133 results on '"Barak LS"'
Search Results
2. beta-Adrenergic receptor kinase 1 mediated phosphoinositide-3-kinase translocation: A process linked to receptor internalization
- Author
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Prasad SVN, Barak LS, Caron MG, Rockman HA, RAPACCIUOLO, ANTONIO, Prasad, Svn, Barak, L, Rapacciuolo, Antonio, Caron, Mg, and Rockman, Ha
- Subjects
internalization ,beta-Adrenergic receptor kinase 1 ,phosphoinositide-3-kinase translocation - Published
- 2000
3. Pleiotropic Role for GRKs and b-Arrestins in Receptor Regulation
- Author
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Ferguson, SSG, primary, Zhang, J, additional, Barak, LS, additional, and Caron, MG, additional
- Published
- 1997
- Full Text
- View/download PDF
4. Diffusion of low density lipoprotein-receptor complex on human fibroblasts
- Author
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Barak, LS and Webb, WW
- Abstract
Diffusion of the complex consisting of low density lipoprotein (LDL) bound to its receptor on the surface of human fibroblasts has been measured with the help of an intensely fluorescent, biologically active LDL derivative, dioctadecylindocarbocyanine LDL (dil(3)-LDL). Fluorescence photobleaching recovering and direct video observations of the Brownian motion of individual LDL-receptor complexes yielded diffusion coefficients for the slow diffusion on cell surfaces and fast diffusion on membrane blebs, respectively. At 10 degrees C, less that 20 percent of the LDL-receptor complex was measurably diffusible either on normal human fibroblasts GM-3348 or on LDL-receptor- internalization-defective J.D. cells GM-2408A. At 21 degrees and 28 degrees C, the diffusion fractions of approximately 75 and 60 percent, respectively, on both cell lines. The lipid analog nitrobenzoxadiazolephosphatidylcholine (NBD-PC) diffused in the GM-2408A cell membrane at 1.5x10(-8) cm(2)/sec at 22 degrees C. On blebs induced in GM-2408A cell membranes, the dil(3)-LDL receptor complex diffusion coefficient increased to approximately 10(-9) cm(2)/s, thus approaching the maximum theoretical predictions for a large protein in the viscous lipid bilayer. Cytoskeletal staining of blebs with NBD- phallacidin, a fluorescent probe specific for F-actin, indicated that loss of the bulk of the F-actin cytoskeleton accompanied the release of the natural constraints on later diffusion observed on blebs. This work shows that the internalization defect of J.D. is not due to immobilization of the LDL-receptor complex since its diffusibility is sufficient to sustain even the internalization rates observed in the native fibroblasts. Nevertheless, as with many other cell membrane receptors, the diffusion coefficient of the LDL-receptor complex is at least two orders of magnitude slower on native membrane than the viscous limit approached on cell membrane blebs where it is released from lateral constraints. However, LDL-receptor diffusion may not limit LDL internalization in normal human fibroblasts.
- Published
- 1982
- Full Text
- View/download PDF
5. Design of allosteric modulators that change GPCR G protein subtype selectivity.
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Moore MN, Person KL, Alwin A, Krusemark C, Foster N, Ray C, Inoue A, Jackson MR, Sheedlo MJ, Barak LS, Fernandez de Velasco EM, Olson SH, and Slosky LM
- Abstract
G protein-coupled receptors (GPCRs), the largest family of drug targets, can signal through 16 subtypes of Gα proteins. Biased compounds that selectively activate therapy-relevant pathways promise to be safer, more effective medications. The determinants of bias are poorly understood, however, and rationally-designed, G protein-subtype-selective compounds are lacking. Here, using the prototypical class A GPCR neurotensin receptor 1 (NTSR1), we find that small molecules binding the intracellular GPCR-transducer interface change G protein coupling by subtype-specific and predictable mechanisms, enabling rational drug design. We demonstrate that the compound SBI-553 switches NTSR1 G protein preference by acting both as a molecular bumper and a molecular glue. Structurally, SBI-553 occludes G protein binding determinants on NTSR1, promoting association with select G protein subtypes for which an alternative, shallow-binding conformation is energetically favorable. Minor modifications to the SBI-553 scaffold produce allosteric modulators with distinct G protein subtype selectivity profiles. Selectivity profiles are probe-independent, conserved across species, and translate to differences in in vivo activity. These studies demonstrate that G protein selectivity can be tailored with small changes to a single chemical scaffold targeting the receptor-transducer interface and, as this pocket is broadly conserved, present a strategy for pathway-selective drug discovery applicable to the diverse GPCR superfamily., Competing Interests: DECLARATION OF INTERESTS. US Patents 9,868,707 and 20,150,329,497 relating to the chemistry of SBI-553 and its derivatives have been issued to the Sanford Burnham Prebys Medical Research Institute (SBP) and Duke University and US Patent 10,118,902 has been issued to SBP. Patent application 63/689,904 related to the use of SBI-553 and its derivatives has been filed by Duke University.
