Chemogenetic Tools for Causal Cellular and Neuronal Biology.

Chemogenetic technologies enable selective pharmacological control of specific cell populations. An increasing number of approaches have been developed that modulate different signaling pathways. Selective pharmacological control over G protein-coupled receptor signaling, ion channel conductances, protein association, protein stability, and small molecule targeting allows modulation of cellular processes in distinct cell types. Here, we review these chemogenetic technologies and instances of their applications in complex tissues in vivo and ex vivo.

Anti-Inflammatory Effect of Synaptamide in Ischemic Acute Kidney Injury and the Role of G-Protein-Coupled Receptor 110.

The development of drugs for the treatment of acute kidney injury (AKI) that could suppress the excessive inflammatory response in damaged kidneys is an important clinical challenge. Recently, synaptamide (N-docosahexaenoylethanolamine) has been shown to exert anti-inflammatory and neurogenic properties. The aim of this study was to investigate the anti-inflammatory effect of synaptamide in ischemic AKI. For this purpose, we analyzed the expression of inflammatory mediators and the infiltration of different leukocyte populations into the kidney after injury, evaluated the expression of the putative synaptamide receptor G-protein-coupled receptor 110 (GPR110), and isolated a population of CD11b/c+ cells mainly representing neutrophils and macrophages using cell sorting. We also evaluated the severity of AKI during synaptamide therapy and the serum metabolic profile. We demonstrated that synaptamide reduced the level of pro-inflammatory interleukins and the expression of integrin CD11a in kidney tissue after injury. We found that the administration of synaptamide increased the expression of its receptor GPR110 in both total kidney tissue and renal CD11b/c+ cells that was associated with the reduced production of pro-inflammatory interleukins in these cells. Thus, we demonstrated that synaptamide therapy mitigates the inflammatory response in kidney tissue during ischemic AKI, which can be achieved through GPR110 signaling in neutrophils and a reduction in these cells' pro-inflammatory interleukin production.

Effects of acute and chronic administration of trace amine-associated receptor 1 (TAAR1) ligands on in vivo excitability of central monoamine-secreting neurons in rats.

Trace amine-associated receptor 1 (TAAR1) has been recently identified as a target for the future antidepressant, antipsychotic, and anti-addiction drugs. Full (e.g. RO5256390) and partial (e.g. RO5263397) TAAR1 agonists showed antidepressant-, antipsychotic- and anti-addiction-like behavioral effects in rodents and primates. Acute RO5256390 suppressed, and RO5263397 stimulated serotonin (5-HT) neurons of the dorsal raphe nucleus (DRN) and dopamine neurons of the ventral tegmental area (VTA) in brain slices, suggesting that the behavioral effects of TAAR1 ligands involve 5-HT and dopamine. For more comprehensive testing of this hypothesis, we examined acute and chronic effects of RO5256390 and RO5263397 on monoamine neurons in in vivo conditions. Excitability of 5-HT neurons of the DRN, noradrenaline neurons of the locus coeruleus (LC), and dopamine neurons of the VTA was assessed using single-unit electrophysiology in anesthetized rats. For acute experiments, RO5256390 and RO5263397 were administered intravenously; neuronal excitability after RO5256390 and RO5263397 administration was compared to the basal activity of the same neuron. For chronic experiments, RO5256390 was administered orally for fourteen days prior to electrophysiological assessments. The neuronal excitability in RO5256390-treated rats was compared to vehicle-treated controls. We found that acute RO5256390 inhibited 5-HT and dopamine neurons. This effect of RO5256390 was reversed by the subsequent and prevented by the earlier administration of RO5263397. Acute RO5256390 and RO5263397 did not alter the excitability of LC noradrenaline neurons in a statistically significant way. Chronic RO5256390 increased excitability of 5-HT neurons of the DRN and dopamine neurons of the VTA. In conclusion, the putative antidepressant and antipsychotic effects of TAAR1 ligands might be mediated, at least in part, via the modulation of excitability of central 5-HT and dopamine neurons.

