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Loss of function of voltage-gated potassium (Kv) channels is linked to a range of lethal or debilitating channelopathies. New pharmacological approaches are warranted to isoform-selectively activate specific Kv channels. One example is KCNA1 Potassium Voltage-Gated Channel Subfamily A Member 1 (KCNA1) (Kv1.1), an archetypal Shaker-type Kv channel, in which loss-of-function mutations cause episodic ataxia type 1 (EA1). EA1 causes constant myokomia and episodic bouts of ataxia and may associate with epilepsy and other disorders. We previously found that the inhibitory neurotransmitter γ-aminobutyric acid and modified versions of glycine directly activate Kv channels within the KCNQ subfamily, a characteristic favored by strong negative electrostatic surface potential near the neurotransmitter carbonyl group. Here, we report that adjusting the number and positioning of fluorine atoms within the fluorophenyl ring of glycine derivatives produces isoform-selective KCNA1 channel openers that are inactive against KCNQ2/3 channels, or even KCNA2, the closest relative of KCNA1. The findings refine our understanding of the molecular basis for KCNQ versus KCNA1 activation and isoform selectivity and constitute, to our knowledge, the first reported isoform-selective KCNA1 opener. SIGNIFICANCE STATEMENT Inherited loss-of-function gene sequence variants in KCNA1, which encodes the KCNA1 (Kv1.1) voltage-gated potassium channel, cause episodic ataxia type 1 (EA1), a movement disorder also linked to epilepsy and developmental delay. We have discovered several isoform-specific KCNA1-activating small molecules, addressing a notable gap in the field and providing possible lead compounds and a novel chemical space for the development of potential future therapeutic drugs for EA1.

Sickle cell disease (SCD) is associated with overactive bladder (OAB). Detrusor overactivity, a component of OAB, is present in an SCD mouse, but the molecular mechanisms for this condition are not well-defined. We hypothesize that nitric oxide (NO)/ ras homolog gene family (Rho) A/Rho-associated kinase (ROCK) dysregulation is a mechanism for detrusor overactivity and that NO-releasing nanoparticles (NO-nps), a novel NO delivery system, may serve to treat this condition. Male adult SCD transgenic, combined endothelial NO synthases (eNOSs) and neuronal NOS (nNOS) gene-deficient (dNOS(−/−)), and wild-type (WT) mice were used. Empty nanoparticle or NO-np was injected into the bladder, followed by cystometric studies. The expression levels of phosphorylated eNOS (Ser-1177), protein kinase B (Akt) (Ser-473), nNOS (Ser-1412), and myosin phosphatase target subunit 1 (MYPT1) (Thr-696) were assessed in the bladder. SCD and dNOS(−/−) mice had a greater (P < 0.05) number of voiding and nonvoiding contractions compared with WT mice, and they were normalized by NO-np treatment. eNOS (Ser-1177) and AKT (Ser-473) phosphorylation were decreased (P < 0.05) in the bladder of SCD compared with WT mice and reversed by NO-np. Phosphorylated MYPT1, a marker of the RhoA/ROCK pathway, was increased (P < 0.05) in the bladder of SCD mice compared with WT and reversed by NO-np. nNOS phosphorylation on positive (Ser-1412) regulatory site was decreased (P < 0.05) in the bladder of SCD mice compared with WT and was not affected by NO-np. NO-nps did not affect any of the measured parameters in WT mice. In conclusion, dysregulation of NO and RhoA/ROCK pathways is associated with detrusor overactivity in SCD mice; NO-np reverses these molecular derangements in the bladder and decreases detrusor overactivity. SIGNIFICANCE STATEMENT: Voiding abnormalities commonly affect patients with sickle cell disease (SCD) but are problematic to treat. Clarification of the science for this condition in an animal model of SCD may lead to improved interventions for it. Our findings suggest that novel topical delivery of a vasorelaxant agent nitric oxide into the bladder of these mice corrects overactive bladder by improving deranged bladder physiology regulatory signaling.

β2-Adrenoceptors (β2ARs) are concentrated in caveolar lipid raft domains of the plasma membrane in airway smooth muscle (ASM) cells, along with adenylyl cyclase type 6 (AC6). This is believed to contribute to how these receptors can selectively regulate certain types of cAMP-dependent responses in these cells. The goal of the present study was to test the hypothesis that β2AR production of cAMP is localized to specific subcellular compartments using fluorescence resonance energy transfer (FRET)-based cAMP biosensors targeted to different microdomains in human ASM cells. Epac2-MyrPalm and Epac2-CAAX biosensors were used to measure responses associated with lipid raft and non-raft regions of the plasma membrane, respectively. Activation of β2ARs with isoproterenol produced cAMP responses most readily detected in lipid raft domains. Furthermore, overexpression of AC6 somewhat paradoxically inhibited β2AR production of cAMP in lipid raft domains, without affecting β2AR responses detected in other subcellular locations or cAMP responses to EP2 prostaglandin receptor activation, which were confined primarily to non-raft domains of the plasma membrane. The inhibitory effect of overexpressing AC6 was blocked by inhibition of phosphodiesterase type 4 (PDE4) activity with rolipram, inhibition of protein kinase A (PKA) activity with H89, and inhibition of A kinase anchoring protein (AKAP) interactions with the peptide inhibitor Ht31. These results support the idea that overexpression of AC6 leads to enhanced feedback activation of PDE4 via phosphorylation by PKA that is part of an AKAP-dependent signaling complex. This provides insight into the molecular basis for localized regulation of cAMP signaling in human ASM cells. SIGNIFICANCE STATEMENT N/A

We have created a novel glutathione S-transferase π1 (gstp1) knockout (KO) zebrafish model and used it for comparative analyses of redox homeostasis and response to drugs that cause endoplasmic reticulum (ER) stress and induce the unfolded protein response (UPR). Under basal conditions, gstp1 KO larvae had higher expression of antioxidant nuclear factor erythroid 2–related factor 2 (Nrf2) accompanied by a more reduced larval environment and a status consistent with reductive stress. Compared with wild type, various UPR markers were decreased in KO larvae, but treatment with drugs that induce ER stress caused greater toxicities and increased expression of Nrf2 and UPR markers in KO. Tunicamycin and 0(2)-{2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl}1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/nitric oxide) activated inositol-requiring protein-1/X-box binding protein 1 pathways, whereas thapsigargin caused greater activation of protein kinase-like ER kinase/activating transcription factor 4/CHOP pathways. These results suggest that this teleost model is useful for predicting how GSTP regulates organismal management of oxidative/reductive stress and is a determinant of response to drug-induced ER stress and the UPR. SIGNIFICANCE STATEMENT: A new zebrafish model has been created to study the importance of glutathione S-transferase π1 in development, redox homeostasis, and response to drugs that enact cytotoxicity through endoplasmic reticulum stress and induction of the unfolded protein response.

Ketogenic diets are effective therapies for refractory epilepsy, yet the underlying mechanisms are incompletely understood. The anticonvulsant efficacy of ketogenic diets correlates positively to the serum concentration of β-hydroxybutyrate (BHB), the primary ketone body generated by ketosis. Voltage-gated potassium channels generated by KCNQ2-5 subunits, especially KCNQ2/3 heteromers, generate the M-current, a therapeutic target for synthetic anticonvulsants. Here, we report that BHB directly activates KCNQ2/3 channels (EC(50) = 0.7 µM), via a highly conserved S5 tryptophan (W265) on KCNQ3. BHB was also acutely effective as an anticonvulsant in the pentylene tetrazole (PTZ) seizure assay in mice. Strikingly, coadministration of γ-amino-β-hydroxybutyric acid, a high-affinity KCNQ2/3 partial agonist that also acts via KCNQ3-W265, similarly reduced the efficacy of BHB in KCNQ2/3 channel activation in vitro and in the PTZ seizure assay in vivo. Our results uncover a novel, unexpected molecular basis for anticonvulsant effects of the major ketone body induced by ketosis. SIGNIFICANCE STATEMENT: Ketogenic diets are used to treat refractory epilepsy but the therapeutic mechanism is not fully understood. Here, we show that clinically relevant concentrations of β-hydroxybutyrate, the primary ketone body generated during ketogenesis, activates KCNQ2/3 potassium channels by binding to a specific site on KCNQ3, an effect known to reduce neuronal excitability. We provide evidence using a mouse chemoconvulsant model that KCNQ2/3 activation contributes to the antiepileptic action of β-hydroxybutyrate.

