Putative Agonist Research Articles

Insight into binding of endogenous neurosteroid ligands to the sigma-1 receptor

The sigma-1 receptor (σ1R) is a non-opioid membrane receptor, which responds to a diverse array of synthetic ligands to exert various pharmacological effects. Meanwhile, candidates for endogenous ligands of σ1R have also been identified. However, how endogenous ligands bind to σ1R remains unknown. Here, we present crystal structures of σ1R from Xenopus laevis (xlσ1R) bound to two endogenous neurosteroid ligands, progesterone (a putative antagonist) and dehydroepiandrosterone sulfate (DHEAS) (a putative agonist), at 2.15-3.09 Å resolutions. Both neurosteroids bind to a similar location in xlσ1R mainly through hydrophobic interactions, but surprisingly, with opposite binding orientations. DHEAS also forms hydrogen bonds with xlσ1R, whereas progesterone interacts indirectly with the receptor through water molecules near the binding site. Binding analyses are consistent with the xlσ1R-neurosteroid complex structures. Furthermore, molecular dynamics simulations and structural data reveal a potential water entry pathway. Our results provide insight into binding of two endogenous neurosteroid ligands to σ1R.

Confirmation of high-throughput screening data and novel mechanistic insights into FXR-xenobiotic interactions by orthogonal assays

Toxicology in the 21st Century (Tox21) is a federal collaboration employing a high-throughput robotic screening system to test 10,000 environmental chemicals. One of the primary goals of the program is prioritizing toxicity evaluations through in vitro high-throughput screening (HTS) assays for large numbers of chemicals already in commercial use for which little or no toxicity data is available. Within the Tox21 screening program, disruption in nuclear receptor (NR) signaling represents a particular area of interest. Given the role of NR’s in modulating a wide range of biological processes, alterations of their activity can have profound biological impacts. Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily that has demonstrated importance in bile acid homeostasis, glucose metabolism, lipid homeostasis and hepatic regeneration. In this study, we re-evaluated 24 FXR agonists and antagonists identified through Tox21 using select orthogonal assays. In transient transactivation assays, 7/8 putative agonists and 4/4 putative inactive compounds were confirmed. Likewise, we confirmed 9/12 antagonists tested. Using a mammalian two hybrid approach we demonstrate that both FXR agonists and antagonists facilitate FXRα-coregulator interactions suggesting that differential coregulator recruitment may mediate activation/repression of FXRα mediated transcription. Additionally, we tested the ability of select FXR agonists and antagonists to facilitate hepatic transcription of FXR gene targets Shp and Bsep in a teleost (Medaka) model. Through application of in vitro cell-based assays, in silico modeling and in vivo gene expressions, we demonstrated the molecular complexity of FXR:ligand interactions and confirmed the ability of diverse ligands to modulate FXRα, facilitate differential coregulator recruitment and activate/repress receptor-mediated transcription. Overall, we suggest a multiplicative approach to assessment of nuclear receptor function may facilitate a greater understanding of the biological and mechanistic complexities of nuclear receptor activities and further our ability to interpret broad HTS outcomes.

Biological activity of interferon gamma and lipopolysaccharide on the nitric oxide production in C6 astroglioma cells and some unexpected effects of potentization

Background: A proinflammatory environment is a hallmark of several neurodegenerative diseases where astrocyte involvement is also well established. Astrocytes and microglia in central nervous system are mainly involved in the release of cytokines, oxygen free radicals and nitric oxide (NO). Several studies on C6 astroglioma cells, a widely used in vitro model for these events, demonstrated that co-stimulation of this cell line with bacterial lipopolysaccharide (LPS) and interferon gamma (IFN-) induces a synergistic nitric oxide synthase (iNOS) expression.1 In our laboratory we are using this versatile cell model in order to carefully investigate dose-response effects of various putative agonists or inhibitors and to assess the possible changes provoked in those agents by different procedures of dilution and succussion (agitation) (potentization according to the homeopathic terminology).

Bitter Taste Receptors in the Airway Cells Functions.