Impact of Classic Adrenal Secretagogues on mRNA Levels of Urotensin II and Its Receptor in Adrenal Gland of Rats.

Urotensin 2 (Uts2) is a biologically active peptide involved in the regulation of a variety of physiological and pathophysiological processes. In both the human and rat adrenal gland, the expressions of the Uts2 gene and its receptor (Uts2r) have been described. This paper focuses on the description of the hormonal control of the mRNA levels of urotensin II and its receptor in the adrenal gland of the rat, both in vitro and in vivo. The initial in vitro experiments were carried out on freshly isolated rat adrenocortical cells and their primary culture. The obtained results indicated a stimulating PKA-independent effect of ACTH on the Uts2 mRNA level in the tested cells, with no changes in the Uts2r transcript. Subsequent in vivo experiments showed that ACTH-induced adrenal growth was accompanied by an elevated level of the Uts2 mRNA, with unchanged expression of Uts2r. In the other types of in vivo gland growth studied, enucleation-induced adrenal regeneration and compensatory growth of the gland, the mRNA levels of the studied genes showed no significant differences. The only exception was hemiadrenalectomy, which led to a significant increase in Uts2 mRNA expression level 24 h after surgery. In 12-week-old rats of both sexes, gonadectomy led to a significant increase in the level of Uts2 mRNA in the adrenal gland, an effect that was prevented by sex hormones' replacement. No changes in Uts2r transcript levels were observed under these conditions. Thus, this study suggests that the regulation of Uts2 and Uts2r mRNA levels differs significantly in the rat adrenal gland. While Uts2 transcript levels appear to be mainly dependent on ACTH action, Uts2r mRNA levels are not under the control of this hormone.

Molecular dynamics-guided discovery of an ago-allosteric modulator for GPR40/FFAR1.

The long-chain fatty acid receptor FFAR1/GPR40 binds agonists in both an interhelical site between the extracellular segments of transmembrane helix (TM)-III and TM-IV and a lipid-exposed groove between the intracellular segments of these helices. Molecular dynamics simulations of FFAR1 with agonist removed demonstrated a major rearrangement of the polar and charged anchor point residues for the carboxylic acid moiety of the agonist in the interhelical site, which was associated with closure of a neighboring, solvent-exposed pocket between the extracellular poles of TM-I, TM-II, and TM-VII. A synthetic compound designed to bind in this pocket, and thereby prevent its closure, was identified through structure-based virtual screening and shown to function both as an agonist and as an allosteric modulator of receptor activation. This discovery of an allosteric agonist for a previously unexploited, dynamic pocket in FFAR1 demonstrates both the power of including molecular dynamics in the drug discovery process and that this specific, clinically proven, but difficult, antidiabetes target can be addressed by chemotypes different from existing ligands.

Ligand-Independent G Protein-Coupled Estrogen Receptor/G Protein-Coupled Receptor 30 Activity: Lack of Receptor-Dependent Effects of G-1 and 17ß-Estradiol.