- Published
- 2024
- Full Text
- View/download PDF
6. Nonlinear progression during the occult transition establishes cancer lethality.
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Ginzel JD, Chapman H, Sills JE, Allen EJ, Barak LS, Cardiff RD, Borowsky AD, Lyerly HK, Rogers BW, and Snyder JC
- Abstract
Cancer screening is based upon a linear model of neoplastic growth and malignant progression. Yet, historical observations suggest that malignant progression is uncoupled from growth which may explain the paradoxical increase in early-stage breast cancer detection without a dramatic reduction in metastatic burden. Here we lineage trace millions of genetically transformed field cells and thousands of screen detectable and symptomatic tumors using a cancer rainbow mouse model of HER2+ breast cancer. Transition rates from field cell to screen detectable tumor and then to symptomatic tumors were estimated from a dynamical model of tumor development. Field cells are orders of magnitude less likely to transition to a screen detectable tumor than the subsequent transition of a screen detectable tumor to a symptomatic tumor. Our model supports a critical occult transition in tumor development during which time a transformed cell becomes a bona fide neoplasm. Lineage tracing and test-by-transplantation reveals that nonlinear progression during or prior to the occult transition gives rise to nascent lethal cancers at screen detection. Simulations illustrate how occult transition rates are a critical determinant of tumor growth and malignancy in the lifetime of a host. Our data provides direct experimental evidence that cancers can deviate from the predictable linear progression model foundational to current screening paradigms.
- Published
- 2024
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7. β-arrestin-biased Allosteric Modulator of Neurotensin Receptor 1 Reduces Ethanol Drinking and Responses to Ethanol Administration in Rodents.
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Gereau GB, Zhou D, Van Voorhies K, Tyler RE, Campbell J, Murray JG, Alvarez-Pamir A, Wykoff LA, Companion MA, Jackson MR, Olson SH, Barak LS, Slosky LM, Vetreno RP, Besheer J, and McElligott ZA
- Abstract
Alcohol use disorders (AUDs) impose an enormous societal and financial burden, and world-wide, alcohol misuse is the 7
th leading cause of premature death1 . Despite this, there are currently only 3 FDA approved pharmacological treatments for the treatment of AUDs in the United States. The neurotensin (Nts) system has long been implicated in modulating behaviors associated with alcohol misuse. Recently, a novel compound, SBI-553, that biases the action of Nts receptor 1 (NTSR1) activation, has shown promise in preclinical models of psychostimulant misuse. Here we investigate the efficacy of this compound to alter ethanol-mediated behaviors in a comprehensive battery of experiments assessing ethanol consumption, behavioral responses to ethanol, sensitivity to ethanol, and ethanol metabolism. Additionally, we investigated behavior in avoidance and cognitive assays to monitor potential side effects of SBI-553. We find that SBI-553 reduces binge-like ethanol consumption in mice without altering avoidance behavior or novel object recognition. We also observe sex-dependent differences in physiological responses to sequential ethanol injections in mice. In rats, we show that SBI-553 attenuates sensitivity to the interoceptive effects of ethanol (using a Pavlovian drug discrimination task). Our data suggest that targeting NTSR1 signaling may be promising to attenuate alcohol misuse, and adds to a body of literature that suggests NTSR1 may be a common downstream target involved in the psychoactive effects of multiple reinforcing substances., Competing Interests: Conflict of interest disclosure ZAM is subcontracted by Epicypher on a project unrelated to this work DA057749. The other authors have nothing to disclose. US Patents 9,868,707 and 10,118,902 related to the chemistry of SBI-553 and its derivatives have been issued to the Sanford Burnham Prebys Medical Research Institute and Duke University (MRJ, LSB).- Published
- 2024
- Full Text
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8. Neurotensin receptor 1-biased ligand attenuates neurotensin-mediated excitation of ventral tegmental area dopamine neurons and dopamine release in the nucleus accumbens.