G protein-coupled receptors (GPCRs) play a central role in regulating the functions of a diverse range of cell types in the airway. Taste 2 receptor (T2R) family of GPCRs is responsible for the transduction of bitter taste; however, recent studies have demonstrated that different subtypes of T2Rs and key components of T2R signaling are expressed in several extra-oral tissues including airways with many physiological roles. In the lung, expression of T2Rs has been confirmed in multiple airway cell types including airway smooth muscle (ASM) cells, various epithelial cell subtypes, and on both resident and migratory immune cells. Most importantly, activation of T2Rs with a variety of putative agonists elicits unique signaling in ASM and specialized airway epithelial cells resulting in the inhibition of ASM contraction and proliferation, promotion of ciliary motility, and innate immune response in chemosensory airway epithelial cells. Here we discuss the expression of T2Rs and the mechanistic basis of their function in the structural cells of the airways with some useful insights on immune cells in the context of allergic asthma and other upper airway inflammatory disorders. Emphasis on T2R biology and pharmacology in airway cells has an ulterior goal of exploiting T2Rs for therapeutic benefit in obstructive airway diseases.

4β-Hydroxycholesterol is a prolipogenic factor that promotes SREBP1c expression and activity through the liver X receptor

Oxysterols are oxidized derivatives of cholesterol that play regulatory roles in lipid biosynthesis and homeostasis. How oxysterol signaling coordinates different lipid classes such as sterols and triglycerides remains incompletely understood. Here, we show that 4β-hydroxycholesterol (HC) (4β-HC), a liver and serum abundant oxysterol of poorly defined functions, is a potent and selective inducer of the master lipogenic transcription factor, SREBP1c, but not the related steroidogenic transcription factor SREBP2. By correlating tracing of lipid synthesis with lipogenic gene expression profiling, we found that 4β-HC acts as a putative agonist for the liver X receptor (LXR), a sterol sensor and transcriptional regulator previously linked to SREBP1c activation. Unique among the oxysterol agonists of the LXR, 4β-HC induced expression of the lipogenic program downstream of SREBP1c and triggered de novo lipogenesis both in primary hepatocytes and in the mouse liver. In addition, 4β-HC acted in parallel to insulin-PI3K–dependent signaling to stimulate triglyceride synthesis and lipid-droplet accumulation. Thus, 4β-HC is an endogenous regulator of de novo lipogenesis through the LXR-SREBP1c axis.

PhyteByte: identification of foods containing compounds with specific pharmacological properties

BackgroundPhytochemicals and other molecules in foods elicit positive health benefits, often by poorly established or unknown mechanisms. While there is a wealth of data on the biological and biophysical properties of drugs and therapeutic compounds, there is a notable lack of similar data for compounds commonly present in food. Computational methods for high-throughput identification of food compounds with specific biological effects, especially when accompanied by relevant food composition data, could enable more effective and more personalized dietary planning. We have created a machine learning-based tool (PhyteByte) to leverage existing pharmacological data to predict bioactivity across a comprehensive molecular database of foods and food compounds.ResultsPhyteByte uses a cheminformatic approach to structure-based activity prediction and applies it to uncover the putative bioactivity of food compounds. The tool takes an input protein target and develops a random forest classifier to predict the effect of an input molecule based on its molecular fingerprint, using structure and activity data available from the ChEMBL database. It then predicts the relevant bioactivity of a library of food compounds with known molecular structures from the FooDB database. The output is a list of food compounds with high confidence of eliciting relevant biological effects, along with their source foods and associated quantities in those foods, where available. Applying PhyteByte to the human PPARG gene, we identified irigenin, sesamin, fargesin, and delta-sanshool as putative agonists of PPARG, along with previously identified agonists of this important metabolic regulator.ConclusionsPhyteByte identifies food-based compounds that are predicted to interact with specific protein targets. The identified relationships can be used to prioritize food compounds for experimental or epidemiological follow-up and can contribute to the rapid development of precision approaches to new nutraceuticals as well as personalized dietary planning.

Stringent Small Molecule Dose Requirements for the Optimal Expansion of Hematopoietic Stem Cells Revealed By Predictive Analytics and Xenotransplants

