Signaling Potency Research Articles

NRG1-Fc improves metabolic health via dual hepatic and central action.

Neuregulins (NRGs) are emerging as an important family of signaling ligands that regulate glucose and lipid homeostasis. NRG1 lowers blood glucose levels in obese mice, whereas the brown fat-enriched secreted factor NRG4 protects mice from high-fat diet-induced insulin resistance and hepatic steatosis. However, the therapeutic potential of NRGs remains elusive, given the poor plasma half-life of the native ligands. Here, we engineered a fusion protein using human NRG1 and the Fc domain of human IgG1 (NRG1-Fc) that exhibited extended half-life in circulation and improved potency in receptor signaling. We evaluated its efficacy in improving metabolic parameters and dissected the mechanisms of action. NRG1-Fc treatment triggered potent AKT activation in the liver, lowered blood glucose, improved insulin sensitivity, and suppressed food intake in obese mice. NRG1-Fc acted as a potent secretagogue for the metabolic hormone FGF21; however, the latter was largely dispensable for its metabolic effects. NRG1-Fc directly targeted the hypothalamic POMC neurons to promote membrane depolarization and increase firing rate. Together, NRG1-Fc exhibits improved pharmacokinetic properties and exerts metabolic benefits through dual inhibition of hepatic gluconeogenesis and caloric intake.

O6C-20-nor-salvinorin A is a stable and potent KOR agonist

Salvinorin A (SalA) is a potent and selective agonist of the kappa-opioid receptor (KOR), but its instability has frustrated medicinal chemistry efforts. Treatment of SalA with weak bases like DBU leads to C8 epimerization with loss of receptor affinity and signaling potency. Here we show that replacement of C20 with H and replacement of O6 with CH2 stabilizes the SalA scaffold relative to its C8 epimer, so much so that epimerization is completely supressed. This new compound, O6C-20-nor-SalA, retains high potency for agonism of KOR.

Mechanical Stretching of Fibronectin Fibers Upregulates Binding of Interleukin-7.

Since evidence is rising that extracellular matrix (ECM) fibers might serve as reservoirs for growth factors and cytokines, we investigated the interaction between fibronectin (FN) and interleukin-7 (IL-7), a cytokine of immunological significance and a target of several immunotherapies. By employing a FN fiber stretch assay and Förster resonance energy transfer (FRET) confocal microscopy, we found that stretching of FN fibers increased IL-7 binding. We localized the FN binding site on the CD loop of IL-7, since a synthetic CD loop peptide also bound stronger to stretched than to relaxed FN fibers. On the basis of a structural model, we propose that the CD loop can bind to FN, while IL-7 is bound to its cognate cell surface receptors. Sequence alignment with bacterial adhesins, which also bind the FN N-terminus, suggests that a conserved motif on the CD loop (110TKSLEEN116 and the truncated 112SLEE115 in human and mouse IL-7, respectively) might bind to the second FN type I module (FnI2) and that additional epitopes enhance the stretch-upregulated binding. FN fiber stretching might thus serve as a mechano-regulated mechanism to locally concentrate IL-7 in an ECM-bound state, thereby upregulating the potency of IL-7 signaling. A feedback model mechanism is proposed that could explain the well-known, but poorly understood, function of IL-7 in ECM homeostasis. Understanding how local IL-7 availability and signaling might be modulated by the tensional state of the ECM niche, which is adjusted by residing stroma cells, is highly relevant for basic science but also for advancing IL-7 based immunotherapies.

1,2,3-Triazole Rings as a Disulfide Bond Mimetic in Chimeric AGRP-Melanocortin Peptides: Design, Synthesis, and Functional Characterization.