T-cell leukemia 1A (TCL1A) single-nucleotide polymorphisms (SNPs) have been associated with aromatase inhibitor-induced musculoskeletal adverse events. We previously demonstrated that TCL1A is inducible by estradiol (E(2)) and plays a critical role in the regulation of cytokines, chemokines, and Toll-like receptors in a TCL1A SNP genotype and estrogen-dependent fashion. Furthermore, TCLIA SNP-dependent expression phenotypes can be “reversed” by exposure to selective estrogen receptor modulators such as 4-hydroxytamoxifen (4OH-TAM). The present study was designed to comprehensively characterize the role of TCL1A in transcriptional regulation across the genome by performing RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) assays with lymphoblastoid cell lines. RNA-seq identified 357 genes that were regulated in a TCL1A SNP- and E(2)-dependent fashion with expression patterns that were 4OH-TAM reversible. ChIP-seq for the same cells identified 57 TCL1A binding sites that could be regulated by E(2) in a SNP-dependent fashion. Even more striking, nuclear factor-κB (NF-κB) p65 bound to those same DNA regions. In summary, TCL1A is a novel transcription factor with expression that is regulated in a SNP- and E(2)-dependent fashion—a pattern of expression that can be reversed by 4OH-TAM. Integrated RNA-seq and ChIP-seq results suggest that TCL1A also acts as a transcriptional coregulator with NF-κB p65, an important immune system transcription factor.

Ser54 of G(s)α binds guanine nucleotide and Mg(2+) as part of a conserved sequence motif in GTP binding proteins. Mutating the homologous residue in small and heterotrimeric G proteins generates dominant-negative proteins, but by protein-specific mechanisms. For α(i/o), this results from persistent binding of α to βγ, whereas for small GTP binding proteins and α(s) this results from persistent binding to guanine nucleotide exchange factor or receptor. This work examined the role of βγ interactions in mediating the properties of the Ser54-like mutants of Gα subunits. Unexpectedly, WT–α(s) or N54-α(s) coexpressed with α(1B)-adrenergic receptor in human embryonic kidney 293 cells decreased receptor stimulation of IP3 production by a cAMP-independent mechanism, but WT-α(s) was more effective than the mutant. One explanation for this result would be that α(s), like Ser47 α(i/o), blocks receptor activation by sequestering βγ; implying that N54-α(S) has reduced affinity for βγ since it was less effective at blocking IP3 production. This possibility was more directly supported by the observation that WT-α(s) was more effective than the mutant in inhibiting βγ activation of phospholipase Cβ2. Further, in vitro synthesized N54-α(s) bound biotinylated-βγ with lower apparent affinity than did WT-α(s). The Cys54 mutation also decreased βγ binding but less effectively than N54-α(s). Substitution of the conserved Ser in α(o) with Cys or Asn increased βγ binding, with the Cys mutant being more effective. This suggests that Ser54 of α(s) is involved in coupling changes in nucleotide binding with altered subunit interactions, and has important implications for how receptors activate G proteins.

Glycogen synthase kinase-3s (GSK3α and GSK3β) are constitutively active protein kinases that target over 100 substrates, incorporate into numerous protein complexes, and regulate such vital cellular functions as proliferation, apoptosis, and inflammation. Cyclin-dependent kinase 9 (CDK9) regulates RNA production as a component of positive transcription elongation factor b and promotes expression of oncogenic and inflammatory genes. Simultaneous inhibition of these signaling nodes is a promising approach for drug discovery, although previous compounds exhibit limited selectivity and clinical efficacy. The novel diaminothiazole ABC1183 is a selective GSK3α/β and CDK9 inhibitor and is growth-inhibitory against a broad panel of cancer cell lines. ABC1183 treatment decreases cell survival through G2/M arrest and modulates oncogenic signaling through changes in GSK3, glycogen synthase, and β-catenin phosphorylation and MCL1 expression. Oral administration, which demonstrates no organ or hematologic toxicity, suppresses tumor growth and inflammation-driven gastrointestinal disease symptoms, owing in part to downregulation of tumor necrosis factor α and interleukin-6 proinflammatory cytokines. Therefore, ABC1183 is strategically poised to effectively mitigate multiple clinically relevant diseases.

The upregulation of kidney injury molecule-1 (KIM-1) has been extensively studied in various renal diseases and following acute injury; however, the initial mechanisms controlling KIM-1 expression remain limited. In this study, KIM-1 expression was examined in mouse renal cell cultures and in two different models of acute kidney injury (AKI), ischemia reperfusion (IR)-induced and lipopolysaccharide (LPS)-induced sepsis. KIM-1 mRNA increased in both AKI models, and pharmacological inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling attenuated injury-induced KIM-1 expression in the renal cortex. Toll-like receptor 4 knockout (TLR4KO) mice exhibited reduced ERK1/2 phosphorylation and attenuated KIM-1 mRNA after LPS exposure. TLR4KO mice were not protected from IR-induced ERK1/2 phosphorylation and upregulation of KIM-1 mRNA. Following renal IR injury, phosphorylation of signal transducer and activator of transcription 3 (STAT3) at serine 727 and tyrosine 705 increased downstream from ERK1/2 activation. Because phosphorylated STAT3 is a transcriptional upregulator of KIM-1 and inhibition of ERK1/2 attenuated increases in STAT3 phosphorylation, we suggest an ERK1/2-STAT3-KIM-1 pathway following renal injury. Finally, ERK1/2 inhibition in naive mice decreased KIM-1 mRNA and nuclear STAT3 phosphorylation in the cortex, indicating homeostatic regulation of KIM-1. These findings reveal renal ERK1/2 as an important initial regulator of KIM-1 expression in IR and septic AKI and at a physiologic level.Visual Abstract.Proposed mechanism of IR, LPS, and ROS-induced renal damage that initiates ERK1/2 and STAT3 phosphorylation. STAT3 then binds to the KIM-1 promoter and increases KIM-1 mRNA. By preventing ERK1/2 phosphorylation following renal injury, STAT3 phosphorylation is decreased, leading to less phosphorylated STAT3 within the nucleus, and subsequently less KIM-1 mRNA increases post injury.

Free intracellular calcium ([Ca2+]i), in addition to being an important second messenger, is a key regulator of many cellular processes including the cell membrane potential, proliferation and apoptosis. In many cases, the mobilization of [Ca2+]i is controlled by intracellular store activation and calcium influx. We have investigated the effect of several ion channel modulators, which have been used to treat a range of human diseases, on [Ca2+]i release, by ratiometric calcium imaging. We show that six such modulators (Amiodarone (Ami), Dofetilide (Dof), Furosemide (Fur), Minoxidil (Min), Loxapine (Lox), and Nicorandil (Nic)) initiate release of [Ca2+]i in prostate and breast cancer cell lines, PC3 and MCF7, respectively. Whole cell currents in PC3 cells were inhibited by the compounds tested in patch clamp experiments in a concentration dependent manner. In all cases [Ca2+]i was increased by modulator concentrations comparable to those used clinically. The increase in [Ca2+]i in response to Ami, Fur, Lox and Min was reduced, significantly (p

Activator of G-protein signaling 4 (AGS4)/G-protein signaling modulator 3 (Gpsm3) contains three G-protein regulatory (GPR) motifs, each of which can bind Gαi-GDP free of Gβγ. We previously demonstrated that the AGS4-Gαi interaction is regulated by seven transmembrane-spanning receptors (7-TMR), which may reflect direct coupling of the GPR-Gαi module to the receptor analogous to canonical Gαβγ heterotrimer. We have demonstrated that the AGS4-Gαi complex is regulated by chemokine receptors in an agonist-dependent manner that is receptor-proximal. As an initial approach to investigate the functional role(s) of this regulated interaction in vivo, we analyzed leukocytes, in which AGS4/Gpsm3 is predominantly expressed, from AGS4/Gpsm3-null mice. Loss of AGS4/Gpsm3 resulted in mild but significant neutropenia and leukocytosis. Dendritic cells, T lymphocytes, and neutrophils from AGS4/Gpsm3-null mice also exhibited significant defects in chemoattractant-directed chemotaxis and extracellular signal-regulated kinase activation. An in vivo peritonitis model revealed a dramatic reduction in the ability of AGS4/Gpsm3-null neutrophils to migrate to primary sites of inflammation. Taken together, these data suggest that AGS4/Gpsm3 is required for proper chemokine signal processing in leukocytes and provide further evidence for the importance of the GPR-Gαi module in the regulation of leukocyte function.

