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A prospective, large library virtual screen against an activated β2-adrenergic receptor (β2AR) structure returned potent agonists to the exclusion of inverse-agonists, providing the first complement to the previous virtual screening campaigns against inver

Colloidal aggregation is the dominant mechanism for artifactual inhibition of soluble proteins, and controls against it are now widely deployed. Conversely, investigating this mechanism for membrane-bound receptors has proven difficult. Here we investigate

Protein classification typically uses structural, sequence or functional similarity. Here we introduce an orthogonal method that organizes proteins by ligand similarity, focusing on the class A G-protein-coupled receptor (GPCR) protein family. Comparing a

Rationale: Acute respiratory distress syndrome (ARDS) has an unacceptably high mortality rate (35%) and is without effective therapy. Orai1 is a Ca 2+ channel involved in store-operated Ca 2+ entry (SOCE), a process that exquisitely regulates inflammation. Orai1 is considered a druggable target, but no Orai1-specific inhibitors exist to date. Objectives: To evaluate whether ELD607, a first-in-class Orai1 antagonist, can treat ARDS caused by bacterial pneumonia in preclinical models. Methods: ELD607 pharmacology was evaluated in HEK293T cells and freshly isolated immune cells from patients with ARDS. A murine acute lung injury model caused by bacterial pneumonia was then used: mice were infected with Pseudomonas aeruginosa , Staphylococcus aureus , methicillin-resistant S. aureus , or multidrug-resistant P. aeruginosa and then treated with ELD607 intranasally. Measurements and Main Results: ELD607 specifically inhibited SOCE in HEK293T cells with a half-maximal inhibitory concentration of 9 nM. ELD607 was stable in ARDS airway secretions and inhibited SOCE in ARDS immune cells. In vivo , inhaled ELD607 significantly reduced neutrophilia and improved survival. Surprisingly, Orai1 inhibition by ELD607 caused a significant reduction in lung bacteria, including methicillin-resistant S. aureus . ELD607 worked as an immunomodulator that reduced cytokine levels, reduced neutrophilia, and promoted macrophage-mediated resolution of inflammation and clearance of bacteria. Indeed, when alveolar macrophages were depleted with inhaled clodronate, ELD607 was no longer able to resolve inflammation or clear bacteria. Conclusions: These data indicate that specific Orai1 inhibition by ELD607 may be a novel approach to reduce multiorgan inflammation and treat antibiotic-resistant bacteria.

Orai1 is a plasma membrane Ca 2+  channel that mediates store-operated Ca 2+  entry (SOCE) and regulates inflammation. Short palate lung and nasal epithelial clone 1 (SPLUNC1) is an asthma gene modifier that inhibits Orai1 and SOCE via its C-terminal α6 region. SPLUNC1 levels are diminished in asthma patient airways. Thus, we hypothesized that inhaled α6 peptidomimetics could inhibit Orai1 and reduce airway inflammation in a murine asthma model. To evaluate α6-Orai1 interactions, we used fluorescent assays to measure Ca 2+  signaling, Förster resonance energy transfer, fluorescent recovery after photobleaching, immunostaining, total internal reflection microscopy, and Western blotting. To test whether α6 peptidomimetics inhibited SOCE and decreased inflammation in vivo , wild-type and SPLUNC1 -/- mice were exposed to house dust mite (HDM) extract with or without α6 peptide. We also performed nebulization, jet milling, and scanning electron microscopy to evaluate α6 for inhalation. SPLUNC1 -/- mice had an exaggerated response to HDM. In BAL-derived immune cells, Orai1 levels increased after HDM exposure in SPLUNC1 -/- but not wild-type mice. Inhaled α6 reduced Orai1 levels in mice regardless of genotype. In HDM-exposed mice, α6 dose-dependently reduced eosinophilia and neutrophilia. In vitro , α6 inhibited SOCE in multiple immune cell types, and α6 could be nebulized or jet milled without loss of function. These data suggest that α6 peptidomimetics may be a novel, effective antiinflammatory therapy for patients with asthma.

Electronic cigarettes (e-cigarettes) are battery powered electronic nicotine delivery systems that use a propylene glycol/vegetable glycerin base to deliver vaporized nicotine and flavorings to the body. E-cigarettes became commercially available without evidence regarding their risks, long-term safety, or utility in smoking cessation. Recent clinical trials suggest that e-cigarette use with counseling may be effective in reducing cigarette use but not nicotine dependence. However, meta-analyses of observational studies demonstrate that e-cigarette use is not associated with smoking cessation. Cardiovascular studies reported sympathetic activation, vascular stiffening, and endothelial dysfunction, which are associated with adverse cardiovascular events. The majority of pulmonary clinical trials in e-cigarette users included standard spirometry as the primary outcome measure, reporting no change in lung function. However, studies reported increased biomarkers of pulmonary disease in e-cigarette users. These studies were conducted in adults, but >30% of high school-age adolescents reported e-cigarette use. The effects of e-cigarette use on cardiopulmonary endpoints in adolescents and young adults remain unstudied. Because of adverse clinical findings and associations between e-cigarette use and increased incidence of respiratory diseases in people who have never smoked, large longitudinal studies are needed to understand the risk profile of e-cigarettes. Consistent with the Centers for Disease Control and Prevention recommendations, clinicians should monitor the health risks of e-cigarette use, discourage nonsmokers and adolescents from using e-cigarettes, and discourage smokers from engaging in dual use without cigarette reduction or cessation.