The melanocortin system is involved in the regulation of complex physiological functions, including energy and weight homeostasis, feeding behavior, inflammation, sexual function, pigmentation, and exocrine gland function. The five melanocortin receptors that belong to the superfamily of G protein-coupled receptors (GPCRs) are regulated by endogenously expressed agonists and antagonists. The aim of this study was to explore the potential of replacing the disulfide bridge in chimeric AGRP-melanocortin peptide Tyr-c[Cys-His-d-Phe-Arg-Trp-Asn-Ala-Phe-Cys]-Tyr-NH2 (1) with 1,2,3-triazole moieties. A series of 1,2,3-triazole-bridged peptidomimetics were designed, synthesized, and pharmacologically evaluated at the mouse melanocortin receptors. The ligands possessed nanomolar to micromolar agonist cAMP signaling potency. A key finding was that the disulfide bond in peptide 1 can be replaced with the monotriazole ring with minimal effect on the functional activity at the melanocortin receptors. The 1,5-disubstituted triazole-bridged peptide 6 showed equipotent functional activity at the mMC3R and modest 5-fold decreased agonist potency at the mMC4R compared to those of 1. Interestingly, the 1,4- and 1,5-disubstituted isomers of the triazole ring resulted in different selectivities at the receptor subtypes, indicating subtle structural features that may be exploited in the generation of selective melanocortin ligands. Introducing cyclic and acyclic bis-triazole moieties into chimeric AGRP template 1 generally decreased agonist activity. These results will be useful for the further design of neuronal chemical probes for the melanocortin receptors as well as in other receptor systems.

IL-6-HaloTag® enables live-cell plasma membrane staining, flow cytometry, functional expression, and de-orphaning of recombinant odorant receptors.

The assignment of cognate odorant/agonist pairs is a prerequisite for an understanding of odorant coding at the receptor level. However, the identification of new ligands for odorant receptors (ORs) in cell-based assays has been challenging, due to their individual and rather sub-optimal plasma membrane expression, as compared with other G protein-coupled receptors. Accessory proteins, such as the chaperone RTP1S, or Ric8b, have improved the surface expression of at least a portion of ORs. Typically, recombinant ORs carry N-terminal tags, which proved helpful for their functional membrane expression. The most common tag is the ‘Rho-tag’, representing an N-terminal part of rhodopsin, but also ‘Lucy-’ or ‘Flag-tag’ extensions have been described. Here, we used a bi-functional N-terminal tag, called ‘interleukin 6 (IL-6)-HaloTag®’, with IL-6 facilitating functional cell surface expression of recombinant ORs, and the HaloTag® protein, serving as a highly specific acceptor for cell-impermeant or cell-permeant, fluorophore-coupled ligands, which enable the quantification of odorant receptor expression by live-cell flow cytometry. Our experiments revealed on average an about four-fold increased surface expression, a four-fold higher signaling amplitude, and a significantly higher potency of odorant-induced cAMP signaling of six different human IL-6-HaloTag®-ORs across five different receptor families in NxG 108CC15 cells, as compared to their Rho-tag–HaloTag® constructs. We observed similar results in HEK-293 cells. Moreover, screening an IL-6–HaloTag®-odorant receptor library with allyl phenyl acetate, revealed both known receptors as best responders for this compound. In summary, the IL-6–HaloTag® represents a promising tool for the de-orphaning of ORs.

CRH Receptor Signalling: Potential Roles in Pathophysiology.

To maintain homeostatic equilibrium, living organisms have evolved complex adaptation systems that control an array of behavioural, autonomic, neuroendocrine and immune responses. One of the important switches of this system is the hypothalamic hormone corticotropin-releasing hormone (CRH), which together with a family of related peptides (urocortins, UCNs) orchestrate stress-coping responses that reinstate homeostasis. Persistent disturbances in the homeostatic equilibrium either due to inadequate or persistently uncontrolled responses have been associated with pathogenic mechanisms of disease. CRH and UCNs exert their actions by activating two receptors of the Class B1 GPCRs, CRH-R1 and CRH-R2. Their signalling versatility allows activation of multiple and diverse signalling pathways characterized by 'cell-specific agonist-dependent signalling' responses. Alternative mRNA splicing, interactions with intracellular protein partners and mechanisms that allow selective regulation of signalling potency and termination, provide additional levels of regulation to fine-tune cellular responses. Although understanding of CRH-R signalling is still incomplete, recent important advances in decoding CRH-R structure and signalling properties uncovered key important functions and roles in physiology and pathobiology.