G protein-coupled receptor 30 (GPR30) is a membrane receptor reported to bind 17ß-estradiol (E2) and mediate rapid nongenomic estrogen responses, hence also named G protein-coupled estrogen receptor. G-1 is a proposed GPR30-specific agonist that has been used to implicate the receptor in several pathophysiological events. However, controversy surrounds the role of GPR30 in G-1 and E2 responses. We investigated GPR30 activity in the absence and presence of G-1 and E2 in several eukaryotic systems ex vivo and in vitro in the absence and presence of the receptor. Ex vivo activity was addressed using the caudal artery from wild-type (WT) and GPR30 knockout (KO) mice, and in vitro activity was addressed using a HeLa cell line stably expressing a synthetic multifunctional promoter (nuclear factor κB, signal transducer and activator of transcription, activator protein 1)-luciferase construct (HFF11 cells) and a human GPR30-inducible T-REx system (T-REx HFF11 cells), HFF11 and human embryonic kidney 293 cells transiently expressing WT GPR30 and GPR30 lacking the C-terminal PDZ (postsynaptic density-95/discs-large /zonula occludens-1 homology) motif SSAV, and yeast Saccharomyces cerevisiae transformed to express GPR30. WT and KO arteries exhibited similar contractile responses to 60 mM KCl and 0.3 µM cirazoline, and G-1 relaxed both arteries with the same potency and efficacy. Furthermore, expression of GPR30 did not introduce any responses to 1 µM G-1 and 0.1 µM E2 in vitro. On the other hand, receptor expression caused considerable ligand-independent activity in vitro, which was receptor PDZ motif-dependent in mammalian cells. We conclude from these results that GPR30 exhibits ligand-independent activity in vitro but no G-1- or E2-stimulated activity in any of the systems used. SIGNIFICANCE STATEMENT: Much controversy surrounds 17ß-estradiol (E2) and G-1 as G protein-coupled receptor 30 (GPR30) agonists. We used several recombinant eukaryotic systems ex vivo and in vitro with and without GPR30 expression to address the role of this receptor in responses to these proposed agonists. Our results show that GPR30 exhibits considerable ligand-independent activity in vitro but no G-1- or E2-stimulated activity in any of the systems used. Thus, classifying GPR30 as an estrogen receptor and G-1 as a specific GPR30 agonist is unfounded.

Thiazolidinediones (TZDs) enhance insulin secretory response via GPR40 and adenylate cyclase (AC).

Thiazolidinediones are synthetic PPARγ ligands that enhance insulin sensitivity, and that could increase insulin secretion from ß-cells. However, the functional role and mechanism(s) of action in pancreatic ß-cells have not been investigated in detail.

Perinatal activation of ERα and ERß but not GPER-1 masculinizes female rat caudate-putamen medium spiny neuron electrophysiological properties.

Exposure to steroid sex hormones such as 17ß-estradiol (estradiol) during early life potentially permanently masculinize neuron electrophysiological phenotype. In rodents, one crucial component of this developmental process occurs in males, with estradiol aromatized in the brain from testes-sourced testosterone. However, it is unknown whether most neuron electrophysiological phenotypes are altered by this early masculinization process, including medium spiny neurons (MSNs) of the rat caudate-putamen. MSNs are the predominant and primary output neurons of the caudate-putamen and exhibit increased intrinsic excitability in females compared to males. Here, we hypothesize that since perinatal estradiol exposure occurs in males, then a comparable exposure in females to estradiol or its receptor agonists would be sufficient to induce masculinization. To test this hypothesis, we injected perinatal female rats with estradiol or its receptor agonists and then later assessed MSN electrophysiology. Female and male rats on postnatal day 0 and 1 were systemically injected with either vehicle, estradiol, the estrogen receptor (ER)α agonist PPT, the ERß agonist DPN, or the G-protein-coupled receptor 1 (GPER-1) agonist G1. On postnatal days 19 ± 2, MSN electrophysiological properties were assessed using whole cell patch clamp recordings. Estradiol exposure abolished increased intrinsic excitability in female compared to male MSNs. Exposure to either an ERα or ERß agonist masculinized female MSN evoked action potential firing rate properties, whereas exposure to an ERß agonist masculinized female MSN inward rectification properties. Exposure to ER agonists minimally impacted male MSN electrophysiological properties. These findings indicate that perinatal estradiol exposure masculinizes MSN electrophysiological phenotype via activation of ERα and ERß.NEW & NOTEWORTHY This study is the first to demonstrate that estradiol and estrogen receptor α and ß stimulation during early development sexually differentiates the electrophysiological properties of caudate-putamen medium spiny neurons, the primary output neuron of the striatal regions. Overall, this evidence provides new insight into the neuroendocrine mechanism by which caudate-putamen neuron electrophysiology is sexually differentiated and demonstrates the powerful action of early hormone exposure upon individual neuron electrophysiology.