Tobacco smoking is the largest risk factor for developing chronic obstructive pulmonary disease (COPD), and is associated with hyperresponsiveness of airway smooth muscle (ASM). Chronic exposure to cigarette smoke (CS) leads to airway inflammation and remodelling. However, the direct effect of gaseous CS or CS extract (CSE) on human airway smooth muscle cell (hASMC) function remains poorly understood. This study investigated the acute effect of CS/CSE on calcium homeostasis, a key regulator of ASM physiology and pathophysiology. Primary hASMC were isolated from non-smoking donor lungs, and subjected to Ca 2+ imaging studies. We found that both CS, and CSE, rapidly elevated cytosolic Ca 2+ in hASMC through stimulation of plasmalemmal Ca 2+ influx, but excluded store-operated and L-type Ca 2+ channels as mediators of this effect. Using a specific pharmacological inhibitor, or shRNA-driven knockdown, we established that both CS and CSE stimulated Ca 2+ influx in hASMC through the neurogenic pain receptor channel, transient receptor potential ankyrin 1 (TRPA1). CS/CSE-dependent, TRPA1-mediated Ca 2+ influx led to myosin light-chain phosphorylation, a key process regulating ASM contractility. We conclude that TRPA1 is likely an important link between CS/CSE exposure and airway hyperresponsiveness, and speculate that acute CS/CSE-induced Ca 2+ influx could lead to exacerbated ASM contraction and potentially initiate further chronic pathological effects of tobacco smoke.

Introduction: The use of flavors in electronic cigarettes appeals to adults and never-smoking youth. Consumption has rapidly increased over the last decade, and in the U.S. market alone, there are over 8000 unique flavors. The U.S. Food and Drug Administration (FDA) has begun to regulate e-liquids, but many have not been tested, and their impact, both at the cellular level, and on human health remains unclear., Methods: We tested e-liquids on the human cell line HEK293T and measured toxicity, mitochondrial membrane potential (ΔΨ  m), reactive oxygen species production (ROS), and cellular membrane potential (Vm) using high-throughput screening (HTS) approaches. Our HTS efforts included single-dose and 16-point dose-response curves, which allowed testing of ≥90 commercially available e-liquids in parallel to provide a rapid assessment of cellular effects as a proof of concept for a fast, preliminary toxicity method. We also investigated the chemical composition of the flavors via gas chromatography-mass spectrometry., Results: We found that e-liquids caused a decrease in ΔΨ  m and Vm and an increase in ROS production and toxicity in a dose-dependent fashion. In addition, the presence of five specific chemical components: vanillin, benzyl alcohol, acetoin, cinnamaldehyde, and methyl-cyclopentenolone, but not nicotine, were linked with the changes observed in the cellular traits studied., Conclusion: Our data suggest that ΔΨ  m, ROS, Vm, and toxicity may be indicative of the extent of cell death upon e-liquid exposure. Further research on the effect of flavors should be prioritized to help policy makers such as the FDA to regulate e-liquid composition., Implications: E-liquid cellular toxicity can be predicted using parameters amenable to HTS. Our data suggest that ΔΨ  m, ROS, Vm, and toxicity may be indicative of the extent of cell death upon e-liquid exposure, and this toxicity is linked to the chemical composition, that is, flavoring components. Further research on the effect of flavors should be prioritized to help policy makers such as the FDA to regulate e-liquid composition., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco.)

Competing Interests: Disclosure: The authors have declared no conflicts of interest.