Priming GPCR Signaling Through the Synergistic Effect of Two G Proteins

While individual GPCRs are known to activate one or more G proteins, the GPCR‐G protein interaction is viewed as a bi‐molecular event involving the formation of a ternary ligand‐GPCR‐G protein complex. We have designed a FRET‐based sensor to monitor and modulate GPCR – G protein interactions in live cells. Using this systematic protein affinity strength modulation (SPASM) approach we present evidence that individual GPCR‐G protein interactions can reinforce each other to enhance signaling through canonical downstream second messengers, a phenomenon we term GPCR priming. Specifically, we find that the presence of non‐cognate Gq protein enhances cAMP stimulated by two Gs coupled receptors, β2‐adrenergic receptor (β2‐AR) and D1 dopamine receptor (D1‐R). The C‐terminus of either the Gαs or Gαq subunit is sufficient to enhance Gα subunit activation and cAMP levels. Interaction of Gαs or Gαq C‐termini with the GPCR increases signaling potency suggesting an altered GPCR conformation as the underlying basis for GPCR priming. We propose three parallel mechanisms involving (1) sequential G protein interactions at the orthosteric site, (2) simultaneous allosteric and orthosteric G protein interactions, and (3) asymmetric GPCR dimers. GPCR priming suggests another layer of regulation in the classic GPCR ternary complex model, with broad implications for the multiplicity inherent in signaling networks.Support or Funding InformationResearch was funded by the American Heart Association Scientist Development Grant (13SDG14270009) & the NIH (1DP2 CA186752‐01 & 1‐R01‐GM‐105646‐01‐A1) to S.S. T.M.G is an NCBS‐inStem Career Development Fellow. R.F.S is a Life Sciences Research Foundation postdoctoral fellow.

Biased Signaling by Agonists of Protease Activated Receptor 2.

Protease activated receptor 2 (PAR2) is associated with metabolism, obesity, inflammatory, respiratory and gastrointestinal disorders, pain, cancer, and other diseases. The extracellular N-terminus of PAR2 is a common target for multiple proteases, which cleave it at different sites to generate different N-termini that activate different PAR2-mediated intracellular signaling pathways. There are no synthetic PAR2 ligands that reproduce the same signaling profiles and potencies as proteases. Structure-activity relationships here for 26 compounds spanned a signaling bias over 3 log units, culminating in three small ligands as biased agonist tools for interrogating PAR2 functions. DF253 (2f-LAAAAI-NH2) triggered PAR2-mediated calcium release (EC50 2 μM) but not ERK1/2 phosphorylation (EC50 > 100 μM) in CHO cells transfected with hPAR2. AY77 (Isox-Cha-Chg-NH2) was a more potent calcium-biased agonist (EC50 40 nM, Ca2+; EC50 2 μM, ERK1/2), while its analogue AY254 (Isox-Cha-Chg-A-R-NH2) was an ERK-biased agonist (EC50 2 nM, ERK1/2; EC50 80 nM, Ca2+). Signaling bias led to different functional responses in human colorectal carcinoma cells (HT29). AY254, but not AY77 or DF253, attenuated cytokine-induced caspase 3/8 activation, promoted scratch-wound healing, and induced IL-8 secretion, all via PAR2-ERK1/2 signaling. Different ligand components were responsible for different PAR2 signaling and functions, clues that can potentially lead to drugs that modulate different pathway-selective cellular and physiological responses.

Kynurenic acid and its analogue can alter the opioid receptor G-protein signaling after acute treatment via NMDA receptor in rat cortex and striatum

Previously, we have shown that the N-methyl d-aspartate (NMDA)-receptor antagonist kynurenic acid (KYNA) and its analogue KYNA1 do not bind directly to mu, kappa and delta opioid receptors in vitro. On the other hand, chronic administration of KYNA and KYNA1 resulted in region (cortex vs striatum) and opioid receptor-type specific alterations in G-protein activation of mouse brain homogenates. Here we describe for the first time the acute effect of KYNA and KYNA1 on opioid receptor function with the possible involvement of the NMDA receptor.The acute 30minute in vivo KYNA1 and KYNA treatments altered opioid receptor G-protein signaling or ligand potency depending on the opioid receptor type and brain region (rat cortex vs striatum) using [35S]GTPγS binding assays. Pretreatment with the NMDA receptor antagonist MK-801 impaired or reversed the effects of KYNA1 and KYNA. These results suggest an NMDA receptor mediated effect. After acute 30minute treatment HPLC measurements revealed a similar KYNA1 and a higher KYNA plasma concentration compared to cerebrospinal fluid concentrations. Finally, KYNA, KYNA1 and MK-801 showed comparable results in opioid receptor G-protein activity and ligand potency with acute in vivo treatments when they were administered in vitro for 30min on isolated cortex and striatum slices.We previously demonstrated that KYNA1 and KYNA acutely altered opioid receptor function in vivo and in vitro through the NMDA receptor depending on the opioid receptor type and brain region. This study may lead to a new, indirect approach to influence opioid receptor signaling.