The function of δ opioid receptors (DOR) expressed by peripheral pain-sensing neurons (nociceptors) is regulated by both cyclooxygenase- and lipoxygenase (LOX)-dependent arachidonic acid (AA) metabolites. Whereas cyclooxygenase metabolites enhance responsiveness, LOX metabolites elicit a refractory, nonsignaling state of the DOR receptor system for antinociceptive signaling. In this study, using high-performance liquid chromatography-tandem mass spectrometry analyses, we have found that the 12-/15-LOX metabolites, 12-hydroxyeicosatetraenoic acid (HETE) and 15-HETE, were elevated after treatment of adult rat primary sensory neuron cultures with AA. Exogenously applied 12-HETE and 15-HETE, but not 5-HETE, completely prevented DOR and κ opioid receptor (KOR) agonist–mediated inhibition of prostaglandin E2 (PGE(2))-stimulated cAMP accumulation, but not inhibition, by the 5-HT(1) receptor agonist 5-carboxamidotryptamine in cultured peripheral sensory neurons and in Chinese hamster ovary (CHO) cells heterologously expressing DOR or KOR. Similarly, intraplantar injection of 12- or 15-HETE, either alone or in combination, prevented DOR agonist-mediated inhibition of PGE(2)-evoked thermal allodynia. Further, both AA- and carrageenan-mediated induction of the nonresponsive state of the DOR system was blocked by an intraplantar coinjection of the 12-/15-LOX inhibitors baicalein and luteolin. In contrast to the regulation of cAMP signaling, pretreatment with 12- and 15-HETE had no effect on either DOR or KOR agonist- mediated activation of extracellular signal-regulated kinase in peripheral sensory neurons or CHO cells. These results suggest that the analgesic efficacy of peripherally restricted opioids for treatment of inflammatory pain may be enhanced by adjunct inhibition of LOX activity.

Synthetic cannabinoids (SCs) are novel psychoactive substances that are easily acquired, widely abused as a substitute for cannabis, and associated with cardiotoxicity and seizures. Although the structural bases of these compounds are scaffolds with known affinity and efficacy at the human cannabinoid type-1 receptor (hCB(1)), upon ingestion or inhalation they can be metabolized to multiple chemical entities of unknown pharmacological activity. A large proportion of these metabolites are hydroxylated on the pentyl chain, a key substituent that determines receptor affinity and selectivity. Thus, the pharmacology of SC metabolites may be an important component in understanding the in vivo effects of SCs. We examined nine SCs (AB-PINACA, 5F-AB-PINACA, ADB/MDMB-PINACA, 5F-ADB, 5F-CUMYL-PINACA, AMB-PINACA, 5F-AMB, APINACA, and 5F-APINACA) and their hydroxypentyl (either 4-OH or 5-OH) metabolites in [(3)H]CP55,940 receptor binding and the [(35)S]GTPγS functional assay to determine the extent to which these metabolites retain activity at cannabinoid receptors. All of the SCs tested exhibited high affinity (

Lysophosphatidic acid (LPA) is a lipid mediator that mediates cellular effects via G protein–coupled receptors (GPCRs). Epidermal growth factor (EGF) is a peptide that acts via a receptor tyrosine kinase. LPA and EGF both induce proliferation of prostate cancer cells and can transactivate each other’s receptors. The LPA receptor LPA1 is particularly important for LPA response in human prostate cancer cells. Previous work in our laboratory has demonstrated that free fatty acid 4 (FFA4), a GPCR activated by ω-3 fatty acids, inhibits responses to both LPA and EGF in these cells. One potential mechanism for the inhibition involves negative interactions between FFA4 and LPA1, thereby suppressing responses to EGF that require LPA1. In the current study, we examined the role of LPA1 in mediating EGF and FFA4 agonist responses in two human prostate cancer cell lines, DU145 and PC-3. The results show that an LPA1-selective antagonist inhibits proliferation and migration to both LPA and EGF. Knockdown of LPA1 expression, using silencing RNA, blocks responses to LPA and significantly inhibits responses to EGF. The partial response to EGF that is observed after LPA1 knockdown is not inhibited by FFA4 agonists. Finally, the role of arrestin-3, a GPCR-binding protein that mediates many actions of activated GPCRs, was tested. Knockdown of arrestin-3 completely inhibits responses to both LPA and EGF in prostate cancer cells. Taken together, these results suggest that LPA1 plays a critical role in EGF responses and that FFA4 agonists inhibit proliferation by suppressing positive cross-talk between LPA1 and the EGF receptor.

The autophagy pathway has been recognized as a mechanism of survival and therapy resistance in cancer, yet the extent of autophagy’s function in metastatic progression is still unclear. Therefore, we used murine models of metastatic cancer to investigate the effect of autophagy modulation on metastasis development. Pharmacologic and genetic autophagy inhibition were able to impede cell proliferation in culture, but did not impact the development of experimentally induced 4T1 and B16-F10 metastases. Similarly, autophagy inhibition by adjuvant chloroquine (CQ) treatment did not delay metastasis in an orthotopic 4T1, tumor-resection model. However, neoadjuvant CQ treatment or genetic autophagy inhibition resulted in delayed metastasis development, whereas stimulation of autophagy by trehalose hastened development. Cisplatin was also administered either as a single agent or in combination with CQ. The combination of cisplatin and CQ was antagonistic. The effects of autophagy modulation on metastasis did not appear to be due to alterations in the intrinsic metastatic capability of the cells, as modulating autophagy had no impact on migration, invasion, or anchorage-independent growth in vitro. To explore the possibility of autophagy’s influence on the metastatic microenvironment, bone marrow–derived cells (BMDCs), which mediate the establishment of the premetastatic niche, were measured in the lung and in circulation. Trehalose-treated mice had significantly more BMDCs than either vehicle- or CQ-treated mice. Autophagy inhibition may be most useful as a treatment to impede early metastatic development. However, modulating autophagy may also alter the efficacy of platinum-based therapies, requiring caution when considering combination therapies.

Sigma-2 receptors are attractive antineoplastic targets due to their ability to induce apoptosis and their upregulation in rapidly proliferating cancer cells compared with healthy tissue. However, this role is inconsistent with overexpression in cancer, which is typically associated with upregulation of prosurvival factors. Here, we report a novel metabolic regulatory function for sigma-2 receptors. CM764 [6-acetyl-3-(4-(4-(2-amino-4-fluorophenyl)piperazin-1-yl)butyl)benzo[d]oxazol-2(3H)-one] binds with Ki values of 86.6 ± 2.8 and 3.5 ± 0.9 nM at the sigma-1 and sigma-2 receptors, respectively. CM764 increased reduction of MTT [3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide] in human SK-N-SH neuroblastoma compared with untreated cells, an effect not due to proliferation. This effect was attenuated by five different sigma antagonists, including CM572 [3-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)-6-isothiocyanatobenzo[d]oxazol-2(3H)-one], which has no significant affinity for sigma-1 receptors. This effect was also observed in MG-63 osteosarcoma and HEK293T cells, indicating that this function is not exclusive to neuroblastoma or to cancer cells. CM764 produced an immediate, robust, and transient increase in cytosolic calcium, consistent with sigma-2 receptor activation. Additionally, we observed an increase in the total NAD(+)/NADH level and the ATP level in CM764-treated SK-N-SH cells compared with untreated cells. After only 4 hours of treatment, basal levels of reactive oxygen species were reduced by 90% in cells treated with CM764 over untreated cells, and HIF1α and VEGF levels were increased after 3-24 hours of treatment. These data indicate that sigma-2 receptors may play a role in induction of glycolysis, representing a possible prosurvival function for the sigma-2 receptor that is consistent with its upregulation in cancer cells compared with healthy tissue.

Sigma-2 receptors, recently identified as TMEM97, have been implicated in cancer and neurodegenerative disease. Structurally distinct sigma-2 receptor ligands induce cell death in tumor cells, linking sigma-2 receptors to apoptotic pathways. Recently, we reported that sigma-2 receptors can also stimulate glycolytic hallmarks, effects consistent with a prosurvival function and upregulation in cancer cells. Both apoptotic and metabolically stimulative effects were observed with compounds related to the canonical sigma-2 antagonist SN79. Here we investigate a series of 6-substituted SN79 analogs to assess the structural determinants governing these divergent effects. Substitutions on the benzoxazolone ring of the core SN79 structure resulted in high-affinity sigma-2 receptor ligands (K(i) = 0.56–17.9 nM), with replacement of the heterocyclic oxygen by N-methyl (producing N-methylbenzimidazolones) generally decreasing sigma-1 affinity and a sulfur substitution (producing benzothiazolones) imparting high affinity at both subtypes, lowering subtype selectivity. Substitution at the 6-position with COCH(3), NO(2), NH(2), or F resulted in ligands that were not cytotoxic. Five of these ligands induced an increase in metabolic activity, as measured by increased reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) in human SK-N-SH neuroblastoma cells, further supporting a role for sigma-2 receptors in metabolism. Substitution with 6-isothiocyanate resulted in ligands that were sigma-2 selective and that irreversibly bound to the sigma-2 receptor, but not to the sigma-1 receptor. These ligands induced cell death upon both acute and continuous treatment (EC(50) = 7.6–32.8 μM), suggesting that irreversible receptor binding plays a role in cytotoxicity. These ligands will be useful for further study of these divergent roles of sigma-2 receptors.