Introduction: Loss of self-renewal of hematopoietic stem cells (HSC) is a major roadblock to cell engineering therapies. Small molecules have been identified that promote HSC expansion. We recently identified UM171, StemReginin1 (SR1) and valproic acid (VPA) as strongest agonist for expansion of cord blood (CB) CD34+CD45RA- and CD34+CD45RA-EpcrHigh (EpcrHg) HSC-enriched cells of 12 molecules tested. In addition, we identified a novel putative stem cell agonist in L-Ascorbic acid 2-phosphate (AA2P), a derivative of vitamin C. Using response surface methodology and machine learning, we identified a series of Stem Cell Agonist Cocktails (SCAC) composed of these 4 agonists at varying concentrations. The objectives of this study were to characterize the in vitro properties of AA2P and SCACs on CB HSC and, test the capacity of AA2P- and SCAC-expanded CB CD34+ cells to support hematopoietic recovery and long-term (LT) engraftment after transplantation. Methods: Predictive models for HSC expansion (CD34+CD45RA- and EpcrHg) promoted by SR1, UM171, VPA, and AA2P were built by design of experiments. The data was then used to train a neural network. These were used as predictive tools to derive a series of SCAC composed of different concentrations of the 4 agonists (Table 1). CB expanded HSPC were characterized after 14 days of culture. Migration of HSPCs toward SDF-1 was tested in a transwell assay. Serial and limit dilution transplant assays in NSG mice were done to characterize the capacity of SCAC to support expansions of short-term (ST) and LT HSC. Results: First, we investigated the capacity of AA2P to act as an HSC agonist. AA2P was unable on its own to expand EpcrHg cells but promoted cell growth and the expansion of CD34+CD45RA- HSPC (2-fold, p<0.05), a property shared by L-Ascorbic acid. Moreover, AA2P-expanded HSPCs enhanced ST platelet engraftment when compared to serum-free medium (SFM) control (p=0.053, n=3). Next, we tested the activity of SCACs presented in Table 1. Varying the concentrations of the small molecules profoundly impacted cell growth and the type of HSPC expanded (Table 1). For instance, SM-2 with high UM171 provided high expansion of EpcrHg, but low level of overall cell growth. SM-A and SM-6 maximized expansion of CD34+CD45RA- cells but had lower expansion of EpcrHg due partly to lower UM171. X2A was unique as it produced balanced expansion of EpcrHg and CD34+CD45RA- cells. Lowering AA2P concentration in X2A significantly reduced the expansion of both HSC-enriched fractions (X2B, Table 1). Moreover, most SCACs enhanced expansion of HSC-enriched cells (CD34+CD45RA-CD38-CD90+CD133+, p<0.05 vs SFM) and that of lymphoid-primed multi-potential progenitors and multipotent progenitors vs. SFM cultures (p<0.05), but not of downstream progenitors. Since homing to the bone marrow (BM) is a key step towards engraftment, we investigated whether SCACs influenced the expression of homing receptors and the migration activity of HSPCs. SCAC expanded HSPCs were characterized with elevated fucosylation of PSGL-1 known to favor homing and engraftment (e.g. 82 ± 2% vs. 42 ± 8% for X2A vs. SFM CD34+ cells, p<0.01, n=4). Also, most SCAC-expanded CD34+ cells showed improved migration towards SDF-1 (e.g. 22 ± 6% vs. 13 ± 9% for X2A vs. SFM CD34+ cells, p<0.05, n=4). The capacity of SCAC-expanded HSPCs to support engraftment is still ongoing. Current results showed that X2A-expanded HSPCs provided the strongest ST recovery of platelets and leucocytes of all SCACs-HSPC, superior also to that seen with UM171-expanded HSPCs and non-cultured HSPCs (p<0.05, n=2-3). Further, LT human BM reconstitution was notably better for X2A- and SM-6-expanded HSPCs than other SCAC-expanded cells (p<0.05 vs SM2, n=2). Moreover, reducing AA2P in X2A resulted in a loss in ST and LT engraftment activity (p<0.05, n=2). Secondary transplants and limit dilution assays are ongoing to provide further insights into the impact of SCACs on the production and self-renewal activity of HSCs. Conclusion: Our study reveals that AA2P promotes cell growth and can synergize with strong stem cell agonists to promote the expansion of ST and LT engrafting HSPCs. The engraftment properties of SCAC-expanded HSPCs was highly dependent on the concentrations of the small molecules due in part to negative interactions amongst some of the agonists. Gene expression studies are ongoing to define the transcriptional landscape of HSPC produced with these SCACs. Disclosures No relevant conflicts of interest to declare.