Chronic obstructive pulmonary disease (COPD), which is most commonly caused by cigarette smoke (CS) exposure, is the third leading cause of death worldwide. The cystic fibrosis transmembrane conductance regulator (CFTR) is an apical membrane anion channel that is widely expressed in epithelia throughout the body. In the airways, CFTR plays an important role in fluid homeostasis and helps flush mucus and inhaled pathogens/toxicants out of the lung. Inhibition of CFTR leads to mucus stasis and severe airway disease. CS exposure also inhibits CFTR, leading to the decreased anion secretion/hydration seen in COPD patients. However, the underlying mechanism is poorly understood. Here, we report that CS causes CFTR to be internalized in a clathrin/dynamin-dependent fashion. This internalization is followed by retrograde trafficking of CFTR to the endoplasmic reticulum. Although this internalization pathway has been described for bacterial toxins and cargo machinery, it has never been reported for mammalian ion channels. Furthermore, the rapid internalization of CFTR is dependent on CFTR dephosphorylation by calcineurin, a protein phosphatase that is upregulated by CS. These results provide new insights into the mechanism of CFTR internalization, and may help in the development of new therapies for CFTR correction and lung rehydration in patients with debilitating airway diseases such as COPD.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated, apical anion channel that regulates ion and fluid transport in many epithelia including the airways. We have previously shown that cigarette smoke (CS) exposure to airway epithelia causes a reduction in plasma membrane CFTR expression which correlated with a decrease in airway surface hydration. The effect of CS on CFTR was dependent on an increase in cytosolic Ca 2+ . However, the underlying mechanism for this Ca 2+ -dependent, internalisation of CFTR is unknown. To gain a better understanding of the effect of Ca 2+ on CFTR, we performed whole cell current recordings to study the temporal effect of raising cytosolic Ca 2+ on CFTR function. We show that an increase in cytosolic Ca 2+ induced a time-dependent reduction in whole cell CFTR conductance, which was paralleled by a loss of cell surface CFTR expression, as measured by confocal and widefield fluorescence microscopy. The decrease in CFTR conductance and cell surface expression were both dynamin-dependent. Single channel reconstitution studies showed that raising cytosolic Ca 2+ per se had no direct effect on CFTR. In fact, the loss of CFTR plasma membrane activity correlated with activation of calcineurin, a Ca 2+ -dependent phosphatase, suggesting that dephosphorylation of CFTR was linked to the loss of surface expression. In support of this, the calcineurin inhibitor, cyclosporin A, prevented the Ca 2+ -induced decrease in cell surface CFTR. These results provide a hitherto unrecognised role for cytosolic Ca 2+ in modulating the residency of CFTR at the plasma membrane through a dynamin- and calcineurin-dependent mechanism.

Chemerin receptor (CMKLR1) is a G protein-coupled receptor (GPCR) implicated in macrophage-mediated inflammation and in several forms of human arthritis. Analogous to other GPCR, CMKLR1 is likely regulated by G protein-coupled receptor kinase (GRK) phosphorylation of intracellular domains in an activation-dependent manner, which leads to recruitment and termination of intracellular signaling via desensitization and internalization of the receptor. The ubiquitously expressed GRK family members include GRK2, GRK3, GRK5, and GRK6, but it is unknown which GRK regulates CMKLR1 cellular and signaling functions. Our data show that activation of CMKLR1 by chemerin in primary macrophages leads to signaling and functional outcomes that are regulated by GRK6 and β-arrestin 2. We show that arrestin recruitment to CMKLR1 following chemerin stimulation is enhanced with co-expression of GRK6. Further, internalization of endogenous CMKLR1, following the addition of chemerin, is decreased in inflammatory macrophages from GRK6- and β-arrestin 2-deficient mice. These GRK6- and β-arrestin 2-deficient macrophages display increased migration toward chemerin and altered AKT and Extracellular-signal Related Kinase (ERK) signaling. Our findings show that chemerin-activated CMKLR1 regulation in inflammatory macrophages is largely GRK6 and β-arrestin mediated, which may impact innate immunity and have therapeutic implications in rheumatic disease., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Currently, there are no established pharmaceutical strategies that effectively treat social deficits in autism spectrum disorder (ASD). Oxytocin, a neurohormone that plays a role in multiple types of social behaviors, has been proposed as a possible therapeutic against social impairment and other symptoms in ASD. However, from the standpoint of pharmacotherapy, oxytocin has several liabilities as a standard clinical treatment, including rapid metabolism, low brain penetrance, and activity at the vasopressin (antidiuretic hormone) receptors. The present studies describe findings from a preclinical screening program to evaluate oxytocin receptor (OXTR) agonists and oxytocin metabolites for potential clinical use as more optimal treatments. We first investigated two synthetic oxytocin analogs, TC-OT-39 and carbetocin, using in vitro cell-based assays for pharmacological characterization and behavioral tests in the BALB/cByJ mouse model of ASD-like social deficits. Although both TC-OT-39 and carbetocin selectively activate the OXTR, neither synthetic agonist had prosocial efficacy in the BALB/cByJ model. We next evaluated two oxytocin metabolites: OT(4-9) and OT(5-9). While OT(5-9) failed to affect social deficits, the metabolite OT(4-9) led to significant social preference in the BALB/cByJ model, in a dose-dependent manner. The increased sociability was observed at both 24 h and 12 days following the end of a subchronic regimen with OT(4-9) (2.0 mg/kg). Overall, these results suggest that the prosocial effects of oxytocin could be mediated by downstream activity of oxytocin metabolites, raising the possibility of new pathways to target for drug discovery relevant to ASD., (Copyright © 2018. Published by Elsevier Ltd.)