Central α4βδ receptors are the most abundant isoform of δ subunit–containing extrasynaptic GABA(A) receptors that mediate tonic inhibition. Although the amplitude of GABA-activated currents through α4βδ receptors is modulated by multiple general anesthetics, the effects of general anesthetics on desensitization and deactivation of α4βδ receptors remain unknown. In the current study, we investigated the effect of etomidate, a potent general anesthetic, on the kinetics and the pseudo steady-state current amplitude of α4β3δ receptors inducibly expressed in human embryonic kidney 293 TetR cells. Etomidate directly activates α4β3δ receptors in a concentration-dependent manner. Etomidate at a clinically relevant concentration (3.2 μM) enhances maximal response without altering the EC(50) of GABA concentration response. Etomidate also increases the extent of desensitization and prolongs the deactivation of α4β3δ receptors in the presence of maximally activating concentrations of GABA (1 mM). To mimic the modulatory effect of etomidate on tonic currents, long pulses (30–60 seconds) of a low GABA concentration (1 μM) were applied to activate α4β3δ receptors in the absence and presence of etomidate. Although etomidate increases the desensitization of α4β3δ receptors, the pseudo steady-state current amplitude at 1 μM GABA is augmented by etomidate. Our data demonstrate that etomidate enhances the pseudo steady-state current of α4β3δ receptors evoked by a GABA concentration comparable to an ambient GABA level, suggesting that α4β3δ receptors may mediate etomidate’s anesthetic effect in the brain.

A clade of New World monkeys (NWMs) exhibits considerable diversity in both oxytocin (OT) ligand and oxytocin receptor (OTR) structure. Most notable is the variant Pro(8)-OT, with proline instead of leucine at the eighth position, resulting in a rigid bend in the peptide backbone. A higher proportion of species that express Pro(8)-OT also engage in biparental care and social monogamy. When marmosets (genus Callithrix), a biparental and monogamous Pro(8)-OT NWM species, are administered the ancestral Leu(8)-OT, there is no change in social behavior compared with saline treatment. However, when Pro(8)-OT is administered, marmosets’ sociosexual and prosocial behaviors are altered. The studies here tested the hypothesis that OTR binding affinities and OT-induced intracellular Ca(2+) potencies would favor the native OT ligand in OTRs from four primate species, each representing a unique combination of ancestral lineage, breeding system, and native OT ligand: humans (Leu(8)-OT, monogamous, apes), macaques (Leu(8)-OT, nonmonogamous, Old World monkey), marmosets (Pro(8)-OT, monogamous, NWM), and titi monkeys (Leu(8)-OT, monogamous, NWM). OTRs were expressed in immortalized Chinese hamster ovary cells and tested for intact-cell binding affinities for Pro(8)-OT, Leu(8)-OT, and arginine vasopressin (AVP), as well as intracellular Ca(2+) signaling after stimulation with Pro(8)-OT, Leu(8)-OT, and AVP. Contrary to our hypothesis, Pro(8)-OT bound at modestly higher affinities and stimulated calcium signaling at modestly higher potencies compared with Leu(8)-OT in all four primate OTRs. Thus, differences downstream from a ligand-receptor binding event are more likely to explain the different behavioral responses to these two ligands.

Ethanol produces changes in GABAA receptor trafficking and function that contribute to ethanol dependence symptomatology. Extrasynaptic γ-aminobutyric acid A receptors (GABAA-R) mediate inhibitory tonic current and are of particular interest because they are potentiated by physiologically relevant doses of ethanol. Here, we isolate GABAA α4δ receptors by western blotting in subsynaptic fractions to investigate protein kinase A (PKA) and protein kinase C (PKC) modulation of ethanol-induced receptor trafficking, while extrasynaptic receptor function is determined by measurement of tonic inhibition and responses evoked by 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP). Rat cerebral cortical neurons were grown for 18 days in vitro and exposed to ethanol and/or PKA/PKC modulators. Ethanol exposure (1 hour) did not alter GABAA α4 receptor abundance, but it increased tonic current amplitude, an effect that was prevented by inhibiting PKA, but not PKC. Direct activation of PKA, but not PKC, increased the abundance and tonic current of extrasynaptic α4δ receptors. In contrast, prolonged ethanol exposure (4 hours) reduced α4δ receptor abundance as well as tonic current, and this effect was also PKA dependent. Finally, PKC activation by ethanol or phorbol-12,13-dibutyrate (PdBu) had no effect on extrasynaptic α4δ subunit abundance or activity. We conclude that ethanol alters extrasynaptic α4δ receptor function and expression in cortical neurons in a PKA-dependent manner, but ethanol activation of PKC does not influence these receptors. These results could have clinical relevance for therapeutic strategies to restore normal GABAergic functioning for the treatment of alcohol use disorders.

Beyond canonical signaling via Gαs and cAMP, the concept of functional selectivity at β2-adrenoceptors (β2ARs) describes the ability of adrenergic drugs to stabilize ligand-specific receptor conformations to initiate further signaling cascades comprising additional G-protein classes or β-arrestins (βarr). A set of 65 adrenergic ligands including 40 agonists and 25 antagonists in either racemic or enantiopure forms was used for βarr recruitment experiments based on a split-luciferase assay in a cellular system expressing β2AR. Many agonists showed only (weak) partial agonism regarding βarr recruitment. Potencies and/or efficacies increased depending on the number of chirality centers in (R) configuration; no (S)-configured distomer was more effective at inducing βarr recruitment other than the eutomer. βarr2 was recruited more effectively than βarr1. The analysis of antagonists revealed no significant effects on βarr recruitment. Several agonists showed preference for activation of Gαs GTPase relative to βarr recruitment, and no βarr-biased ligand was identified. In conclusion: 1) agonists show strong bias for Gαs activation relative to βarr recruitment; 2) agonists recruit βarr1 and βarr2 with subtle differences; and 3) there is no evidence for βarr recruitment by antagonists.

Sphingosine 1-phosphate (S1P) levels are significantly higher in blood and lymph than in tissues. This S1P concentration difference is necessary for proper lymphocyte egress from secondary lymphoid tissue and to maintain endothelial barrier integrity. Studies with mice lacking either sphingosine kinase (SphK) type 1 and 2 indicate that these enzymes are the sole biosynthetic source of S1P, but they play different roles in setting S1P blood levels. We have developed a set of drug-like SphK inhibitors, with differing selectivity for the two isoforms of this enzyme. Although all SphK inhibitors tested decrease S1P when applied to cultured U937 cells, only those inhibitors with a bias for SphK2 drove a substantial increase in blood S1P in mice and this rise was detectable within minutes of administration of the inhibitor. Blood S1P also increased in response to SphK2 inhibitors in rats. Mass-labeled S1P was cleared more slowly after intravenous injection into SphK2 inhibitor–treated mice or mice lacking a functional SphK2 gene; thus, the increased accumulation of S1P in the blood appears to result from the decreased clearance of S1P from the blood. Therefore, SphK2 appears to have a function independent of generating S1P in cells. Our results suggest that differential SphK inhibition with a drug might afford a method to manipulate blood S1P levels in either direction while lowering tissue S1P levels.

In rat sympathetic neurons from the superior cervical ganglia (SCG) expressing metabotropic glutamate receptor mGluR1 or mGluR5, overexpression of scaffolding Homer proteins, which bind to a Homer ligand in their C termini, cause receptor clustering and uncoupling from ion channel modulation. In the absence of recombinant Homer protein overexpression, uncoupling of mGluRs from voltage-dependent channels can be induced by expression of Preso1, an adaptor of proline-directed kinases that phosphorylates the Homer ligand and recruits binding of endogenous Homer proteins. Here we show that in SCG neurons expressing mGluR1 and the tyrosine receptor kinase B, treatment with brain-derived neurotrophic factor (BDNF) produces a similar uncoupling of the receptors from calcium channels. We investigated the pathways that mediate this uncoupling and compared it with uncoupling observed with Preso1 expression. Both BDNF- and Preso1-induced uncoupling require residues T1151 and S1154 in the mGluR1 Homer ligand (TPPSPF). Uncoupling via Preso1 but not BDNF was prevented by expression of a dominant negative Cdk5, suggesting that endogenous Cdk5 mediates Preso1-dependent phosphorylation of mGluR1. Dominant negative Cdk5 did not block the BDNF effect but this was sensitive to inhibitors of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase cascade. Interestingly, the BDNF pathway appeared to require native Preso1 binding to mGluR, because overexpression of the Preso1 FERM domain, which mediates the Preso1-mGluR interaction, prevented BDNF-induced uncoupling. These data suggest that the BDNF/tyrosine receptor kinase B and Cdk5 pathways converge at the level of mGluR to similarly induce Homer ligand phosphorylation, recruit Homer binding, and uncouple mGluRs from channel regulation.