Abstract 1490: Novel microbiome-derived peptides activate the host innate immune system by regulation of TLR signaling

Abstract The gut microbiome is a key contributor to the maintenance of host physiology. At the same time, increasing evidence implicates microbiome dysbiosis as a key determinant of numerous metabolic and inflammatory disorders. It is, therefore, of paramount importance to understand the interactions between the host and gut microbiota. Second Genome has developed a proprietary, bioinformatics-driven discovery platform to identify novel microbiome-derived peptides with the potential to modulate host immune responses. We have leveraged this platform to evaluate immunomodulatory functions of novel, secreted peptides derived from gut-resident bacterial species Bifidobacterium longum and Bifidobacterium breve. Previously, the microbial genus Bifidobacterium (‘Bifido’) has been shown to be associated with enhanced dendritic cell functions and antitumor effects. Here, we identify Bifido-derived peptides that are putative agonists of certain murine and human Toll-like receptors (TLRs). These peptides induce secretion of pro-inflammatory cytokines such as TNF-α and IL-6 by in vitro-generated mouse dendritic cells and peritoneal macrophages in a TLR4 and CD14-dependent manner. One of the peptides, SG-A, also induces secretion of Th1-type cytokines and chemokines such as IL-21, GRO-α, and IP-10 by human monocytes and monocyte-derived dendritic cells. Moreover, in vivo administration of SG-A in mice elicited a systemic pro-inflammatory immune response in the absence of any secondary stimulus. Collectively, our results demonstrate the ability of a novel bacterial-derived peptide to engage the host innate immune system. Moreover, our findings validate the potential of the Second Genome discovery platform for identification of novel microbiome-derived agents that: i) have the ability to modulate host cell functions; and ii) may be developed into therapeutic agents for immune and inflammatory disorders. Citation Format: Dhwani Haria, Helena Kiefel, Yuliya Katlinskaya, Sunit Jain, Thomas Weinmaier, Shoko Iwai, Todd DeSantis, Toshi Takeuchi, Karim Dabbagh, Kareem Graham. Novel microbiome-derived peptides activate the host innate immune system by regulation of TLR signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1490.

2,4-Thiazolidinedione in Well-Fed Lactating Dairy Goats: II. Response to Intra-Mammary Infection.

In a prior experiment, treatment of goats with the putative PPARγ agonist 2,4-thiazolidinedione (2,4-TZD) ameliorated the response to intramammary infection without evidence of PPARγ activation. The lack of PPARγ activation was possibly due to deficiency of vitamin A and/or a poor body condition of the animals. Therefore, the present study hypothesized that activation of PPARγ by 2,4-TZD in goats supplemented with adequate amounts of vitamin A can improve the response to sub-clinical mastitis. Lactating goats receiving a diet that met National Research Council requirements, including vitamin A, were injected with 8 mg/kg BW of 2,4-TZD (n = 6) or saline (n = 6; control (CTR)) daily. Two weeks into treatment, all goats received Streptococcus uberis (IMI) in the right mammary gland. Blood biomarkers of metabolism, inflammation, and oxidative status plus leukocytes phagocytosis were measured. Mammary epithelial cells (MEC) and macrophages were isolated from milk and liver tissue collected for gene expression analysis. Milk fat was maintained by treatment with 2,4-TZD, but decreased in CTR, after IMI. Haptoglobin was increased after IMI only in 2,4-TZD without any effect on negative acute phase proteins, indicating an improved liver function. 2,4-TZD vs. CTR had a greater amount of globulin. The expression of inflammation-related genes was increased by IMI in both macrophages and MEC. Except for decreasing expression of SCD1 in MEC, 2,4-TZD did not affect the expression of measured genes. Results confirmed the successful induction of sub-clinical mastitis but did not confirm the positive effect of 2,4-TZD on the response to IMI in well-fed goats.

2,4-Thiazolidinedione in Well-Fed Lactating Dairy Goats: I. Effect on Adiposity and Milk Fat Synthesis.

Background: In a prior experiment, treatment of goats with the putative PPARγ agonist 2,4-thiazolidinedione (2,4-TZD) did not affect milk fat or expression of milk-fat related genes. The lack of response was possibly due to deficiency of vitamin A and/or a poor body condition of the animals. In the present experiment, we tested the hypothesis that PPARγ activation affects milk fat synthesis in goats with a good body condition and receiving adequate levels of vitamin A. Methods: Lactating goats receiving a diet that met NRC requirements, including vitamin A, were injected with 8 mg/kg BW of 2,4-TZD (n = 6) or saline (n = 6; CTR) daily for 26 days. Blood metabolic profiling and milk yield and components were measured including fatty acid profile. Expression of genes related to glucose and lipid metabolism was measured in adipose tissue and in mammary epithelial cells (MEC). Size of adipocytes was assessed by histological analysis. Results: NEFA, BHBA, and fatty acids available in plasma decreased while glucose increased in 2,4-TZD vs. CTR. Size of cells and expression of insulin signaling and glucose metabolism-related genes were larger in 2,4-TZD vs. CTR in adipose tissue. In MEC, expression of SCD1 and desaturation of stearate was lower in 2,4-TZD vs. CTR. Conclusions: Overall data revealed a lack of PPARγ activation by 2,4-TZD and no effect on milk fat synthesis despite a strong anti-lipolysis effect on adipose tissue.