Lentiviral-mediated overexpression of KCTD12 inhibits the proliferation of human uveal melanoma OCM-1 cells.

Human potassium channel tetramerization domain containing12 (KCTD12, also known as Pfetin) is a member of the KCTD family which consists of 26members. It has been reported that KCTD12 regulates agonist potency and kinetics of GABAB receptor signaling. Proteomic analysis indicates that KCTD12 may be a potential biomarker for the diagnosis and prognosis of gastrointestinal stromal tumors. However, little has been reported concerning the role of KCTD12 in the other tumor types. In the present study, we designed and subcloned N-terminally Flag-tagged human KCTD12 into the pLVX‑Puro vector. We then generated a human uveal melanoma cell line (OCM-1) stably expressing KCTD12. Using this stable cell line, we performed a series of experiments including colony formation, invasion, migration and wound-healing assays, flow cytometry and western blotting. Based on the experimental results, we first demonstrated that KCTD12 effectively suppressed the proliferation of OCM-1 cells and limited the spread of OCM-1 cells. In the flow cytometric analysis, prolongation of the progression of G2/M to G1phase in the KCTD12-overexpressing OCM-1 cells was observed. In addition, inhibition of KCTD12-overexpressing OCM-1 cell xenograft growth in nude mice was observed. Taken together, KCTD12 may serve as a novel therapeutic target for patients with uveal melanoma.

Interferon-γ Perturbs Key Signaling Pathways Induced By Thrombopoietin, but Not Eltrombopag, in Human Hematopoietic Stem/Progenitor Cells