Efavirenz (EFV) is a non-nucleoside reverse-transcriptase inhibitor in wide use for the treatment of human immunodeficiency virus infection. Although EFV is generally well tolerated, neuropsychiatric toxicity has been well documented. The toxic effects of EFV in hepatocytes and keratinocytes have been linked to mitochondrial perturbations and changes in autophagy. Here, we studied the effect of EFV on mitochondria and autophagy in neuronal cell lines and primary neurons. In SH-SY5Y cells, EFV induced a drop in ATP production, which coincided with increased autophagy, mitochondrial fragmentation, and mitochondrial depolarization. EFV-induced mitophagy was also detected by colocalization of mitochondria and autophagosomes and use of an outer mitochondrial membrane tandem fluorescent vector. Pharmacologic inhibition of autophagy with 3-methyladenine increased the cytotoxic effect of EFV, suggesting that autophagy promotes cell survival. EFV also reduces ATP production in primary neurons, induces autophagy, and changes mitochondrial morphology. Overall, EFV is able to acutely induce autophagy and mitochondrial changes in neurons. These changes may be involved in the mechanism leading to central nervous system toxicity observed in clinical EFV use.

Numerous studies have demonstrated that females have a higher risk of experiencing several pain disorders with either greater frequency or severity than males. Although the mechanisms that underlie this sex disparity remain unclear, several studies have shown an important role for sex steroids, such as estrogen, in the modulation of nociception. Receptors for estrogen are present in primary afferent neurons in the trigeminal and dorsal root ganglia, and brief exposure to estrogen increases responses to the inflammatory mediator bradykinin (BK). However, the mechanism for estrogen-mediated enhancement of BK signaling is not fully understood. The aim of the present study was to evaluate the relative contributions of estrogen receptor α (ERα), ERβ, and G protein-coupled estrogen receptor 1 (GPER) to the enhanced signaling of the inflammatory mediator BK by 17β-estradiol (17β-E2) in primary sensory neurons from female rats in culture (ex vivo) and in behavioral assays of nociception in vivo. The effects of 17β-E2 on BK responses were mimicked by ERα-selective agonists and blocked by ERα-selective antagonists and by small interfering RNA knockdown of ERα. The data indicate that ERα is required for 17β-E2-mediated enhancement of BK signaling in peripheral sensory neurons in female rats.

Nicotine, the primary psychoactive component in tobacco smoke, produces its behavioral effects through interactions with neuronal nicotinic acetylcholine receptors (nAChRs). α 4 β 2 nAChRs are the most abundant in mammalian brain, and converging evidence shows that this subtype mediates the rewarding and reinforcing effects of nicotine. A number of rare variants in the CHRNA4 gene that encode the α 4 nAChR subunit have been identified in human subjects and appear to be underrepresented in a cohort of smokers. We compared three of these variants ( α 4R336C, α 4P451L, and α 4R487Q) to the common variant to determine their effects on α 4 β 2 nAChR pharmacology. We examined [ 3 H]epibatidine binding, interacting proteins, and phosphorylation of the α 4 nAChR subunit with liquid chromatography and tandem mass spectrometry (LC-MS/MS) in HEK 293 cells and voltage-clamp electrophysiology in Xenopus laevis oocytes. We observed significant effects of the α 4 variants on nAChR expression, subcellular distribution, and sensitivity to nicotine-induced receptor upregulation. Proteomic analysis of immunopurified α 4 β 2 nAChRs incorporating the rare variants identified considerable differences in the intracellular interactomes due to these single amino acid substitutions. Electrophysiological characterization in X. laevis oocytes revealed alterations in the functional parameters of activation by nAChR agonists conferred by these α 4 rare variants, as well as shifts in receptor function after incubation with nicotine. Taken together, these experiments suggest that genetic variation at CHRNA4 alters the assembly and expression of human α 4 β 2 nAChRs, resulting in receptors that are more sensitive to nicotine exposure than those assembled with the common α 4 variant. The changes in nAChR pharmacology could contribute to differences in responses to smoked nicotine in individuals harboring these rare variants.

Opioid receptors expressed by peripheral pain-sensing neurons are functionally inactive for antinociceptive signaling under most basal conditions; however, tissue damage or exposure to inflammatory mediators (e.g., bradykinin) converts these receptors from a nonresponsive state to a functionally competent state. Here we tested the hypothesis that the basal, nonresponsive state of the mu- and delta-opioid receptors (MOR and DOR, respectively) is the result of constitutive receptor activity that activates desensitization mechanisms, resulting in MOR and DOR receptor systems that are constitutively desensitized. Consistent with our previous findings, under basal conditions, neither the MOR agonist [d-Ala2,N-MePhe4,Gly-ol5]-enkephalin nor the DOR agonist [d-Pen2,5]-enkephalin, inhibited prostaglandin E2 (PGE2)-stimulated cAMP accumulation in peripheral sensory neurons in culture (ex vivo) or inhibited PGE2-stimulated thermal allodynia in the rat hind paw in vivo. Prolonged treatment with naloxone induced MOR and DOR responsiveness both in vivo and ex vivo to a similar magnitude as that produced by bradykinin. Also similar to bradykinin, the effect of naloxone persisted for 60 minutes after washout of the ligand. By contrast, prolonged treatment with 6β-naltrexol, did not induce functional competence of MOR or DOR but blocked the effect of naloxone. Treatment with siRNA for β-arrestin-2, but not β-arrestin-1, also induced MOR and DOR functional competence in cultured peripheral sensory neurons. These data suggest that the lack of responsiveness of MOR and DOR to agonist for antinociceptive signaling in peripheral sensory neurons is due to constitutive desensitization that is likely mediated by β-arrestin-2.

Interleukin-1β (IL-1β) is a proinflammatory cytokine that is implicated in many autoinflammatory disorders, but is also important in defense against pathogens. Thus, there is a need to safely and effectively modulate IL-1β activity to reduce pathology while maintaining function. Gevokizumab is a potent anti–IL-1β antibody being developed as a treatment for diseases in which IL-1β has been associated with pathogenesis. Previous data indicated that gevokizumab negatively modulates IL-1β signaling through an allosteric mechanism. Because IL-1β signaling is a complex, dynamic process involving multiple components, it is important to understand the kinetics of IL-1β signaling and the impact of gevokizumab on this process. In the present study, we measured the impact of gevokizumab on the IL-1β system using Schild analysis and surface plasmon resonance studies, both of which demonstrated that gevokizumab decreases the binding affinity of IL-1β for the IL-1 receptor type I (IL-1RI) signaling receptor, but not the IL-1 counter-regulatory decoy receptor (IL-1 receptor type II). Gevokizumab inhibits both the binding of IL-1β to IL-1RI and the subsequent recruitment of IL-1 accessory protein primarily by reducing the association rates of these interactions. Based on this information and recently published structural data, we propose that gevokizumab decreases the association rate for binding of IL-1β to its receptor by altering the electrostatic surface potential of IL-1β, thus reducing the contribution of electrostatic steering to the rapid association rate. These data indicate, therefore, that gevokizumab is a unique inhibitor of IL-1β signaling that may offer an alternative to current therapies for IL-1β–associated autoinflammatory diseases.

The second messenger cAMP is involved in a number of cellular signaling pathways. In mammals, cAMP is produced by either the hormonally responsive, G protein–regulated transmembrane adenylyl cyclases (tmACs) or by the bicarbonate- and calcium-regulated soluble adenylyl cyclase (sAC). To develop tools to differentiate tmAC and sAC signaling, we determined the specificity and potency of commercially available adenylyl cyclase inhibitors. In cellular systems, two inhibitors, KH7 and catechol estrogens, proved specific for sAC, and 2′,5′-dideoxyadenosine proved specific for tmACs. These tools provide a means to define the specific contributions of the different families of adenylyl cyclases in cells and tissues, which will further our understanding of cell signaling.

Monocytic cells enhance neovascularization by releasing proangiogenic mediators and/or by transdifferentiating into endothelial-like cells. However, the mechanisms that govern this transdifferentiation process are largely unknown. Recently, monocyte chemotactic protein-1 (MCP-1)-induced protein (MCPIP) has been identified as a novel CCCH-type zinc-finger protein expressed primarily in monocytic cells. Here, we analyzed whether MCPIP might exert angiogenic effects by promoting differentiation of monocytic cells into endothelial cell (EC)-like phenotype. The expression of MCPIP increased during MCP-1-induced transdifferentiation in human bone marrow mononuclear cells (BMNCs). Knockdown of MCPIP with small interfering RNA (siRNA) abolished MCP-1-induced expression of EC markers Flk-1 and Tie-2 in human BMNCs. BMNCs transfected with MCPIP expression vector displayed EC-like morphology accompanied by downregulation of monocytic markers CD14 and CD11b, upregulation of EC markers Flk-1 and Tie-2, induction of cadherin (cdh)-12 and -19, activation of endoplasmic reticulum (ER) stress, and autophagy. Knockdown of cdh-12 or cdh-19 markedly inhibited MCPIP-induced enhancement of cell attachment and EC-marker expression. Inhibition of ER stress by tauroursodeoxycholate abolished MCPIP-induced expression of EC markers. Inhibition of autophagy by knockdown of Beclin-1 with siRNA or by an autophagy inhibitor 3'-methyladenine inhibited MCPIP-induced expression of EC markers. Expression of MCPIP in BMNCs enhanced uptake of acetylated low-density lipoprotein (acLDL), formation of EC-colony, incorporation of cells into capillary-like structure on Matrigel, and exhibited increased neovascularization in the ischemic hindlimb in mice. These results demonstrate that MCPIP may be an important regulator of inflammatory angiogenesis and provide novel mechanistic insights into the link between MCP-1 and cardiovascular diseases.