Phosphatidylinositol-3 kinase mediates the sweet suppressive effect of leptin in mouse taste cells.

Leptin is known to selectively suppress neural and taste cell responses to sweet compounds. The sweet suppressive effect of leptin is mediated by the leptin receptor Ob-Rb, and the ATP-gated K+ (KATP ) channel expressed in some sweet-sensitive, taste receptor family 1 member 3 (T1R3)-positive taste cells. However, the intracellular transduction pathway connecting Ob-Rb to KATP channel remains unknown. Here we report that phosphoinositide 3-kinase (PI3K) mediates leptin's suppression of sweet responses in T1R3-positive taste cells. In in situ taste cell recording, systemically administrated leptin suppressed taste cell responses to sucrose in T1R3-positive taste cells. Such leptin's suppression of sucrose responses was impaired by co-administration of PI3K inhibitors (wortmannin or LY294002). In contrast, co-administration of signal transducer and activator of transcription 3 inhibitor (Stattic) or Src homology region 2 domain-containing phosphatase-2 inhibitor (SHP099) had no effect on leptin's suppression of sucrose responses, although signal transducer and activator of transcription 3 and Src homology region 2 domain-containing phosphatase-2 were expressed in T1R3-positive taste cells. In peeled tongue epithelium, phosphatidylinositol (3,4,5)-trisphosphate production and phosphorylation of AKT by leptin were immunohistochemically detected in some T1R3-positive taste cells but not in glutamate decarboxylase 67-positive taste cells. Leptin-induced phosphatidylinositol (3,4,5)-trisphosphate production was suppressed by LY294002. Thus, leptin suppresses sweet responses of T1R3-positive taste cells by activation of Ob-Rb-PI3K-KATP channel pathway.

Downregulation of TGR5 (GPBAR1) in biliary epithelial cells contributes to the pathogenesis of sclerosing cholangitis.

BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) is characterized by chronic inflammation and progressive fibrosis of the biliary tree. The bile acid receptor TGR5 (GPBAR1) is found on biliary epithelial cells (BECs), where it promotes secretion, proliferation and tight junction integrity. Thus, we speculated that changes in TGR5-expression in BECs may contribute to PSC pathogenesis. METHODS: TGR5-expression and -localization were analyzed in PSC livers and liver tissue, isolated bile ducts and BECs from Abcb4-/-, Abcb4-/-/Tgr5Tg and ursodeoxycholic acid (UDCA)- or 24-norursodeoxycholic acid (norUDCA)-fed Abcb4-/- mice. The effects of IL8/IL8 homologues on TGR5 mRNA and protein levels were studied. BEC gene expression was analyzed by single-cell transcriptomics (scRNA-seq) from distinct mouse models. RESULTS: TGR5 mRNA expression and immunofluorescence staining intensity were reduced in BECs of PSC and Abcb4-/- livers, in Abcb4-/- extrahepatic bile ducts, but not in intrahepatic macrophages. No changes in TGR5 BEC fluorescence intensity were detected in liver tissue of other liver diseases, including primary biliary cholangitis. Incubation of BECs with IL8/IL8 homologues, but not with other cytokines, reduced TGR5 mRNA and protein levels. BECs from Abcb4-/- mice had lower levels of phosphorylated Erk and higher expression levels of Icam1, Vcam1 and Tgfß2. Overexpression of Tgr5 abolished the activated inflammatory phenotype characteristic of Abcb4-/- BECs. NorUDCA-feeding restored TGR5-expression levels in BECs in Abcb4-/- livers. CONCLUSIONS: Reduced TGR5 levels in BECs from patients with PSC and Abcb4-/- mice promote development of a reactive BEC phenotype, aggravate biliary injury and thus contribute to the pathogenesis of sclerosing cholangitis. Restoration of biliary TGR5-expression levels represents a previously unknown mechanism of action of norUDCA. LAY SUMMARY: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease-associated with progressive inflammation of the bile duct, leading to fibrosis and end-stage liver disease. Bile acid (BA) toxicity may contribute to the development and disease progression of PSC. TGR5 is a membrane-bound receptor for BAs, which is found on bile ducts and protects bile ducts from BA toxicity. In this study, we show that TGR5 levels were reduced in bile ducts from PSC livers and in bile ducts from a genetic mouse model of PSC. Our investigations indicate that lower levels of TGR5 in bile ducts may contribute to PSC development and progression. Furthermore, treatment with norUDCA, a drug currently being tested in a phase III trial for PSC, restored TGR5 levels in biliary epithelial cells.