CC-chemokine receptor 7 (CCR7) is a G protein-coupled receptor expressed on a variety of immune cells. CCR7 plays a critical role in the migration of lymphocytes into secondary lymphoid tissues. CCR7 expression, however, has been linked to numerous disease states. Due to its therapeutic relevance and absence of available CCR7 inhibitors, we undertook a high-throughput screen (HTS) to identify small-molecule antagonists of the receptor. Here, we describe a robust HTS approach using a commercially available β-galactosidase enzyme fragment complementation system and confirmatory transwell chemotaxis assays. This work resulted in the identification of several compounds with activity against CCR7. The most potent of these was subsequently determined to be cosalane, a cholesterol derivative previously designed as a therapeutic for human immunodeficiency virus. Cosalane inhibited both human and murine CCR7 in response to both CCL19 and CCL21 agonists at physiologic concentrations. Furthermore, cosalane produced durable inhibition of the receptor following a cellular incubation period with subsequent washout. Overall, our work describes the development of an HTS-compatible assay, completion of a large HTS campaign, and demonstration for the first time that cosalane is a validated CCR7 antagonist. These efforts could pave the way for new approaches to address CCR7-associated disease processes.

To investigate large library docking's ability to find molecules with joint activity against on-targets and selectivity versus antitargets, the dopamine D 2 and serotonin 5-HT 2A receptors were targeted, seeking selectivity against the histamine H 1 receptor. In a second campaign, κ-opioid receptor ligands were sought with selectivity versus the μ-opioid receptor. While hit rates ranged from 40% to 63% against the on-targets, they were just as good against the antitargets, even though the molecules were selected for their putative lack of binding to the off-targets. Affinities, too, were often as good or better for the off-targets. Even though it was occasionally possible to find selective molecules, such as a mid-nanomolar D 2 /5-HT 2A ligand with 21-fold selectivity versus the H 1 receptor, this was the exception. Whereas false-negatives are tolerable in docking screens against on-targets, they are intolerable against antitargets; addressing this problem may demand new strategies in the field.

Rationale: E-cigarettes vaporize propylene glycol/vegetable glycerin (PG/VG), nicotine, and flavorings. However, the long-term health effects of exposing lungs to vaped e-liquids are unknown., Objectives: To determine the effects of chronic vaping on pulmonary epithelia., Methods: We performed research bronchoscopies on healthy nonsmokers, cigarette smokers, and e-cigarette users (vapers) and obtained bronchial brush biopsies and lavage samples from these subjects for proteomic investigation. We further employed in vitro and murine exposure models to support our human findings., Measurements and Main Results: Visual inspection by bronchoscopy revealed that vaper airways appeared friable and erythematous. Epithelial cells from biopsy samples revealed approximately 300 proteins that were differentially expressed in smoker and vaper airways, with only 78 proteins being commonly altered in both groups and 113 uniquely altered in vapers. For example, CYP1B1 (cytochrome P450 family 1 subfamily B member 1), MUC5AC (mucin 5 AC), and MUC4 levels were increased in vapers. Aerosolized PG/VG alone significantly increased MUC5AC protein in human airway epithelial cultures and in murine nasal epithelia in vivo. We also found that e-liquids rapidly entered cells and that PG/VG reduced membrane fluidity and impaired protein diffusion., Conclusions: We conclude that chronic vaping exerts marked biological effects on the lung and that these effects may in part be mediated by the PG/VG base. These changes are likely not harmless and may have clinical implications for the development of chronic lung disease. Further studies will be required to determine the full extent of vaping on the lung.

The e-liquids used in electronic cigarettes (E-cigs) consist of propylene glycol (PG), vegetable glycerin (VG), nicotine, and chemical additives for flavoring. There are currently over 7,700 e-liquid flavors available, and while some have been tested for toxicity in the laboratory, most have not. Here, we developed a 3-phase, 384-well, plate-based, high-throughput screening (HTS) assay to rapidly triage and validate the toxicity of multiple e-liquids. Our data demonstrated that the PG/VG vehicle adversely affected cell viability and that a large number of e-liquids were more toxic than PG/VG. We also performed gas chromatography-mass spectrometry (GC-MS) analysis on all tested e-liquids. Subsequent nonmetric multidimensional scaling (NMDS) analysis revealed that e-liquids are an extremely heterogeneous group. Furthermore, these data indicated that (i) the more chemicals contained in an e-liquid, the more toxic it was likely to be and (ii) the presence of vanillin was associated with higher toxicity values. Further analysis of common constituents by electron ionization revealed that the concentration of cinnamaldehyde and vanillin, but not triacetin, correlated with toxicity. We have also developed a publicly available searchable website (www.eliquidinfo.org). Given the large numbers of available e-liquids, this website will serve as a resource to facilitate dissemination of this information. Our data suggest that an HTS approach to evaluate the toxicity of multiple e-liquids is feasible. Such an approach may serve as a roadmap to enable bodies such as the Food and Drug Administration (FDA) to better regulate e-liquid composition.