Induction of apoptosis by the death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising antitumor therapy. However, not all tumor cells are sensitive to TRAIL, highlighting the need for strategies to overcome TRAIL resistance. Inhibitor of apoptosis family member survivin is constitutively activated in various cancers and blocks apoptotic signaling. Recently, we demonstrated that YM-155 [3-(2-methoxyethyl)-2-methyl-4,9-dioxo-1-(pyrazin-2-ylmethyl)-4,9-dihydro-3H-naphtho[2,3-d]imidazol-1-ium bromide], a small molecule inhibitor, downregulates not only survivin in gliomas but also myeloid cell leukemia sequence 1 (Mcl-1), and it upregulates proapoptotic Noxa levels. Because Mcl-1 and survivin are critical mediators of resistance to various anticancer therapies, we questioned whether YM-155 could sensitize resistant glioma cells to TRAIL. To address this hypothesis, we combined YM-155 with TRAIL and examined the effects on cell survival and apoptotic signaling. TRAIL or YM-155 individually induced minimal killing in highly resistant U373 and LNZ308 cell lines, but combining TRAIL with YM-155 triggered a synergistic proapoptotic response, mediated through mitochondrial dysfunction via activation of caspases-8, -9, -7, -3, poly-ADP-ribose polymerase, and Bid. Apoptosis induced by combination treatments was blocked by caspase-8 and pan-caspase inhibitors. In addition, knockdown of Mcl-1 by RNA interference overcame apoptotic resistance to TRAIL. Conversely, silencing Noxa by RNA interference reduced the combined effects of YM-155 and TRAIL on apoptosis. Mechanistically, these findings indicate that YM-155 plays a role in counteracting glioma cell resistance to TRAIL-induced apoptosis by downregulating Mcl-1 and survivin and amplifying mitochondrial signaling through intrinsic and extrinsic apoptotic pathways. The significantly enhanced antitumor activity of the combination of YM-155 and TRAIL may have applications for therapy of malignant glioma.

Activation of the cannabinoid CB1 receptor (CB1) is modulated by aspartate residue D2.63(176) in transmembrane helix (TMH) 2. Interestingly, D2.63 does not affect the affinity for ligand binding at the CB1 receptor. Studies in class A G protein-coupled receptors have suggested an ionic interaction between residues of TMH2 and 7. In this report, modeling studies identified residue K373 in the extracellular-3 (EC-3) loop in charged interactions with D2.63. We investigated this possibility by performing reciprocal mutations and biochemical studies. D2.63(176)A, K373A, D2.63(176)A-K373A, and the reciprocal mutant with the interacting residues juxtaposed D2.63(176)K-K373D were characterized using radioligand binding and guanosine 5'-3-O-(thio)triphosphate functional assays. None of the mutations resulted in a significant change in the binding affinity of N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A) or (-)-3cis -[2-hydroxyl-4-(1,1-dimethyl-heptyl)phenyl]-trans-4-[3-hydroxyl-propyl] cyclohexan-1-ol (CP55,940). Modeling studies indicated that binding-site interactions and energies of interaction for CP55,940 were similar between wild-type and mutant receptors. However, the signaling of CP55,940, and (R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]-pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)-methanone mesylate (WIN55,212-2) was impaired at the D2.63(176)A-K373A and the single-alanine mutants. In contrast, the reciprocal D2.63(176)K-K373D mutant regained function for both CP55,940 and WIN55,212-2. Computational results indicate that the D2.63(176)-K373 ionic interaction strongly influences the conformation(s) of the EC-3 loop, providing a structure-based rationale for the importance of the EC-3 loop to signal transduction in CB1. The putative ionic interaction results in the EC-3 loop pulling over the top (extracellular side) of the receptor; this EC-3 loop conformation may serve protective and mechanistic roles. These results suggest that the ionic interaction between D2.63(176) and K373 is important for CB1 signal transduction.

Drugs targeting the orphan receptor GPR35 have potential therapeutic application in a number of disease areas, including inflammation, metabolic disorders, nociception, and cardiovascular disease. Currently available surrogate GPR35 agonists identified from pharmacologically relevant compound libraries have limited utility due to the likelihood of off-target effects in vitro and in vivo and the variable potency that such ligands exhibit across species. We sought to identify and characterize novel GPR35 agonists to facilitate studies aimed at defining the physiologic role of GPR35. PathHunter β-arrestin recruitment technology was validated as a human GPR35 screening assay, and a high-throughput screen of 100,000 diverse low molecular weight compounds was conducted. Confirmed GPR35 agonists from five distinct chemotypes were selected for detailed characterization using both β-arrestin recruitment and G protein-dependent assays and each of the human, mouse, and rat GPR35 orthologs. These studies identified 4-{(Z)-[(2Z)-2-(2-fluorobenzylidene)-4-oxo-1,3-thiazolidin-5-ylidene]methyl}benzoic acid (compound 1) as the highest potency full agonist of human GPR35 yet described. As with certain other GPR35 agonists, compound 1 was markedly selective for human GPR35, but displayed elements of signal bias between β-arrestin-2 and G protein-dependent assays. Compound 1 also displayed competitive behavior when assessed against the human GPR35 antagonist, ML-145 (2-hydroxy-4-[4-(5Z)-5-[(E)-2-methyl-3-phenylprop-2-enylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]butanoylamino]benzoic acid). Of the other chemotypes studied, compounds 2 and 3 were selective for the human receptor, but compounds 4 and 5 demonstrated similar activity at human, rat, and mouse GPR35 orthologs. Further characterization of these compounds and related analogs is likely to facilitate a better understanding of GPR35 in health and disease.

Population studies, preclinical, and clinical trials suggest a role for cyclooxygenase-2 (COX-2, PTGS2) in tumor formation and progression. The downstream prostanoid receptor signaling pathways involved in tumorigenesis are poorly understood, although prostaglandin E2 (PGE(2)), a major COX-2 metabolite which is usually upregulated in the involved tissues, presumably plays important roles in tumor biology. Taking advantage of our recently identified novel selective antagonist for the EP2 (PTGER2) subtype of PGE(2) receptor, we demonstrated that EP2 receptor activation could promote prostate cancer cell growth and invasion in vitro, accompanied by upregulation of the tumor-promoting inflammatory cytokines, such as IL-1β and IL-6. Our results suggest the involvement of prostaglandin receptor EP2 in cancer cell proliferation and invasion possibly via its inflammatory actions, and indicate that selective blockade of the PGE(2)-EP2 signaling pathway via small molecule antagonists might represent a novel therapy for tumorigenesis.

Inhibitors of zinc-dependent histone deacetylases (HDACs) profoundly affect cellular function by altering gene expression via changes in nucleosomal histone tail acetylation. Historically, investigators have employed pan-HDAC inhibitors, such as the hydroxamate trichostatin A (TSA), which simultaneously targets members of each of the three zinc-dependent HDAC classes (classes I, II, and IV). More recently, class- and isoform-selective HDAC inhibitors have been developed, providing invaluable chemical biology probes for dissecting the roles of distinct HDACs in the control of various physiologic and pathophysiological processes. For example, the benzamide class I HDAC-selective inhibitor, MGCD0103 [N-(2-aminophenyl)-4-[[(4-pyridin-3-ylpyrimidin-2-yl)amino]methyl] benzamide], was shown to block cardiac fibrosis, a process involving excess extracellular matrix deposition, which often results in heart dysfunction. Here, we compare the mechanisms of action of structurally distinct HDAC inhibitors in isolated primary cardiac fibroblasts, which are the major extracellular matrix–producing cells of the heart. TSA, MGCD0103, and the cyclic peptide class I HDAC inhibitor, apicidin, exhibited a common ability to enhance histone acetylation, and all potently blocked cardiac fibroblast cell cycle progression. In contrast, MGCD0103, but not TSA or apicidin, paradoxically increased expression of a subset of fibrosis-associated genes. Using the cellular thermal shift assay, we provide evidence that the divergent effects of HDAC inhibitors on cardiac fibroblast gene expression relate to differential engagement of HDAC1- and HDAC2-containing complexes. These findings illustrate the importance of employing multiple compounds when pharmacologically assessing HDAC function in a cellular context and during HDAC inhibitor drug development.