Profiling novel pharmacology of receptor complexes using Receptor-HIT.

Many receptors are able to undergo heteromerisation, leading to the formation of receptor complexes that may have pharmacological profiles distinct from those of the individual receptors. As a consequence of this, receptor heteromers can be classed as new drug targets, with the potential for achieving greater specificity and selectivity over targeting their constituent receptors. We have developed the Receptor-Heteromer Investigation Technology (Receptor-HIT), which enables the detection of receptor heteromers using a proximity-based reporter system such as bioluminescence resonance energy transfer (BRET). Receptor-HIT detects heteromers in live cells and in real time, by utilising ligand-induced signals that arise from altered interactions with specific biomolecules, such as ligands or proteins. Furthermore, monitoring the interaction between the receptors and the specific biomolecules generates functional information about the heteromer that can be pharmacologically quantified. This review will discuss various applications of Receptor-HIT, including its use with different classes of receptors (e.g. G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and others), its use to monitor receptor interactions both intracellularly and extracellularly, and also its use with genome-edited endogenous proteins.

MRGPRX2 activation in mast cells by neuromuscular blocking agents and other agonists: Modulation by sugammadex.

BACKGROUND: Neuromuscular-blocking agents (NMBAs) can cause both IgE-dependent and IgE-independent anaphylactic reactions, with activation of the mast cell receptor MRGPRX2 being important to the latter. Sugammadex, a reversal agent for certain aminosteroid NMBAs, has been proposed as an antidote for these anaphylactic events with conflicting outcomes. OBJECTIVE: We further characterize the involvement of MRGPRX2 in NMBA-induced mast cell activation and determine how this is influenced by sugammadex. We then apply these in vitro results to infer the possible utility of sugammadex in the acute management of non-IgE-dependent anaphylaxis. METHODS: The LAD2 human mast cell line and a MRGPRX2 knock-down derivative were used to validate the involvement of MRGPRX2 and to test the effect of sugammadex on mast cell activation by NMBAs and other MRGPRX2 agonists. RESULTS: All MRGPRX2 agonists tested were shown to induce MRGPRX2-dependent LAD2 mast cell calcium mobilization and cytokine release and all, apart from rocuronium, induced degranulation. Co-treatment of mast cells with sugammadex and some MRGPRX2 agonists significantly reduced cell activation, but if sugammadex was administered a few minutes following stimulation, degranulation was not attenuated. However, addition of sugammadex up to 180 min following LAD2 MRGPRX2 stimulation, significantly reduced CCL2 mRNA and protein induction. CONCLUSIONS AND CLINICAL RELEVANCE: We show that sugammadex, known to reverse muscle blockade by certain NMBAs, is also able to reduce MRGPRX2 activation by NMBAs and other, but not all, MRGPRX2 agonists. As sugammadex was ineffective in attenuating mast cell degranulation when added rapidly post MRGPRX2 activation, this suggests against the agent having efficacy in controlling acute symptoms of anaphylaxis to NMBAs caused by MRGPRX2 activation. Interestingly, however, sugammadex did impair MRGPRX2-induced CCL2 release, suggesting that it may have some benefit in perhaps dampening less well-defined adverse effects of MRGPRX2-dependent anaphylaxis associated with the more slowly elaborated mast cell mediators.