▪Thrombopoietin (TPO) is the main regulator of hematopoietic stem and progenitor cell (HSPC) self-renewal and survival. Upon binding to its receptor, c-MPL, TPO activates cell signaling, through JAK-STAT and other pathways, which is tightly balanced by negative regulatory signaling processes. Recent studies indicate that chronic exposure of HSPCs to IFNγ, as exemplified in subjects with severe aplastic anemia (SAA), impairs self-renewal by perturbing TPO signaling pathways. Despite elevated levels of TPO in subjects with SAA, the TPO receptor agonist Eltrombopag (Epag) improves trilineage hematopoiesis in refractory SAA, suggesting that it may activate signaling within HSPC in a way that is distinct from TPO under inflammatory conditions. To address the paradox of Epag efficacy despite high endogenous TPO levels in bone marrow failure, G-CSF mobilized human CD34+ cells from 6 healthy donors were cultured in the presence of SCF, FLT3 and either TPO 5 ng/ml (TPO5) or Epag 3 μg/ml (Epag), with or without IFNγ 100 ng/ml. After 7 days in culture, cells were characterized via flow cytometry, CFU assay and transplantation in immunodeficient (NSG) mice. The percentages of CD34+ cells in cultures containing TPO5 or Epag alone were similar (83.3 ± 9.7% and 87.6 ± 7.1%, respectively), but were better preserved with Epag than TPO5 in the presence of IFNγ (46.7 ± 16.1% and 24.6 ± 15.0% respectively, p<0.05). Accordingly, when comparing 7-day cultures with and without IFNγ, the absolute numbers of CD34+ cells were markedly reduced with TPO5 (average 7.6-fold, p<0.005) but only minimally decreased with Epag (average 1.6-fold, p = n.s.). The adjusted numbers of CFUs after 7 days in the presence of IFNγ similarly decreased 2.7-fold with TPO5 but remained unchanged with Epag compared to cultures without IFNγ. When the 7-day expanded progeny of an equal starting number of CD34+ cells was transplanted in NSG mice, human cell engraftment was superior with Epag (34 ± 3.8% human CD45+ cells) than with TPO5 (21 ± 1.8% human CD45+ cells, p<0.05) cultures in the presence of IFNγ, suggesting an impact of Epag on the most primitive long-term repopulating HSPCs. To investigate potential mechanisms by which Epag positively affects maintenance of HSPCs under inflammatory conditions, we examined cell signaling pathways induced upon binding of TPO, Epag and IFNγ to their respective receptors in human CD34+ cells. At a concentration of 5ng/mL, TPO induced a rapid (peak < 1 hour) and high potency rise in STAT5 phosphorylation followed by a rapid (< 2 hours) decay in signal. In contrast, Epag induced a slow (peak 4 hours) low potency rise in STAT5 phosphorylation, and the signal persisted for at least 10 hours. The difference in cell signaling potency and kinetics between TPO and Epag is likely related to their binding to distinct regions of c-MPL, resulting in alternate receptor conformational changes. We next investigated the impact of IFNγ on TPO and Epag-induced STAT5 phosphorylation at the signal peak (<1 and 4 hours, respectively). As previously shown in murine HSPCs, IFNγ impaired TPO signaling in human HSPCs (Figure, panels A, C). In contrast, Epag-induced STAT5 phosphorylation was preserved or increased in the presence of IFNγ (Figure, panels B, C). When Epag and TPO were combined, inhibition of TPO signaling by IFNγ was partially restored (Figure, panel D). By reducing the dose of TPO from 5 to 1ng/mL, and therefore reducing the potency of signaling to levels similar to Epag, the inhibitory effect of IFNγ on TPO signaling was abolished (Figure, panel E). Activation of IFNγ receptor by its ligand induces phosphorylation of STAT1 and subsequent expression of suppressor of cytokine signaling-1 (SOCS-1), a negative regulator of both IFNγ and c-MPL receptors via inhibition of STAT1 and STAT5 phosphorylation, respectively. We found that IFNγ-induced phosphorylation of STAT1 was increased in the presence of TPO 5ng/mL (1.5-fold increase, p<0.05) but unaffected by Epag (1.1-fold increase, p = n.s.) or TPO 1ng/mL (1.1-fold increase, p = n.s.). Our data indicate that Epag counters IFNγ-induced perturbation of TPO signaling in human HSPCs. Epag produces an unopposed low potency, slow kinetic positive signal and activates c-Mpl above a threshold level critical for HSPC self-renewal. Epag’s evasion of IFN blockade of a critical pathway of growth factor cell signaling may explain its efficacy in improving hematopoiesis in SAA. [Display omitted] DisclosuresCheng:Novartis: Research Funding. Cheruku:Novartis: Research Funding. Alvarado:Novartis: Research Funding. Cash:Novartis: Research Funding. Dunbar:Novartis: Research Funding. Young:Novartis: Research Funding. Larochelle:Novartis: Research Funding.

138 - Inflammatory Generation and Pro-Resolving Signaling Actions of Conjugated Nitro-Linoleic Acid

Inflammatory responses result in the activation of pro-oxidant enzymes such as the inducible isoform of nitric oxide synthase (iNOS) and NAPDH oxidases, leading to the generation of the nitrating species nitrogen dioxide (●NO2). Conjugated linoleic acid is a primary substrate for biological nitration in the gastric compartment giving rise to the formation of conjugated nitro-linoleic acid (NO2-CLA). Herein we show that macrophage activation leads to NO2-CLA formation both in vitro and in vivo, as determined by selective reaction monitoring analysis using isotopically labeled internal standards and confirmed by high resolution mass spectrometry. CLA nitration by activated RAW264.7 cells results not only in the dose- and time-dependent formation of the parent NO2-CLA compound but also in the generation of secondary electrophilic and non-electrophilic β-oxidation metabolites. The formation of two positional NO2-CLA isomers with different metabolization rates but similar signaling potency was also observed. Addition of either mixed or pure NO2-CLA positional isomers to cells in culture led to antioxidant response element (ARE) activation and to dose-dependent expression of Nrf2-regulated genes. Similarly, NO2-CLA also attenuated TNF-α mediated NF-κB response element activation and decreased iNOS expression and pro-inflammatory cytokine release in LPS/IFN-γ activated RAW264.7 cells. To test the physiological relevance of these observations, a mouse zymosan A peritonitis model that promotes CLA nitration was utilized. Animals were administered vehicle, CLA or synthetic NO2-CLA and then polymorphonuclear cell (PMN) recruitment and clearance from the peritoneal cavity was monitored. Both synthetic and in situ CLA-derived NO2-CLA led to decreased recruitment and enhanced PMN clearance, which coincided with higher expression of Nrf2-dependent genes in peritoneal cells. Our work demonstrates that NO2-CLA generation might constitute an endogenous adaptive mechanism capable of modulating the severity of the inflammatory response by favoring resolution and inducing the expression of both cytoprotective and antioxidant genes.