Pervasive Positive and Negative Feedback Regulation of Insulin-Like Signaling in Caenorhabditis elegans.

The Caenorhabditis elegans insulin-like signaling network supports homeostasis and developmental plasticity. The genome encodes 40 insulin-like peptides and one known receptor. Feedback regulation has been reported, but the extent of feedback and its effect on signaling dynamics in response to changes in nutrient availability has not been determined. We measured messenger RNA expression for each insulin-like peptide, the receptor daf-2, components of the PI3K pathway, and its transcriptional effectors daf-16/FoxO and skn-1/Nrf at high temporal resolution during transition from a starved, quiescent state to a fed, growing state in wild type and mutants affecting daf-2/InsR and daf-16/FoxO. We also analyzed the effect of temperature on insulin-like gene expression. We found that most PI3K pathway components and insulin-like peptides are affected by signaling activity, revealing pervasive positive and negative feedback regulation at intra- and intercellular levels. Reporter gene analysis demonstrated that the daf-2/InsR agonist daf-28 positively regulates its own transcription and that the putative agonist ins-6 cross-regulates DAF-28 protein expression through feedback. Our results show that positive and negative feedback regulation of insulin-like signaling is widespread, giving rise to an organismal FoxO-to-FoxO signaling network that supports homeostasis during fluctuations in nutrient availability.

GABAA Modulation of S100B Secretion in Acute Hippocampal Slices and Astrocyte Cultures.

Astrocytes are the major glial cells in brain tissue and are involved, among many functions, ionic and metabolic homeostasis maintenance of synapses. These cells express receptors and transporters for neurotransmitters, including GABA. GABA signaling is reportedly able to affect astroglial response to injury, as evaluated by specific astrocyte markers such as glial fibrillary acid protein and the calcium-binding protein, S100B. Herein, we investigated the modulatory effects of the GABAA receptor on astrocyte S100B secretion in acute hippocampal slices and astrocyte cultures, using the agonist, muscimol, and the antagonists pentylenetetrazol (PTZ) and bicuculline. These effects were analyzed in the presence of tetrodotoxin (TTX), fluorocitrate (FLC), cobalt and barium. PTZ positively modify S100B secretion in hippocampal slices and astrocyte cultures; in contrast, bicuculline inhibited S100B secretion only in hippocampal slices. Muscimol, per se, did not change S100B secretion, but prevented the effects of PTZ and bicuculline. Moreover, PTZ-induced S100B secretion was prevented by TTX, FLC, cobalt and barium indicating a complex GABAA communication between astrocytes and neurons. The effects of two putative agonists of GABAA, β-hydroxybutyrate and methylglyoxal, on S100B secretion were also evaluated. In view of the neurotrophic role of extracellular S100B under conditions of injury, our data reinforce the idea that GABAA receptors act directly on astrocytes, and indirectly on neurons, to modulate astroglial response.

Study of μ- and δ-Opioid Activities in Agents with Various κ-Receptor Selectivity.

A putative opioid agonist RU-1205 was ineffective within in vitro model of electrically induced contractions of rat ileum assessing the μ- and δ-opioid receptor pathways, while morphine inhibited these contractions in a dose-dependent and naloxone-reversible manners with EC50=2.6×10-7 M. In vivo experiments revealed no significant effects of RU-1205 on respiration and gastrointestinal tract contractile activity. In contrast, butorphanol decreased respiration rate by 25% (25-100 mg/kg) and slowed down the transit of labeled particles along the small intestine by 77.1% (1 mg/kg) and by 45.5% (10 mg/kg). Morphine-induced inhibition of peristalsis was dose-dependent with maximum effect (by 68.6%) observed in the dose of 10 mg/kg. It was concluded that the effects of RU-1205 are not related to activation μ- and δ-opioid receptors known to mediate the effects of non-selective opioid agonist morphine and agonist-antagonist butorphanol.

Kynurenine pathway metabolites in migraine.

