma tumor xenografts with minimal adverse effects on the host. It also induced a temporary survival benefit in an intracranial model of glioma but had no effect in a model of melanoma metastasis to the brain. Mechanistically, KCN1 did not downregulate the levels of HIF-1α or other components of the HIF transcriptional complex; rather, it antagonized hypoxia-inducible transcription by disrupting the interaction of HIF-1α with transcriptional coactivators p300/CBP. CONCLUSIONS: Our results suggest that the new HIF pathway inhibitor KCN1 has antitumor activity in mouse models, supporting its further translation for the treatment of human tumors displaying hypoxia or HIF overexpression.

Design and in vitro activities of N-alkyl-N-[(8-R-2,2-dimethyl-2H-chromen-6-yl)methyl]heteroarylsulfonamides, novel, small-molecule hypoxia inducible factor-1 pathway inhibitors and anticancer agents.

The hypoxia inducible factor (HIF) pathway is an attractive target for cancer, as it controls tumor adaptation to growth under hypoxia and mediates chemotherapy and radiation resistance. We previously discovered 3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-phenylbenzenesulfonamide as a novel, small-molecule HIF-1 pathway inhibitor in a high-throughput cell-based assay, but its in vivo delivery is hampered by poor aqueous solubility (0.009 µM in water; log P(7.4) = 3.7). Here we describe the synthesis of 12 N-alkyl-N-[(8-R-2,2-dimethyl-2H-chromen-6-yl)methyl]heteroarylsulfonamides, which were designed to possess optimal lipophilicities and aqueous solubilities by in silico calculations. Experimental log P(7.4) values of 8 of the 12 new analogs ranged from 1.2-3.1. Aqueous solubilities of three analogs were measured, among which the most soluble N-[(8-methoxy-2,2-dimethyl-2H-chromen-6-yl)methyl]-N-(propan-2-yl)pyridine-2-sulfonamide had an aqueous solubility of 80 µM, e.g., a solubility improvement of ∼9000-fold. The pharmacological optimization had limited impact on drug efficacy as the compounds retained IC(50) values at or below 5 µM in our HIF-dependent reporter assay.

Structure-activity relationship of 2,2-dimethyl-2H-chromene based arylsulfonamide analogs of 3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-phenylbenzenesulfonamide, a novel small molecule hypoxia inducible factor-1 (HIF-1) pathway inhibitor and anti-cancer agent.

We have discovered that 3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-phenylbenzenesulfonamide, a novel small molecule HIF-1 pathway inhibitor, can antagonize tumor growth in animal models of cancer, but the treatment necessitates its delivery in a formulation, due to poor water solubility (<15 µg/mL; pH 7.4), evidencing that the chemotype needs further exploration of its amenability to additional chemical modifications for ultimate optimization of function and pharmacology. As a first step towards this goal we investigated the structure-activity relationships of 15 lipophilic 2,2-dimethyl-2H-chromene based arylsulfonamide analogs of 3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-phenylbenzenesulfonamide to find out strategies of modification. A 3,4-dimethoxybenzenesulfonyl group in region 1 showed the strongest inhibition among five arylsulfonyl groups tested. The presence of propan-2-amine in region 2 conferred the strongest inhibitory effect of the compound on HIF-1 activated transcription in a reporter assay. These findings are important as they help define the structural motifs where the 3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-phenylbenzenesulfonamide can be chemically modified to improve its pharmacological properties towards development as a cancer therapeutic.