In the past 5 years, e-cigarette use has been increasing rapidly, particularly in youth and young adults. Due to the novelty of e-cigarettes (e-cigs) and e-cigarette liquids (e-liquids), research on their chemo-physical properties is still in its infancy. Here, we describe a previously unknown and potentially useful property of e-liquids, namely their autofluorescence. We performed an emission scan at 9 excitation wavelengths common to fluorescent microscopy and found (i) that autofluorescence differs widely between e-liquids, (ii) that e-liquids are most fluorescent in the UV range (between 350 and 405 nm) and (iii) fluorescence intensity wanes as the emission wavelength increases. Furthermore, we used the autofluorescence of e-liquids as a marker for tracking e-cig aerosol deposition in the laboratory. Using linear regression analysis, we were able to quantify the deposition of a "vaped" e-liquid onto hard surfaces. Using this technique, we found that every 70 mL puff of an e-cigarette deposited 0.019% e-liquid (v/v) in a controlled environment. Finally, we vaped a surface in the laboratory and used our method to detect e-cig aerosol third-hand exposure. In conclusion, our data suggest that e-cigarette autofluorescence can be used as a marker of e-cigarette deposition.

Cytosolic Ca 2+ is a universal second messenger that is involved in many processes throughout the body, including the regulation of cell growth/cell division, apoptosis, and the secretion of both ions, and macromolecules. Tobacco smoke exerts multiple effects on airway epithelia and we have previously shown that Kentucky reference cigarette smoke exposure elevated the second messenger Ca 2+ , leading to dysfunctional ion secretion. In this study, we tested whether little cigar and commercial cigarette smoke exposure exerts similar effects on intracellular Ca 2+ levels. Indeed, Swisher Sweets, Captain Black, and Cheyenne little cigars, as well as Camel, Marlboro, and Newport cigarettes, triggered a comparable increase in intracellular Ca 2+ as seen with Kentucky reference cigarettes in human bronchial epithelia. We also found that Kentucky reference cigarette smoke exposure caused increases in Ca 2+ in HEK293T cells and that similar increases in Ca 2+ were seen with the tobacco smoke metabolites 1-NH 2 -naphthalene, formaldehyde, nicotine, and nicotine-derived nitrosamine ketone. Given the large number of physiological processes governed by changes in cytosolic Ca 2+ , our data suggest that Ca 2+ signaling is a useful and reproducible assay that can be used to probe the propensity of tobacco products and their constituents to cause toxicity., Competing Interests: No competing financial interests exist.

Morphine is an alkaloid from the opium poppy used to treat pain. The potentially lethal side effects of morphine and related opioids-which include fatal respiratory depression-are thought to be mediated by μ-opioid-receptor (μOR) signalling through the β-arrestin pathway or by actions at other receptors. Conversely, G-protein μOR signalling is thought to confer analgesia. Here we computationally dock over 3 million molecules against the μOR structure and identify new scaffolds unrelated to known opioids. Structure-based optimization yields PZM21-a potent G i activator with exceptional selectivity for μOR and minimal β-arrestin-2 recruitment. Unlike morphine, PZM21 is more efficacious for the affective component of analgesia versus the reflexive component and is devoid of both respiratory depression and morphine-like reinforcing activity in mice at equi-analgesic doses. PZM21 thus serves as both a probe to disentangle μOR signalling and a therapeutic lead that is devoid of many of the side effects of current opioids., Competing Interests: The authors declare competing financial interests: details are available in the online version of the paper. Readers are welcome to comment on the online version of the paper.

CFTR is an apical membrane anion channel that regulates fluid homeostasis in many organs including the airways, colon, pancreas and sweat glands. In cystic fibrosis, CFTR dysfunction causes significant morbidity/mortality. Whilst CFTR's function as an ion channel has been well described, its ability to regulate other proteins is less understood. We have previously shown that plasma membrane CFTR increases the surface density of the adenosine 2B receptor (A2BR), but not of the β2 adrenergic receptor (β2AR), leading to an enhanced, adenosine-induced cAMP response in the presence of CFTR. In this study, we have found that the C-terminal PDZ-domain of both A2BR and CFTR were crucial for this interaction, and that replacing the C-terminus of A2BR with that of β2AR removed this CFTR-dependency. This observation extended to intact epithelia and disruption of the actin cytoskeleton prevented A2BR-induced but not β2AR-induced airway surface liquid (ASL) secretion. We also found that CFTR expression altered the organization of the actin cytoskeleton and PDZ-binding proteins in both HEK293T cells and in well-differentiated human bronchial epithelia. Furthermore, removal of CFTR's PDZ binding motif (ΔTRL) prevented actin rearrangement, suggesting that CFTR insertion in the plasma membrane results in local reorganization of actin, PDZ binding proteins and certain GPCRs.

Triple negative breast cancer (TNBC) is a heterogeneous disease that has a poor prognosis and limited treatment options. Chemokine receptor interactions are important modulators of breast cancer metastasis; however, it is now recognized that quantitative surface expression of one important chemokine receptor, CXCR4, may not directly correlate with metastasis and that its functional activity in breast cancer may better inform tumor pathogenicity. G protein coupled receptor kinase 3 (GRK3) is a negative regulator of CXCR4 activity, and we show that GRK expression correlates with tumorigenicity, molecular subtype, and metastatic potential in human tumor microarray analysis. Using established human breast cancer cell lines and an immunocompetent in vivo mouse model, we further demonstrate that alterations in GRK3 expression levels in tumor cells directly affect migration and invasion in vitro and the establishment of distant metastasis in vivo. The effects of GRK3 modulation appear to be specific to chemokine-mediated migration behaviors without influencing tumor cell proliferation or survival. These data demonstrate that GRK3 dysregulation may play an important part in TNBC metastasis.