Kynurenine pathway (KP), the quantitatively main branch of tryptophan metabolism, has long been considered a source of nicotinamide adenine dinucleotide, although several of its products, the so-called kynurenines, are endowed with the capacity to activate glutamate receptors, thus potentially influencing a large group of functions in the central nervous system (CNS). In fact, Kynurenic Acid and Quinolinic Acid are able to interact with ionotropic glutamate receptors and Cinnabarinic Acid has been reported as an orthosteric agonist of metabotropic glutamate receptors (mGlu4), and Xanthurenic Acid has been recently demonstrated to be a putative agonist of metabotropic glutamate receptors 2/3 (mGlu2/3). Moreover, 3-HK and 3-HANA have mainly been studied, since they have been shown to induce neurotoxic effects by increasing oxidative stress and the production of free radicals or through excitotoxicity. Migraine has a complex pathophysiology in which both central and peripheral components of the trigeminal pain pathway play a central role. The trigemino-vascular activation during the attack has largely been described, and recently the brainstem nuclei, called “migraine generators”, have been reported to be involved in migraine. Moreover, a series of destabilizing events within the brain trigger a cortical spreading depression (CSD), responsible for the aura phenomena and for trigeminal activation. The role of glutamate is heavily supported both in the trigemino-vascular as well as in brainstem nuclei activation, and furthermore in the CSD initiation and propagation. Some of the KP metabolites able to interact both with ionotropic and metabotropic glutamate receptors might be involved in migraine pathophysiology. Despite the large number of studies conducted on migraine etiopathology, the KP has only been recently linked to this disease. Nonetheless, some evidence suggests an intriguing role for some kynurenines, and an exploratory study on the serum kynurenine levels has been helpful to better understand possible alterations of the kynurenine pathway in patients suffering from migraine.

Optoactivation of parvalbumin neurons in the spinal dorsal horn evokes GABA release that is regulated by presynaptic GABAB receptors

Among heterogeneous neural cells in the spinal dorsal horn, parvalbumin (PV)-positive neurons are one subtype of GABA (γ-aminobutyric acid)-containing interneurons. Using an optogenetic approach, we expressed blue light-sensitive cation channel channelrhodopsin-2 (ChR2) via a viral vector on PV neurons in the spinal dorsal horn. Combined with in vitro whole-cell recordings, we activated ChR2 expressed on PV neurons by blue light and recorded GABAA receptor-mediated light-evoked inhibitory postsynaptic currents (L-IPSCs). The L-IPSCs were action potential-dependent and abolished by the GABAA receptor antagonist picrotoxin, indicating a synchronic GABA release from presynaptic terminals. Activation of GABAB receptors (the metabotropic receptors of GABA) on presynaptic terminals by a putative agonist, baclofen, depressed the amplitude of L-IPSCs. This depression was largely occluded by pretreatment with the highly selective Cav2.1 (P/Q-type) Ca2+ channel blocker ω-agatoxin IVA. N-type Ca2+ channel blocker ω-conotoxin GVIA showed less effects on either L-IPSCs or baclofen depression. We conclude that optoactivation of PV-ChR2 neurons in the spinal dorsal horn induces GABA release from presynaptic terminals, which is modulated by presynaptic GABAB receptors that are coupled to P/Q-type Ca2+ channels. Importantly, our studies provide a simple and reliable optogenetic approach to study dorsal horn neural circuits.

Novel agonists for serotonin 5-HT7 receptors reverse metabotropic glutamate receptor-mediated long-term depression in the hippocampus of wild-type and Fmr1 KO mice, a model of Fragile X Syndrome.