Truncated Glucagon-like Peptide-1 and Exendin-4 α-Conotoxin pl14a Peptide Chimeras Maintain Potency and α-Helicity and Reveal Interactions Vital for cAMP Signaling in Vitro

Glucagon-like peptide-1 (GLP-1) signaling through the glucagon-like peptide 1 receptor (GLP-1R) is a key regulator of normal glucose metabolism, and exogenous GLP-1R agonist therapy is a promising avenue for the treatment of type 2 diabetes mellitus. To date, the development of therapeutic GLP-1R agonists has focused on producing drugs with an extended serum half-life. This has been achieved by engineering synthetic analogs of GLP-1 or the more stable exogenous GLP-1R agonist exendin-4 (Ex-4). These synthetic peptide hormones share the overall structure of GLP-1 and Ex-4, with a C-terminal helical segment and a flexible N-terminal tail. Although numerous studies have investigated the molecular determinants underpinning GLP-1 and Ex-4 binding and signaling through the GLP-1R, these have primarily focused on the length and composition of the N-terminal tail or on how to modulate the helicity of the full-length peptides. Here, we investigate the effect of C-terminal truncation in GLP-1 and Ex-4 on the cAMP pathway. To ensure helical C-terminal regions in the truncated peptides, we produced a series of chimeric peptides combining the N-terminal portion of GLP-1 or Ex-4 and the C-terminal segment of the helix-promoting peptide α-conotoxin pl14a. The helicity and structures of the chimeric peptides were confirmed using circular dichroism and NMR, respectively. We found no direct correlation between the fractional helicity and potency in signaling via the cAMP pathway. Rather, the most important feature for efficient receptor binding and signaling was the C-terminal helical segment (residues 22-27) directing the binding of Phe(22) into a hydrophobic pocket on the GLP-1R.

A virtual lymph node model to dissect the requirements for T-cell activation by synapses and kinapses.

The initiation of T-cell responses in lymph nodes requires T cells to integrate signals delivered by dendritic cells (DCs) during long-lasting contacts (synapses) or more transient interactions (kinapses). However, it remains extremely challenging to understand how a specific sequence of contacts established by T cells ultimately dictates T-cell fate. Here, we have coupled a computational model of T-cell migration and interactions with DCs with a real-time, flow cytometry-like representation of T-cell activation. In this model, low-affinity peptides trigger T-cell proliferation through kinapses but we show that this process is only effective under conditions of high DC densities and prolonged antigen availability. By contrast, high-affinity peptides favor synapse formation and a vigorous proliferation under a wide range of antigen presentation conditions. In line with the predictions, decreasing the DC density in vivo selectively abolished proliferation induced by the low-affinity peptide. Finally, our results suggest that T cells possess a biochemical memory of previous stimulations of at least 1–2 days. We propose that the stability of T-cell–DC interactions, apart from their signaling potency, profoundly influences the robustness of T-cell activation. By offering the ability to control parameters that are difficult to manipulate experimentally, the virtual lymph node model provides new possibilities to tackle the fundamental mechanisms that regulate T-cell responses elicited by infections or vaccines.

An in Vitro and in Vivo Investigation of Bivalent Ligands That Display Preferential Binding and Functional Activity for Different Melanocortin Receptor Homodimers.