The G protein-coupled estrogen receptor (GPER) regulates recognition and aversively-motivated memory in male rats.

Estrogens, particularly 17ß-estradiol (estradiol, E2), regulate memory formation. E2 acts through its intracellular receptors, estrogen receptors (ER) ERα and ERß, as well as a recently identified G protein-coupled estrogen receptor (GPER). Although the effects of E2 on memory have been investigated, studies examining the effects of GPER stimulation are scarce. Selective GPER agonism improves memory in ovariectomized female rats, but little information is available regarding the effects of GPER stimulation in male rodents. The aim of the present study was to investigate the effects of the GPER agonist, G1, on consolidation and reconsolidation of inhibitory avoidance (IA) and object recognition (OR) memory in male rats. Animals received vehicle, G1 (15, 75, 150 µg/kg; i.p.), or the GPER antagonist G15 (100 µg/kg; i.p.) immediately after training, or G1 (150 µg/kg; i.p.) 3 or 6 h after training. To investigate reconsolidation, G1 was administered immediately after IA retention Test 1. Results indicated that G1 administered immediately after training at the highest dose enhanced both OR and IA memory consolidation, while GPER blockade immediately after training impaired OR. No effects of GPER stimulation were observed when G1 was given 3 or 6 h after training or after Test 1. The present findings provide evidence that GPER is involved in the early stages of memory consolidation in both neutral and emotional memory tasks in male adult rats.

Ultra low doses and biological amplification: Approaching Avogadro's number.

This paper describes evidence establishing that ultra-low doses of diverse chemical agents at concentrations from 10-18 to 10-24 M (e.g., approaching and/or less than 1 atom or molecule of a substance/cell based on Avogadro's constant - 6.022×1023/mole) are capable of engaging receptor and intracellular signaling systems to elicit reproducible effects in a variety of species, from unicellular organisms to humans. Multiple experimental studies have shown that only one or very few molecules are needed to activate a cell and/or entire organism via cascade(s) of amplification mechanisms and processes. For example, ultra-low dose ligand exposure was able to activate both an individual cell, and ~3000 to 25,000 neighboring cells on average, by about 50%. Such activation of cells and whole organisms typically displayed hormetic-biphasic dose responses. These findings indicate that numerous, diverse phylogenetic systems have evolved highly sensitive detection and signaling mechanisms to enhance survival functions, such as defense against infectious agents, responses to diverse types of pheromone communications (e.g., alarm, sexual attraction), and development of several types of cellular protection/resilience processes. This suggests that ultra-low dose effects may be far more common than have been recognized to date. We posit that such findings have important implications for evolutionary theory, ecological and systems biology, and clinical medicine.

The role of compartmentalized signaling pathways in the control of mitochondrial activities in cancer cells.

Mitochondria are the powerhouse organelles present in all eukaryotic cells. They play a fundamental role in cell respiration, survival and metabolism. Stimulation of G-protein coupled receptors (GPCRs) by dedicated ligands and consequent activation of the cAMP·PKA pathway finely couple energy production and metabolism to cell growth and survival. Compartmentalization of PKA signaling at mitochondria by A-Kinase Anchor Proteins (AKAPs) ensures efficient transduction of signals generated at the cell membrane to the organelles, controlling important aspects of mitochondrial biology. Emerging evidence implicates mitochondria as essential bioenergetic elements of cancer cells that promote and support tumor growth and metastasis. In this context, mitochondria provide the building blocks for cellular organelles, cytoskeleton and membranes, and supply all the metabolic needs for the expansion and dissemination of actively replicating cancer cells. Functional interference with mitochondrial activity deeply impacts on cancer cell survival and proliferation. Therefore, mitochondria represent valuable targets of novel therapeutic approaches for the treatment of cancer patients. Understanding the biology of mitochondria, uncovering the molecular mechanisms regulating mitochondrial activity andmapping the relevant metabolic and signaling networks operating in cancer cells will undoubtly contribute to create a molecular platform to be used for the treatment of proliferative disorders. Here, we will highlight the emerging roles of signaling pathways acting downstream to GPCRs and their intersection with the ubiquitin proteasome system in the control of mitochondrial activity in different aspects of cancer cell biology.