Serotonin 5-HT7 receptors are expressed in the hippocampus and modulate the excitability of hippocampal neurons. We have previously shown that 5-HT7 receptors modulate glutamate-mediated hippocampal synaptic transmission and long-term synaptic plasticity. In particular, we have shown that activation of 5-HT7 receptors reversed metabotropic glutamate receptor-mediated long-term depression (mGluR-LTD) in wild-type (wt) and in Fmr1 KO mice, a mouse model of Fragile X Syndrome in which mGluR-LTD is abnormally enhanced, suggesting that 5-HT7 receptor agonists might be envisaged as a novel therapeutic strategy for Fragile X Syndrome. In this perspective, we have characterized the basic in vitro pharmacokinetic properties of novel molecules with high binding affinity and selectivity for 5-HT7 receptors and we have tested their effects on synaptic plasticity using patch clamp on acute hippocampal slices. Here we show that LP-211, a high affinity selective agonist of 5-HT7 receptors, reverses mGluR-LTD in wt and Fmr1 KO mice, correcting a synaptic malfunction in the mouse model of Fragile X Syndrome. Among novel putative agonists of 5-HT7 receptors, the compound BA-10 displayed improved affinity and selectivity for 5-HT7 receptors and improved in vitro pharmacokinetic properties with respect to LP-211. BA-10 significantly reversed mGluR-LTD in the CA3-CA1 synapse in wt and Fmr1KO mice, indicating that BA-10 behaved as a highly effective agonist of 5-HT7 receptors and reduced exaggerated mGluR-LTD in a mouse model of Fragile X Syndrome. On the other side, the compounds RA-7 and PM-20, respectively arising from in vivo metabolism of LP-211 and BA-10, had no effect on mGluR-LTD thus did not behave as agonists of 5-HT7 receptors in our conditions. The present results provide information about the structure-activity relationship of novel 5-HT7 receptor agonists and indicate that LP-211 and BA-10 might be used as novel pharmacological tools for the therapy of Fragile X Syndrome.

Cannabinoid and lipid-mediated vasorelaxation in retinal microvasculature

The endocannabinoid system plays a role in regulation of vasoactivity in the peripheral vasculature; however, little is known about its role in regulation of the CNS microvasculature. This study investigated the pharmacology of cannabinoids and cannabimimetic lipids in the retinal microvasculature, a CNS vascular bed that is autoregulated. Vessel diameter (edge detector) and calcium transients (fura-2) were recorded from segments of retinal microvasculature isolated from adult, male Fischer 344 rats. Results showed that abnormal cannabidiol (Abn-CBD), an agonist at the putative endothelial cannabinoid receptor, CBe, inhibited endothelin 1 (ET-1) induced vasoconstriction in retinal arterioles. These actions of Abn-CBD were independent of CB1/CB2 receptors and were not mediated by agonists for GPR55 or affected by nitric oxide synthase (NOS) inhibition. However, the vasorelaxant effects of Abn-CBD were abolished when the endothelium was removed and were inhibited by the small Ca2+-sensitive K channel (SKCa) blocker, apamin. The effects of the endogenous endocannabinoid metabolite, N-arachidonyl glycine (NAGly), a putative agonist for GPR18, were virtually identical to those of Abn-CBD. GPR18 mRNA and protein were present in the retina, and immunohistochemistry demonstrated that GPR18 was localized to the endothelium of retinal vessels. These findings demonstrate that Abn-CBD and NAGly inhibit ET-1 induced vasoconstriction in retinal arterioles by an endothelium-dependent signaling mechanism that involves SKCa channels. The endothelial localization of GPR18 suggests that GPR18 could contribute to cannabinoid and lipid-mediated retinal vasoactivity.

11C-CUMI-101, a PET Radioligand, Behaves as a Serotonin 1A Receptor Antagonist and Also Binds to α1 Adrenoceptors in Brain

The PET radioligand (11)C-CUMI-101 was previously suggested as a putative agonist radioligand for the serotonin 1A (5-hydroxytryptamine 1A [5-HT1A]) receptor in recombinant cells expressing human 5-HT1A receptor. However, a recent study showed that CUMI-101 behaved as a potent 5-HT1A receptor antagonist in rat brain. CUMI-101 also has moderate affinity (Ki = 6.75 nM) for α1 adrenoceptors measured in vitro. The current study examined the functional properties and selectivity of CUMI-101, both in vitro and in vivo. The functional assay was performed using (35)S-GTPγS (GTP is guanosine triphosphate) in primate brains. The cross-reactivity of CUMI-101 with α1 adrenoceptors was performed using in vitro radioligand binding studies in rat, monkey, and human brains as well as in vivo PET imaging in mouse, rat, and monkey brains. CUMI-101 did not stimulate (35)S-GTPγS binding in primate brain, in contrast to 8-OH-DPAT, a potent 5-HT1A receptor agonist. Instead, CUMI-101 behaved as a potent 5-HT1A receptor antagonist by dose-dependently inhibiting 8-OH-DPAT-stimulated (35)S-GTPγS binding. Both in vitro and in vivo studies showed that CUMI-101 had significant α1 adrenoceptor cross-reactivity. On average, across all 3 species examined, cross-reactivity was highest in the thalamus (>45%) and lowest in the neocortex and cerebellum (<10%). PET imaging further confirmed that only preblocking with WAY-100635 plus prazosin decreased (11)C-CUMI-101 brain uptake to that of self-block. CUMI-101 behaves as a 5-HT1A receptor antagonist in primate brain, with significant, regional-dependent α1 adrenoceptor cross-reactivity, limiting its potential use as a PET radioligand in humans.