Small molecules that target the adrenergic family of G protein–coupled receptors (GPCRs) show promising therapeutic efficacy for the treatment of various cancers. In this study, we report that human colon cancer cell line SW480 expresses low-density functional α1B-adrenergic receptors (ARs) as revealed by label-free dynamic mass redistribution (DMR) signaling technology and confirmed by quantitative reverse-transcriptase polymerase chain reaction analysis. Remarkably, although endogenous α1B-ARs are not detectable via either [3H]-prazosin–binding analysis or phosphoinositol hydrolysis assays, their activation leads to robust DMR and enhanced cell viability. We provide pharmacological evidence that stimulation of α1B-ARs enhances SW480 cell viability without affecting proliferation, whereas stimulating β-ARs diminishes both viability and proliferation of SW480 cells. Our study illustrates the power of label-free DMR technology for identifying and characterizing low-density GPCRs in cells and suggests that drugs targeting both α1B- and β-ARs may represent valuable small-molecule therapeutics for the treatment of colon cancer.

Inhibition of the major lysosomal proteases, cathepsins B, D, and L, impairs growth of several cell types but leads to apoptosis in neuroblastoma. The goal of this study was to examine the mechanisms by which enzyme inhibition could cause cell death. Cathepsin inhibition caused cellular accumulation of fragments of the insulin growth factor 1 (IGF-1) receptor. The fragments were located in dense organelles that were characterized as autophagosomes. This novel discovery provides the first clear link between lysosomal function, autophagy, and IGF-1- mediated cell proliferation. A more in-depth analysis of the IGF1 signaling pathway revealed that the mitogen-activated protein kinase (MAPK) cell-proliferation pathway was impaired in inhibitor treated cells, whereas the Akt cell survival pathway remained functional. Shc, an adapter protein that transmits IGF-1 signaling through the MAPK pathway, was sequestered in autophagosomes; whereas IRS-2, an adapter protein that transmits IGF-1 signaling through the Akt pathway, was unaffected by cathepsin inhibition. Furthermore, Shc was sequestered in autophagosomes as its active form, indicating that autophagy is a key mechanism for downregulating IGF-1-induced cell proliferation. Cathepsin inhibition had a greater effect on autophagic sequestration of the neuronal specific adapter protein, Shc-C, than ubiquitously expressed Shc-A, providing mechanistic support for the enhanced sensitivity of neuronally derived tumor cells. We also observed impaired activation of MAPK by epidermal growth factor treatment in inhibitor-treated cells. The Shc adapter proteins are central to transducing proliferation signaling by a range of receptor tyrosine kinases; consequently, cathepsin inhibition may become an important therapeutic approach for treating neuroblastoma and other tumors of neuronal origin.

Variation in pharmacology and function of ligands at species orthologs can be a confounding feature in understanding the biology and role of poorly characterized receptors. Substantial selectivity in potency of a number of GPR35 agonists has previously been demonstrated between human and rat orthologs of this G protein-coupled receptor. Via a bioluminescence resonance energy transfer-based assay of induced interactions between GPR35 and β-arrestin-2, addition of the mouse ortholog to such studies indicated that, as for the rat ortholog, murine GPR35 displayed very low potency for pamoate, whereas potency for the reference GPR35 agonist zaprinast was intermediate between the rat and human orthologs. This pattern was replicated in receptor internalization and G protein activation assays. The effectiveness and mode of action of two recently reported GPR35 antagonists, methyl-5-[(tert-butylcarbamothioylhydrazinylidene)methyl]-1-(2,4-difluorophenyl)pyrazole-4-carboxylate (CID-2745687) and 2-hydroxy-4-[4-(5Z)-5-[(E)-2-methyl-3-phenylprop-2-enylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]butanoylamino)benzoic acid (ML-145), were investigated. Both CID-2745687 and ML-145 competitively inhibited the effects at human GPR35 of cromolyn disodium and zaprinast, two agonists that share an overlapping binding site. By contrast, although ML-145 also competitively antagonized the effects of pamoate, CID-2745687 acted in a noncompetitive fashion. Neither ML-145 nor CID-2745687 was able to effectively antagonize the agonist effects of either zaprinast or cromolyn disodium at either rodent ortholog of GPR35. These studies demonstrate that marked species selectivity of ligands at GPR35 is not restricted to agonists and considerable care is required to select appropriate ligands to explore the function of GPR35 in nonhuman cells and tissues.

Prenatal exposure to diethylstilbestrol (DES) is known to cause altered immune functions and increased susceptibility to autoimmune disease in humans. In the current study, we investigated the effect of prenatal exposure to DES on thymocyte differentiation involving apoptotic pathways. Prenatal DES exposure caused thymic atrophy, apoptosis, and up-regulation of Fas and Fas ligand (FasL) expression in thymocytes. To examine the mechanism underlying DES-mediated regulation of Fas and FasL, we performed luciferase assays using T cells transfected with luciferase reporter constructs containing full-length Fas or FasL promoters. There was significant luciferase induction in the presence of Fas or FasL promoters after DES exposure. Further analysis demonstrated the presence of several cis-regulatory motifs on both Fas and FasL promoters. When DES-induced transcription factors were analyzed, estrogen receptor element (ERE), nuclear factor κB (NF-κB), nuclear factor of activated T cells (NF-AT), and activator protein-1 motifs on the Fas promoter, as well as ERE, NF-κB, and NF-AT motifs on the FasL promoter, showed binding affinity with the transcription factors. Electrophoretic mobility-shift assays were performed to verify the binding affinity of cis-regulatory motifs of Fas or FasL promoters with transcription factors. There was shift in mobility of probes (ERE or NF-κB2) of both Fas and FasL in the presence of nuclear proteins from DES-treated cells, and the shift was specific to DES because these probes failed to shift their mobility in the presence of nuclear proteins from vehicle-treated cells. Together, the current study demonstrates that prenatal exposure to DES triggers significant alterations in apoptotic molecules expressed on thymocytes, which may affect T-cell differentiation and cause long-term effects on the immune functions.

Leflunomide, flutamide, nimodipine, mexiletine, sulindac, tranilast, 4-hydroxytamoxifen, and omeprazole are pharmaceuticals previously characterized as aryl hydrocarbon receptor (AHR) agonists in various cell lines and animal models. In this study, the eight AHR-active pharmaceuticals were investigated in highly aggressive aryl hydrocarbon (Ah)-responsive BT474 and MDA-MB-468 breast cancer cell lines, and their effects on AHR protein, CYP1A1 (protein and mRNA), CYP1B1 (mRNA), and cell migration were determined. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was used as a positive control. The AHR agonist activities of the pharmaceuticals depended on structure, response, and cell context. Most compounds induced one or more AHR-mediated responses in BT474 cells, whereas in Ah-responsive MDA-MB-468 cells effects of the AHR-active pharmaceuticals were highly variable. 4-Hydroxytamoxifen, mexiletine, and tranilast did not induce CYP1A1 in MDA-MB-468 cells; moreover, in combination with TCDD, mexiletine was a potent AHR antagonist, tranilast was a partial antagonist, and 4-hydroxytamoxifen also exhibited some AHR antagonist activity. Omeprazole and, to a lesser extent, sulindac and leflunomide were full and partial AHR agonists, respectively, in both breast cancer cell lines. These data indicate that the AHR-active pharmaceuticals are selective AHR modulators, and applications of these drugs for targeting the AHR must be confirmed by studies using the most relevant cell context.

Glucocorticoids are standard of care for many inflammatory conditions, but chronic use is associated with a broad array of side effects. This has led to a search for dissociative glucocorticoids--drugs able to retain or improve efficacy associated with transrepression [nuclear factor-κB (NF-κB) inhibition] but with the loss of side effects associated with transactivation (receptor-mediated transcriptional activation through glucocorticoid response element gene promoter elements). We investigated a glucocorticoid derivative with a Δ-9,11 modification as a dissociative steroid. The Δ-9,11 analog showed potent inhibition of tumor necrosis factor-α-induced NF-κB signaling in cell reporter assays, and this transrepression activity was blocked by 17β-hydroxy-11β-[4-dimethylamino phenyl]-17α-[1-propynyl]estra-4,9-dien-3-one (RU-486), showing the requirement for the glucocorticoid receptor (GR). The Δ-9,11 analog induced the nuclear translocation of GR but showed the loss of transactivation as assayed by GR-luciferase constructs as well as mRNA profiles of treated cells. The Δ-9,11 analog was tested for efficacy and side effects in two mouse models of muscular dystrophy: mdx (dystrophin deficiency), and SJL (dysferlin deficiency). Daily oral delivery of the Δ-9,11 analog showed a reduction of muscle inflammation and improvements in multiple muscle function assays yet no reductions in body weight or spleen size, suggesting the loss of key side effects. Our data demonstrate that a Δ-9,11 analog dissociates the GR-mediated transcriptional activities from anti-inflammatory activities. Accordingly, Δ-9,11 analogs may hold promise as a source of safer therapeutic agents for chronic inflammatory disorders.