Ion channels and G protein-coupled receptors as targets for invertebrate pest control: from past challenges to practical insecticides.

In the late 1970s, we discovered that toxic bicyclic phosphates inhibit the generation of miniature inhibitory junction potentials, implying their antagonism of γ-aminobutyric acid (GABA) receptors (GABARs; GABA-gated chloride channels). This unique mode of action provided a strong incentive for our research on GABARs in later years. Furthermore, minor structural changes conferred insect GABAR selectivity to this class of compounds, convincing us of the possibility of GABARs as targets for insecticides. Forty years later, third-generation insecticides acting as allosteric modulator antagonists at a distinctive site of action in insect GABARs were developed. G protein-coupled receptors (GPCRs) are also promising targets for pest control. We characterized phenolamine receptors functionally and pharmacologically. Of the tested receptors, ß-adrenergic-like octopamine receptors were revealed to be the most sensitive to the acaricide/insecticide amitraz. Given the presence of multiple sites of action, ion channels and GPCRs remain potential targets for invertebrate pest control.

"Metalloestrogenic" effects of cadmium downstream of G protein-coupled estrogen receptor and mitogen-activated protein kinase pathways in human uterine fibroid cells.

Cadmium (Cd) is a toxic metal reported to act as an estrogen "mimic" in the rat uterus and in vitro. We have reported that Cd stimulates proliferation of estrogen-responsive human uterine leiomyoma (ht-UtLM; fibroid) cells through nongenomic signaling involving the G protein-coupled estrogen receptor (GPER), with activation of epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase (pMAPK44/42). In this study, we explored Cd-induced mechanisms downstream of MAPK and whether Cd could stimulate phosphorylation of Histone H3 at serine 10 (H3Ser10ph) through activated Aurora B kinase (pAurora B), a kinase important in activation of histone H3 at serine 10 during mitosis, and if this occurs via Fork head box M1 (FOXM1) and cyclin D1 immediately downstream of MAPK. We found that Cd increased proliferating cell nuclear antigen (PCNA) and H3Ser10ph expression by immunofluorescence, and that H3ser10ph and pAurora B were coexpressed along the metaphase plate in ht-UtLM cells. In addition, Cd-exposed cells showed higher expression of pMAPK44/42, FOXM1, pAurora B, H3ser10ph, and Cyclin D1 by western blotting. Immunoprecipitation and proximity ligation assays further indicated an association between FOXM1 and Cyclin D1 in Cd-exposed cells. These effects were attenuated by MAPK kinase (MEK1/2) inhibitor. In summary, Cd-induced proliferation of ht-UtLM cells occurred through activation of Histone H3 and Aurora B via FOXM1/Cyclin D1 interactions downstream of MAPK. This provides a molecular mechanism of how Cd acts as an "estrogen mimic" resulting in mitosis in hormonally responsive cells.

Effect of Sodium Valproate on the Conformational Stability of the Visual G Protein-Coupled Receptor Rhodopsin.