A series of N-benzylated-5-methoxytryptamine analogues was prepared and investigated, with special emphasis on substituents in the meta position of the benzyl group. A parallel series of several N-benzylated analogues of 2,5-dimethoxy-4-iodophenethylamine (2C-I) also was included for comparison of the two major templates (i.e., tryptamine and phenethylamine). A broad affinity screen at serotonin receptors showed that most of the compounds had the highest affinity at the 5-HT2 family receptors. Substitution at the para position of the benzyl group resulted in reduced affinity, whereas substitution in either the ortho or the meta position enhanced affinity. In general, introduction of a large lipophilic group improved affinity, whereas functional activity often followed the opposite trend. Tests of the compounds for functional activity utilized intracellular Ca(2+) mobilization. Function was measured at the human 5-HT2A, 5-HT2B, and 5-HT2C receptors, as well as at the rat 5-HT2A and 5-HT2C receptors. There was no general correlation between affinity and function. Several of the tryptamine congeners were very potent functionally (EC50 values from 7.6 to 63 nM), but most were partial agonists. Tests in the mouse head twitch assay revealed that many of the compounds induced the head twitch and that there was a significant correlation between this behavior and functional potency at the rat 5-HT2A receptor.

It has been suggested that the evolution of vertebrate opioid receptors (ORs) follow a vector of increased functionality. Here, we test this idea by comparing human and frog ORs. Interestingly, some of the most potent opioid peptides known have been isolated from amphibian skin secretions. Here we show that such peptides (dermorphin and deltorphin) are highly potent in the human receptors and inactive in frog ORs. The molecular basis for the insensitivity of the frog ORs to these peptides was studied using chimeras and molecular modeling. The insensitivity of the delta OR (DOR) to deltorphin was due to variation of a single amino acid, Trp7.35, which is a leucine in mammalian DORs. Notably, Trp7.35 is completely conserved in all known DOR sequences from lamprey, fish, and amphibians. The deltorphin-insensitive phenotype was verified in fish. Our results provide a molecular explanation for the species selectivity of skin-derived opioid peptides., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

G protein-coupled receptors (GPCRs) are essential mediators of cellular signaling and are important targets of drug action. Of the approximately 350 nonolfactory human GPCRs, more than 100 are still considered to be 'orphans' because their endogenous ligands remain unknown. Here, we describe a unique open-source resource that allows interrogation of the druggable human GPCRome via a G protein-independent β-arrestin-recruitment assay. We validate this unique platform at more than 120 nonorphan human GPCR targets, demonstrate its utility for discovering new ligands for orphan human GPCRs and describe a method (parallel receptorome expression and screening via transcriptional output, with transcriptional activation following arrestin translocation (PRESTO-Tango)) for the simultaneous and parallel interrogation of the entire human nonolfactory GPCRome.

TLQP-21, a VGF-encoded peptide is emerging as a novel target for obesity-associated disorders. TLQP-21 is found in the sympathetic nerve terminals in the adipose tissue and targets the G-protein-coupled receptor complement-3a receptor1 (C3aR1). The mechanisms of TLQP-21-induced receptor activation remain unexplored. Here, we report that TLQP-21 is intrinsically disordered and undergoes a disorder-to-order transition, adopting an α-helical conformation upon targeting cells expressing the C3aR1. We determined that the hot spots for TLQP-21 are located at the C terminus, with mutations in the last four amino acids progressively reducing the bioactivity and, a single site mutation (R21A) or C-terminal amidation abolishing its function completely. Additionally, the human TLQP-21 sequence carrying a S20A substitution activates the human C3aR1 receptor with lower potency compared to the rodent sequence. These studies reveal the mechanism of action of TLQP-21 and provide molecular templates for designing agonists and antagonists to modulate C3aR1 functions., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Summary: We report on the development of the high-throughput screening (HTS) Navigator software to analyze and visualize the results of HTS of chemical libraries. The HTS Navigator processes output files from different plate readers' formats, computes the overall HTS matrix, automatically detects hits and has different types of baseline navigation and correction features. The software incorporates advanced cheminformatics capabilities such as chemical structure storage and visualization, fast similarity search and chemical neighborhood analysis for retrieved hits. The software is freely available for academic laboratories., Availability and Implementation: http://fourches.web.unc.edu/