Pharmacological probes for the melanocortin receptors have been utilized for studying various disease states including cancer, sexual function disorders, Alzheimer's disease, social disorders, cachexia, and obesity. This study focused on the design and synthesis of bivalent ligands to target melanocortin receptor homodimers. Lead ligands increased binding affinity by 14- to 25-fold and increased cAMP signaling potency by 3- to 5-fold compared to their monovalent counterparts. Unexpectedly, different bivalent ligands showed preferences for particular melanocortin receptor subtypes depending on the linker that connected the binding scaffolds, suggesting structural differences between the various dimer subtypes. Homobivalent compound 12 possessed a functional profile that was unique from its monovalent counterpart providing evidence of the discrete effects of bivalent ligands. Lead compound 7 significantly decreased feeding in mice after intracerebroventricular administration. To the best of our knowledge, this is the first report of a melanocortin bivalent ligand's in vivo physiological effects.

Getting a Handle on RAS-targeted Therapies: Cysteine Directed Inhibitors.

Directly inhibiting oncogenic RAS proteins has proven to be an arduous task, as after more than thirty years of intensive investigation, no clinically relevant therapies exist. Recently, two classes of selective small molecule inhibitors that target a cysteine-containing RAS mutant have been developed, representing the first directed approaches to specifically inhibit an oncogenic KRAS mutant. In this mini-review, we first assess the development and targeting strategies associated with novel cysteine-directed RAS inhibitors. Next, we describe the variable oncogenic potency of the KRAS G12C mutant when compared to other KRAS G12 mutants. Lastly, we evaluate how the redox properties of KRAS G12C may play a role in differential signaling and tumorigenic potency of the oncogene, the efficacy of small molecules targeting this specific RAS mutant and further development of directed oncogenic RAS inhibitors.

Retrograde Melanopsin Signaling Increases With Age in Retinal Degenerate Mice Lacking Rods and the Majority of Cones.

Following on from reports of retrograde retinal signaling in mice, we sought to investigate the influence of age and retinal location on this phenomenon using mice that lack rods and the majority of cones. We used functional anatomy for c-fos (Fos) and tyrosine hydroxylase (TH) to measure light-driven activation of dopamine neurons along a dorsal-ventral transect in C3H/He wild-type and rodless-coneless rd/rd cl (rdcl) mice aged 3, 5, and >14 months. A parallel series of retinae from 3-month-old mice was also stained for cone opsins and melanopsin. Analysis by confocal microscopy revealed light-driven Fos activation in TH cells residing in the middorsal retina of the youngest rdcl mice. This region was largely devoid of residual cones but contained a large number of intrinsically photosensitive retinal ganglion cells (ipRGCs) and the highest density of melanopsin neurites. With advancing age, there was a paradoxical increase in retrograde signaling from ∼3% Fos-positive (Fos+) TH cells at 3 months to ∼36% in rdcl mice >14 months. This increased activation occurred in more central and peripheral retinal regions. Our data provide new insights into the anatomy and plasticity of retrograde melanopsin signaling in mice with severe rod/cone dystrophy. The increased retrograde signaling we detect may result from either an increased potency of melanopsin signaling with advancing age and/or postsynaptic modification to dopaminergic neurons.

Vitamin Para-Aminobenzoic Acid (PABA) Controls Generation of Nitric Oxide (NO) In Vitro and its Biological Functions in the Bacterial Cells