Rhodopsin is the G protein-coupled receptor of rod photoreceptor cells that mediates vertebrate vision at low light intensities. Mutations in rhodopsin cause inherited retinal degenerative diseases such as retinitis pigmentosa. Several therapeutic strategies have attempted to address and counteract the deleterious effect of rhodopsin mutations on the conformation and function of this photoreceptor protein, but none has been successful in efficiently preventing retinal degeneration in humans. These approaches include, among others, the use of small molecules, known as pharmacological chaperones, that bind to the receptor stabilizing its proper folded conformation. Valproic acid, in its sodium valproate form, has been used as an anticonvulsant in epileptic patients and in the treatment of several psychiatric disorders. More recently, this compound has been tested as a potential therapeutic agent for the treatment of retinal degeneration associated with retinitis pigmentosa caused by rhodopsin mutations. We now report on the effect of sodium valproate on the conformational stability of heterologously expressed wild-type rhodopsin and a rhodopsin mutant, I307N, which has been shown to be an appropriate model for studying retinal degeneration in mice. We found no sign of enhanced stability for the dark inactive conformation of the I307N mutant. Furthermore, the photoactivated conformation of the mutant appears to be destabilized by sodium valproate as indicated by a faster decay of its active conformation. Therefore, our results support a destabilizing effect of sodium valproate on rhodopsin I307N mutant associated with retinal degeneration. These findings, at the molecular level, agree with recent clinical studies reporting negative effects of sodium valproate on the visual function of retinitis pigmentosa patients.

Emerging Molecular Receptors for the Specific-Target Delivery of Ruthenium and Gold Complexes into Cancer Cells.

Cisplatin and derivatives are highly effective in the treatment of a wide range of cancer types; however, these metallodrugs display low selectivity, leading to severe side effects. Additionally, their administration often results in the development of chemoresistance, which ultimately results in therapeutic failure. This scenario triggered the study of other transition metals with innovative pharmacological profiles as alternatives to platinum, ruthenium- (e.g., KP1339 and NAMI-A) and gold-based (e.g., Auranofin) complexes being among the most advanced in terms of clinical evaluation. Concerning the importance of improving the in vivo selectivity of metal complexes and the current relevance of ruthenium and gold metals, this review article aims to survey the main research efforts made in the past few years toward the design and biological evaluation of target-specific ruthenium and gold complexes. Herein, we give an overview of the inorganic and organometallic molecules conjugated to different biomolecules for targeting membrane proteins, namely cell adhesion molecules, G-protein coupled receptors, and growth factor receptors. Complexes that recognize the progesterone receptors or other targets involved in metabolic pathways such as glucose transporters are discussed as well. Finally, we describe some complexes aimed at recognizing cell organelles or compartments, mitochondria being the most explored. The few complexes addressing targeted gene therapy are also presented and discussed.

GPCR Pharmacological Profiling of Aaptamine from the Philippine Sponge Stylissa sp. Extends Its Therapeutic Potential for Noncommunicable Diseases.

We report the first isolation of the alkaloid aaptamine from the Philippine marine sponge Stylissa sp. Aaptamine possessed weak antiproliferative activity against HCT116 colon cancer cells and inhibited the proteasome in vitro at 50 µM. These activities may be functionally linked. Due to its known, more potent activity on certain G-protein coupled receptors (GPCRs), including α-adrenergic and δ-opioid receptors, the compound was profiled more broadly at sub-growth inhibitory concentrations against a panel of 168 GPCRs to potentially reveal additional targets and therapeutic opportunities. GPCRs represent the largest class of drug targets. The primary screen at 20 µM using the ß-arrestin functional assay identified the antagonist, agonist, and potentiators of agonist activity of aaptamine. Dose-response analysis validated the α-adrenoreceptor antagonist activity of aaptamine (ADRA2C, IC50 11.9 µM) and revealed the even more potent antagonism of the ß-adrenoreceptor (ADRB2, IC50 0.20 µM) and dopamine receptor D4 (DRD4, IC50 6.9 µM). Additionally, aaptamine showed agonist activity on selected chemokine receptors, by itself (CXCR7, EC50 6.2 µM; CCR1, EC50 11.8 µM) or as a potentiator of agonist activity (CXCR3, EC50 31.8 µM; CCR3, EC50 16.2 µM). These GPCRs play a critical role in the treatment of cardiovascular disease, diabetes, cancer, and neurological disorders. The results of this study may thus provide novel preventive and therapeutic strategies for noncommunicable diseases (NCDs).