Phenotypic screens can identify molecules that are at once penetrant and active on the integrated circuitry of a whole cell or organism. These advantages are offset by the need to identify the targets underlying the phenotypes. Additionally, logistical considerations limit screening for certain physiological and behavioral phenotypes to organisms such as zebrafish and C. elegans. This further raises the challenge of elucidating whether compound-target relationships found in model organisms are preserved in humans. To address these challenges we searched for compounds that affect feeding behavior in C. elegans and sought to identify their molecular mechanisms of action. Here, we applied predictive chemoinformatics to small molecules previously identified in a C. elegans phenotypic screen likely to be enriched for feeding regulatory compounds. Based on the predictions, 16 of these compounds were tested in vitro against 20 mammalian targets. Of these, nine were active, with affinities ranging from 9 nM to 10 µM. Four of these nine compounds were found to alter feeding. We then verified the in vitro findings in vivo through genetic knockdowns, the use of previously characterized compounds with high affinity for the four targets, and chemical genetic epistasis, which is the effect of combined chemical and genetic perturbations on a phenotype relative to that of each perturbation in isolation. Our findings reveal four previously unrecognized pathways that regulate feeding in C. elegans with strong parallels in mammals. Together, our study addresses three inherent challenges in phenotypic screening: the identification of the molecular targets from a phenotypic screen, the confirmation of the in vivo relevance of these targets, and the evolutionary conservation and relevance of these targets to their human orthologs., Competing Interests: The authors have declared that no competing interests exist.

G-protein coupled receptors (GPCRs) are the primary target class of currently marketed drugs, accounting for about a quarter of all drug targets of approved medicines. However, almost all the screening efforts for novel ligand discovery rely exclusively on cellular systems overexpressing the receptors. An alternative ligand discovery strategy is a fragment-based drug discovery, where low molecular weight compounds, known as fragments, are screened as initial starting points for optimization. However, the screening of fragment libraries usually employs biophysical screening methods, and as such, it has not been routinely applied to membrane proteins. We present here a surface plasmon resonance biosensor approach that enables, cell-free, label-free, fragment screening that directly measures fragment interactions with wild-type GPCRs. We exemplify the method by the discovery of novel, selective, high affinity antagonists of human β2 adrenoceptor.

A prospective, large library virtual screen against an activated β2-adrenergic receptor (β2AR) structure returned potent agonists to the exclusion of inverse-agonists, providing the first complement to the previous virtual screening campaigns against inverse-agonist-bound G protein coupled receptor (GPCR) structures, which predicted only inverse-agonists. In addition, two hits recapitulated the signaling profile of the co-crystal ligand with respect to the G protein and arrestin mediated signaling. This functional fidelity has important implications in drug design, as the ability to predict ligands with predefined signaling properties is highly desirable. However, the agonist-bound state provides an uncertain template for modeling the activated conformation of other GPCRs, as a dopamine D2 receptor (DRD2) activated model templated on the activated β2AR structure returned few hits of only marginal potency.

Colloidal aggregation is the dominant mechanism for artifactual inhibition of soluble proteins, and controls against it are now widely deployed. Conversely, investigating this mechanism for membrane-bound receptors has proven difficult. Here we investigate the activity of four well-characterized aggregators against three G protein-coupled receptors (GPCRs) recognizing peptide and protein ligands. Each of the aggregators was active at micromolar concentrations against the three GPCRs in cell-based assays. This activity could be attenuated by either centrifugation of the inhibitor stock solution or by addition of Tween-80 detergent. In the absence of agonist, the aggregators acted as inverse agonists, consistent with a direct receptor interaction. Meanwhile, several literature GPCR ligands that resemble aggregators themselves formed colloids, by both physical and enzymological tests. These observations suggest that some GPCRs may be artifactually antagonized by colloidal aggregates, an effect that merits the attention of investigators in this field.

Protein classification typically uses structural, sequence or functional similarity. Here we introduce an orthogonal method that organizes proteins by ligand similarity, focusing on the class A G-protein-coupled receptor (GPCR) protein family. Comparing a ligand-based dendrogram to a sequence-based one, we identified GPCRs that were distantly linked by sequence but were neighbors by ligand similarity. Experimental testing of the ligands predicted to link three of these new pairs confirmed the predicted association, with potencies ranging from low nanomolar to low micromolar. We also predicted hundreds of non-GPCRs closely related to GPCRs by ligand similarity and confirmed several cases experimentally. Ligand similarities among these targets may reflect the conservation of identical ligands among unrelated receptors, which signal in different time domains. Our method integrates these apparently disparate receptors into chemically coherent circuits and suggests which of these receptors may be targeted by individual ligands.

The clinical efficacy and safety of a drug is determined by its activity profile across many proteins in the proteome. However, designing drugs with a specific multi-target profile is both complex and difficult. Therefore methods to design drugs rationally a priori against profiles of several proteins would have immense value in drug discovery. Here we describe a new approach for the automated design of ligands against profiles of multiple drug targets. The method is demonstrated by the evolution of an approved acetylcholinesterase inhibitor drug into brain-penetrable ligands with either specific polypharmacology or exquisite selectivity profiles for G-protein-coupled receptors. Overall, 800 ligand-target predictions of prospectively designed ligands were tested experimentally, of which 75% were confirmed to be correct. We also demonstrate target engagement in vivo. The approach can be a useful source of drug leads when multi-target profiles are required to achieve either selectivity over other drug targets or a desired polypharmacology.