Nitric oxide (NO) serves either a universal signaling molecule or extremely toxic agent, depending on the dose. Up to date there have been a very limited number of natural compounds serving as effective regulators of NO signaling and toxic potencies. NO acts in concert with H2S to coordinate cell responses; however, how exactly this interaction is achieved is not known. Both agents have an effect on the accumulation of both reactive chemical species, ROS and RNS and can give rise to other reactive species. Para-aminobenzoic acid (PABA) is an essential metabolite for certain organisms. Once considered a vitamin, PABA, functions as an effective inhibitor of inducible SOS DNA repair processes in E. coli. In the present study we focus on the genetic and physiological evidence in favor of interference of NO-donors and PABA in bacterial cells with DNA repair gene expression and biofilm formation, depending on the rate of NOdonating in vitro and intracellular ROS/RNS accumulation in the cells. The crystalline dinitrosyl iron complexes (NO- 29 and NO-33) with thiourea as the ligands and 3 crystalline tetranitrosyl iron complexes with thiosulfate (TNICthio) - and with sulfur-containing aliphatic ligands – cysteamine and penicillamine were studied first as the NO-donors in pure solutions and in the combination with PABA. In E. coli cells with the combined action of PABA (0.01-5 mM) with nitric oxide donors we observed an inhibition of NO-signaling potency in the SOS (sfiA gene)- and the SoxRS (soxS gene) DNA repair pathway up to 3.5 fold, depending on the dose of PABA. PABA tested at 0.5 mM afforded 24% protection against the level of biofilm formation induced by TNICthio. Using the antioxidant-capacity assay, we observed a many-fold decrease in the ROS/RNS level production in the samples of E. coli cells with PABA and NO-donor-TNICthio.

Instructive roles for cytokine-receptor binding parameters in determining signaling and functional potency.

Cytokines dimerize cell surface receptors to activate signaling and regulate many facets of the immune response. Many cytokines have pleiotropic effects, inducing a spectrum of redundant and distinct effects on different cell types. This pleiotropy has hampered cytokine-based therapies, and the high doses required for treatment often lead to off-target effects, highlighting the need for a more detailed understanding of the parameters controlling cytokine-induced signaling and bioactivities. Using the prototypical cytokine interleukin-13 (IL-13), we explored the interrelationships between receptor binding and a wide range of downstream cellular responses. We applied structure-based engineering to generate IL-13 variants that covered a spectrum of binding strengths for the receptor subunit IL-13Rα1. Engineered IL-13 variants representing a broad range of affinities for the receptor exhibited similar potencies in stimulating the phosphorylation of STAT6 (signal transducer and activator of transcription 6). Delays in the phosphorylation and nuclear translocation of STAT6 were only apparent for those IL-13 variants with markedly reduced affinities for the receptor. From these data, we developed a mechanistic model that quantitatively reproduced the kinetics of STAT6 phosphorylation for the entire spectrum of binding affinities. Receptor endocytosis played a key role in modulating STAT6 activation, whereas the lifetime of receptor-ligand complexes at the plasma membrane determined the potency of the variant for inducing more distal responses. This complex interrelationship between extracellular ligand binding and receptor function provides the foundation for new mechanism-based strategies that determine the optimal cytokine dose to enhance therapeutic efficacy.

What we learned from bench to bedside with the neuroblastoma targeting CD171-specific CAR

s of the 51st Workshop for Pediatric Research 51st Workshop for Pediatric Research Gottingen, Germany 16-17 April 2015 This supplement has not been sponsored. Meeting abstracts Meeting abstract Despite the therapeutic efficacy of chimeric antigen receptor (CAR) redirected T cell immunotherapy in leukemia and lymphoma patients, similar clinical responses in solid tumor patients is, to date, an unrealized objective. We developed a CAR specific for CD171, an antigen expressed in several solid tumors including neuroblastoma, the most common extracranial tumor in childhood with an overall survival of less than 50% in high-risk patients. Since CD171 is also expressed in healthy tissues, including cerebellum and kidney, we proved the safety of targeting CD171 with CAR T cells in a non-human primate study. Further, we showed that CAR extracellular spacer and cytoplasmic signaling domain variants can be combined to tune the magnitude of cytotoxic CD8 + T lymphocyte (CTL) activation for tumor cell cytolysis and cytokine secretion. CAR constructs displaying the highest in vitro activity unexpectedly displayed the lowest in vivo anti-tumor activity, whereas CARs tuned for moderate signaling potency mediated tumor eradication. Recursively triggering hyperactive CARs rendered CTLs highly susceptible to activation-induced cell death resulting from augmented FasL expression, indicating that activation-induced cell death may be a critical parameter for achieving clinical efficacy against solid tumors. Our preclinical results assisted the design of a clinical trial comparing two CARs with different cytoplasmic signaling domains in patients with primary refractory or relapsed neuroblastoma, which was launched October 2014 at the Seattle Children's Hospital.