UM171 Is a Novel and Potent Agonist Of Human Hematopoietic Stem Cell Renewal

▪Various combinations of soluble factors have been reported to promote the ex vivo proliferation of human HSCs but few of them are capable of promoting self-renewal. Characterization of StemRegenin (SR1), an aryl hydrocarbon receptor (AhR) antagonist that promotes expansion of CD34+ cells ex vivo, provided a proof of principle that low molecular weight (LMW) compounds have the ability to modulate cellular pathways possibly leading to HSC expansion. In an attempt to identify novel putative agonists of HSC self-renewal, we tested a library comprising > 5,000 LMW molecules in a phenotypical screen based on in vitro expansion of CD34+CD45RA- cells. Eight hits were identified, the majority of which were AhR antagonists. Validation studies identified one hit synthesized in our institute which potently expanded human CD34+CD45RA- cord blood cells ex vivo. Importantly this molecule does not antagonize AhR. Structure activity relationship (SAR) generated a compound called UM171 which is 14-25 folds more potent than the starting molecule with an EC50 of 8-15 nM on CD34+ human cord blood (CB). Using the optimal dose of this analog, CD34+, CD34+CD45RA-, and CD34+CD38-CD90+CD45RA-CD49f+ were expanded 115±15, 62±3 and 121±6 fold respectively over the input values after 12 day culture in fed-batch system. The expansion of these phenotypic HSC populations was enhanced, in an additive manner by including SR1. Washout of UM171 led to a rapid loss of the CD34+CD45RA- population suggesting that its effect is reversible. UM171 is not a mitogen and cannot compensate for the absence of cytokines added to the media (Flt3L, SCF, and TPO). Our cell division studies revealed no effect of the compound on division rate compared to control indicating that UM171 cultured cells retain the CD34+CD45RA- phenotype. Apoptosis and cell phonotype analysis showed significantly less cell differentiation and cell death for UM171 cultured cells compared to controls or SR1 supplemented cultures suggesting that the compound acts by anti-differentiation/apoptosis. Similar to SR1, UM171 treated cells showed a 75±20 fold net increase in the numbers of multilineage colony forming cell (CFU-GEMM) and 150±20 fold when both compounds were present in the 12 day culture.To further understand the impact of UM171 on more primitive cell compartments, we transplanted 100, 500, 1000 and 10,000 uncultured CB CD34+ cells and their progeny after a 12-day culture (with or without UM171) in NSG mice and verified the human hematopoietic reconstitution in their bone marrow 20 weeks later. Mice transplanted with UM171 expanded cells showed a much higher level of human CD45 engraftment compared to uncultured or control expanded cells. The NSG Repopulating Cells (NRC) showed a net expansion ex vivo only in the presence of UM171 with values ranging from 3.1 to 5.5 fold increase over input value in 6 independent experiments. Interestingly, the co-treatment with SR1 did not significantly enhance the impact of UM171 on NRC expansion. In these fed-batch conditions SR1 treatment maintained the NRC population at input values. Importantly, we observed a linear relationship between the number of UM171-treated cells transplanted in vivo and level of human CD45 engraftment. Moreover, at limit dilution, these cells were capable of human myeloid and lymphoid differentiation in NSG BM environment suggesting that UM171 expanded cells remain multipotent. Together, our studies have identified a novel compound which truly expands human progenitors while amplifying the more primitive stem cell population. UM171 has successfully passed toxicity studies and is in the final phase of GMP production for a phase I study which will be launched shortly. In addition, preliminary evidence suggests that UM171 strikingly enhances gene transfer efficiency to primitive human CD34 CB cells. Disclosures:No relevant conflicts of interest to declare.

DPV-001 an autophagosome-enriched cancer vaccine in phase II clinical trials contains 25 putative cancer antigens, DAMPS, HSPS and agonists for TLR 2, 3, 4, 7 and 9

Meeting abstracts Generation of a therapeutic immune response against a diverse set of antigens expressed by a patient's cancer is a central goal of cancer immunotherapy. This goal is important because diverse responses may prevent escape of antigen loss variants. To accomplish this goal we have