Functionally selective G protein-coupled receptor (GPCR) ligands, which differentially modulate canonical and noncanonical signaling, are extremely useful for elucidating key signal transduction pathways essential for both the therapeutic actions and side effects of drugs. However, few such ligands have been created, and very little purposeful attention has been devoted to studying what we term: "structure-functional selectivity relationships" (SFSR). We recently disclosed the first β-arrestin-biased dopamine D(2) receptor (D(2)R) agonists UNC9975 (44) and UNC9994 (36), which have robust in vivo antipsychotic drug-like activities. Here we report the first comprehensive SFSR studies focused on exploring four regions of the aripiprazole scaffold, which resulted in the discovery of these β-arrestin-biased D(2)R agonists. These studies provide a successful proof-of-concept for how functionally selective ligands can be discovered.

Telomeres are protein and DNA complexes located atchromosome ends. Telomeric DNA is composed of a double stranded region of repetitive DNA followed by single-stranded 3' extension of aG-rich sequence. Single-stranded G-rich sequencescan fold into G-quadruplex structures,and molecules that stabilize G-quadruplexes are known to inhibit the enzyme telomerase and disrupt telomere maintenance. Because telomere maintenance is required for proliferation of cancer cells, G-quadruplex stabilizers have become attractive prospects for anticancer drug discovery.However, telomere-targeting G-quadruplex ligands have yet to enter the clinic owing in part to poor pharmacokinetics and target selectivity. Increasing the pharmacophore diversity of G-quadruplex and specifically telomeric-DNA targeting agents should assist in overcoming these shortcomings. In this work, we report the identification and validation ofligands that bind telomeric DNA and induce G-quadruplex formationusing the NCI Diversity Set I, providing validation of anextremely simple, rapid and high-throughput screen using FRET technology. Hits from the screen were validated by examining telomerase inhibition and G-quadruplex inductionusing CD spectroscopy and DNA polymerase stop assays. We show that two known DNA binding molecules, ellipticine derivativeNSC 176327 (apyridocarbazole) and NSC 305831 (an antiparasitic hetero-cyclediamidine referred to as furamidine and DB75),are selective induceG-quadruplex formation in the human telomeric sequence and bind telomeric DNA quadruplexes in the absence of stabilizing monovalent cations with molar ratios(molecule: DNA)of 4:1and 1.5:1, respectively.

The 5-HT(2C) receptor is an attractive drug target in the quest for new therapeutics to treat a variety of human disorders. We have previously undertaken a structural optimization campaign that has led to some potent and moderately selective 5-HT(2C) receptor agonists. After expanding our structure-function library, we were able to combine our datasets so as to allow the design of compounds of improved selectivity and potency. We disclose herein the structural optimization of our previously reported 5-HT(2B)/5-HT(2C) agonists, which has led to the identification of a highly selective 5-HT(2C) agonist, (+)-trans-[2-(2-cyclopropylmethoxyphenyl)cyclopropyl]methylamine hydrochloride, with an EC(50) of 55 nM and no detectable agonism at the 5-HT(2B) receptor.

Elucidating the key signal transduction pathways essential for both antipsychotic efficacy and side-effect profiles is essential for developing safer and more effective therapies. Recent work has highlighted noncanonical modes of dopamine D(2) receptor (D(2)R) signaling via β-arrestins as being important for the therapeutic actions of both antipsychotic and antimanic agents. We thus sought to create unique D(2)R agonists that display signaling bias via β-arrestin-ergic signaling. Through a robust diversity-oriented modification of the scaffold represented by aripiprazole (1), we discovered UNC9975 (2), UNC0006 (3), and UNC9994 (4) as unprecedented β-arrestin-biased D(2)R ligands. These compounds also represent unprecedented β-arrestin-biased ligands for a G(i)-coupled G protein-coupled receptor (GPCR). Significantly, UNC9975, UNC0006, and UNC9994 are simultaneously antagonists of G(i)-regulated cAMP production and partial agonists for D(2)R/β-arrestin-2 interactions. Importantly, UNC9975 displayed potent antipsychotic-like activity without inducing motoric side effects in inbred C57BL/6 mice in vivo. Genetic deletion of β-arrestin-2 simultaneously attenuated the antipsychotic actions of UNC9975 and transformed it into a typical antipsychotic drug with a high propensity to induce catalepsy. Similarly, the antipsychotic-like activity displayed by UNC9994, an extremely β-arrestin-biased D(2)R agonist, in wild-type mice was completely abolished in β-arrestin-2 knockout mice. Taken together, our results suggest that β-arrestin signaling and recruitment can be simultaneously a significant contributor to antipsychotic efficacy and protective against motoric side effects. These functionally selective, β-arrestin-biased D(2)R ligands represent valuable chemical probes for further investigations of D(2)R signaling in health and disease.