Modeling the binding sites for spermine and ifenprodil on the regulatory (R) domains of the N -methyl-d-aspartate receptor GluN1 and GluN2B subunits was carried out after measuring spermine stimulation and ifenprodil inhibition at receptors containing GluN1 and GluN2B R domain mutants. Models were constructed based on the published crystal structure of the GluN1 and GluN2B R domains, which form a heterodimer ( Nature 475: 249–253, 2011). The experimental results and modeling suggest that a binding site for spermine was formed by the residues near the cleft between the R1 and R2 lobes of the GluN1 R domain (GluN1R) together with residues on the surface of the R2 (C-terminal side) lobe of the GluN2B R domain (GluN2BR). The ifenprodil binding site included residues on the surface of the R1 lobe (N-terminal side) of GluN1R together with residues near the cleft between the R1 and R2 lobes of GluN2BR. It was confirmed using a Western blot analysis that GluN1R and GluN2BR formed a heterodimer. Models of spermine and ifenprodil binding to the heterodimer were constructed. The modeling suggests that an open space between the two R1 lobes of GluN1R and GluN2BR is promoted through spermine binding and that the R1 lobes of GluN1R and GluN2BR approach each other through ifenprodil binding—an effect opposite to that seen with the binding of spermine.

In an effort to delineate how specific molecular interactions of dopamine receptor ligand classes vary between D2-like dopamine receptor subtypes, a conserved threonine in transmembrane (TM) helix 7 (Thr7.39), implicated as a key ligand interaction site with biogenic amine G protein-coupled receptors, was substituted with alanine in D2 and D4 receptors. Interrogation of different ligand chemotypes for sensitivity to this substitution revealed enhanced affinity in the D4, but not the D2 receptor, specifically for substituted benzamides (SBAs) having polar 4- (para) and/or 5- (meta) benzamide ring substituents. D4-T7.39A was fully functional, and the mutation did not alter the sodium-mediated positive and negative allostery observed with SBAs and agonists, respectively. With the exception of the non-SBA ligand (+)-butaclamol, which, in contrast to certain SBAs, had decreased affinity for the D4-T7.39A mutant, the interactions of numerous other ligands were unaffected by this mutation. SBAs were docked into D4 models in the same mode as observed for eticlopride in the D3 crystal structure. In this mode, interactions with TM5 and TM6 residues constrain the SBA ring position that produces distal steric crowding between pyrrolidinyl/diethylamine moieties and D4-Thr7.39. Ligand-residue interaction energy profiles suggest this crowding is mitigated by substitution with a smaller alanine. The profiles indicate sites that contribute to the SBA binding interaction and site-specific energy changes imparted by the D4-T7.39A mutation. Substantial interaction energy changes are observed at only a few positions, some of which are not conserved among the dopamine receptor subtypes and thus seem to account for this D4 subtype-specific structure-activity relationship.

We observed that glioma cells are differentially sensitive to N-{4-[4-(4'-chloro-biphenyl-2-ylmethyl)-piperazin-1-yl]-benzoyl}-4-(3-dimethylamino-1-phenylsulfanylmethyl-propylamino)-3-nitro-benzenesulfonamide (ABT-737) and administration of ABT-737 at clinically achievable doses failed to induce apoptosis. Although elevated Bcl-2 levels directly correlated with sensitivity to ABT-737, overexpression of Bcl-2 did not influence sensitivity to ABT-737. To understand the molecular basis for variable and relatively modest sensitivity to the Bcl-2 homology domain 3 mimetic drug ABT-737, the abundance of Bcl-2 family members was assayed in a panel of glioma cell lines. Bcl-2 family member proteins, Bcl-xL, Bcl-w, Mcl-1, Bax, Bak, Bid, and Noxa, were found to be expressed ubiquitously at similar levels in all cell lines tested. We then examined the contribution of other apoptosis-resistance pathways to ABT-737 resistance. Bortezomib, an inhibitor of nuclear factor-kappaB (NF-κB), was found to enhance sensitivity of ABT-737 in phosphatase and tensin homolog on chromosome 10 (PTEN)-wild type, but not PTEN-mutated glioma cell lines. We therefore investigated the association between phosphatidylinositol 3-kinase (PI3K)/Akt activation and resistance to the combination of ABT-737 and bortezomib in PTEN-deficient glioma cells. Genetic and pharmacological inhibition of PI3K inhibition sensitized PTEN-deficient glioma cells to bortezomib- and ABT-737-induced apoptosis by increasing cleavage of Bid protein, activation and oligomerization of Bax, and loss of mitochondrial membrane potential. Our data further suggested that PI3K/Akt-dependent protection may occur upstream of the mitochondria. This study demonstrates that interference with multiple apoptosis-resistance signaling nodes, including NF-κB, Akt, and Bcl-2, may be required to induce apoptosis in highly resistant glioma cells, and therapeutic strategies that target the PI3K/Akt pathway may have a selective role for cancers lacking PTEN function.

Increased vascular 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 arachidonic acid metabolite, promotes vascular dysfunction, injury, and hypertension that is dependent, in part, on the renin angiotensin system (RAS). We have shown that, in human microvascular endothelial cells, 20-HETE increases angiotensin-converting enzyme (ACE) mRNA, protein, and ACE activity via an epidermal growth factor receptor (EGFR)/tyrosine kinase/mitogen-activated protein kinase (MAPK)/inhibitor of κB kinase (IKK)β-mediated signaling pathway. In this work, we show that, similar to epidermal growth factor (EGF), 20-HETE (10 nM) activates EGFR by stimulating tyrosine phosphorylation; however, unlike 20-HETE, EGF does not induce ACE expression, and pretreatment with a neutralizing antibody against EGF does not prevent the 20-HETE-mediated ACE induction. Inhibition of nuclear factor κB (NF-κB) activation prevented the 4.58-fold (±0.78; P < 0.05) 20-HETE-mediated induction of ACE. The 20-HETE increased NF-κB-binding activity in nuclear extracts and the activity of both the somatic and germinal ACE promoters by 4.37-fold (±0.18; P < 0.05) and 2.53-fold (± 0.24; P < 0.05), respectively. The 20-HETE-stimulated ACE promoter activity was abrogated by the 20-HETE antagonist 20-hydroxy-6,15-eicosadienoic acid and by inhibitors of EGFR, MAPK, IKKβ, and NF-κB activation. Sequence analysis demonstrated the presence of two and one putative NF-κB binding sites on the human somatic and germinal ACE promoters, respectively. Chromatin immunoprecipitation assay indicated that 20-HETE stimulates the translocation and subsequent binding of NF-κB to each of the putative binding sites (S1, 3.43 ± 0.3-fold enrichment versus vehicle; S2, 3.72 ± 0.68-fold enrichment versus vehicle; S3, 3.20 ± 0.18-fold enrichment versus vehicle; P < 0.05). This is the first study to identify NF-κB as a transcriptional factor for ACE and to implicate a distinct EGFR/MAPK/IKK/NF-κB signaling cascade underlying 20-HETE-mediated transcriptional activation of ACE mRNA and stimulation of ACE activity.

Ethanol alters GABAA receptor trafficking and function through activation of protein kinases, and these changes may underlie ethanol dependence and withdrawal. In this study, we used subsynaptic fraction techniques and patch-clamp electrophysiology to investigate the biochemical and functional effects of protein kinase A (PKA) and protein kinase C (PKC) activation by ethanol on synaptic GABAA α4 receptors, a key target of ethanol-induced changes. Rat cerebral cortical neurons were grown for 18 days in vitro and exposed to ethanol and/or kinase modulators for 4 hours, a paradigm that recapitulates GABAergic changes found after chronic ethanol exposure in vivo. PKA activation by forskolin or rolipram during ethanol exposure prevented increases in P2 fraction α4 subunit abundance, whereas inhibiting PKA had no effect. Similarly, in the synaptic fraction, activation of PKA by rolipram in the presence of ethanol prevented the increase in synaptic α4 subunit abundance, whereas inhibiting PKA in the presence of ethanol was ineffective. Conversely, PKC inhibition in the presence of ethanol prevented the ethanol-induced increases in synaptic α4 subunit abundance. Finally, we found that either activating PKA or inhibiting PKC in the presence of ethanol prevented the ethanol-induced decrease in GABA miniature inhibitory postsynaptic current decay τ1, whereas inhibiting PKA had no effect. We conclude that PKA and PKC have opposing effects in the regulation of synaptic α4 receptors, with PKA activation negatively modulating, and PKC activation positively modulating, synaptic α4 subunit abundance and function. These results suggest potential targets for restoring normal GABAergic functioning in the treatment of alcohol use disorders.