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Singhal SM, Zell V, Faget L, Slosky LM, Barak LS, Caron MG, Pinkerton AB, and Hnasko TS
- Subjects
- Male, Female, Animals, Mice, Mice, Inbred C57BL, Presynaptic Terminals metabolism, Presynaptic Terminals physiology, Action Potentials drug effects, Ligands, Ventral Tegmental Area metabolism, Ventral Tegmental Area physiology, Dopaminergic Neurons drug effects, Dopaminergic Neurons metabolism, Dopaminergic Neurons physiology, Nucleus Accumbens metabolism, Dopamine metabolism, Receptors, Neurotensin antagonists & inhibitors, Receptors, Neurotensin metabolism, Neurotensin metabolism, Neurotensin pharmacology, Dopamine D2 Receptor Antagonists metabolism, Dopamine D2 Receptor Antagonists pharmacology
- Abstract
Strong expression of the G protein-coupled receptor (GPCR) neurotensin receptor 1 (NTR1) in ventral tegmental area (VTA) dopamine (DA) neurons and terminals makes it an attractive target to modulate DA neuron activity and normalize DA-related pathologies. Recent studies have identified a novel class of NTR1 ligand that shows promising effects in preclinical models of addiction. A lead molecule, SBI-0654553 (SBI-553), can act as a positive allosteric modulator of NTR1 β-arrestin recruitment while simultaneously antagonizing NTR1 Gq protein signaling. Using cell-attached recordings from mouse VTA DA neurons we discovered that, unlike neurotensin (NT), SBI-553 did not independently increase spontaneous firing. Instead, SBI-553 blocked the NT-mediated increase in firing. SBI-553 also antagonized the effects of NT on dopamine D2 auto-receptor signaling, potentially through its inhibitory effects on G-protein signaling. We also measured DA release directly, using fast-scan cyclic voltammetry in the nucleus accumbens and observed antagonist effects of SBI-553 on an NT-induced increase in DA release. Further, in vivo administration of SBI-553 did not notably change basal or cocaine-evoked DA release measured in NAc using fiber photometry. Overall, these results indicate that SBI-553 blunts NT's effects on spontaneous DA neuron firing, D2 auto-receptor function, and DA release, without independently affecting these measures. In the presence of NT, SBI-553 has an inhibitory effect on mesolimbic DA activity, which could contribute to its efficacy in animal models of psychostimulant use., Competing Interests: Declaration of competing interest US patents 9 868 707 and 10 118 902 relating to the chemistry of SBI-553 and its derivatives have been issued to the Sanford Burnham Prebys Medical Research Institute (A.B·P.) and Duke University (M.G.C., L.S·B.)., (Published by Elsevier Ltd.)
- Published
- 2023
- Full Text
- View/download PDF
9. Neurotensin Receptor Allosterism Revealed in Complex with a Biased Allosteric Modulator.
- Author
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Krumm BE, DiBerto JF, Olsen RHJ, Kang HJ, Slocum ST, Zhang S, Strachan RT, Huang XP, Slosky LM, Pinkerton AB, Barak LS, Caron MG, Kenakin T, Fay JF, and Roth BL
- Subjects
- Signal Transduction, Peptides metabolism, beta-Arrestins metabolism, Receptors, Neurotensin chemistry, Receptors, Neurotensin metabolism, Neurotensin metabolism
- Abstract
The NTSR1 neurotensin receptor (NTSR1) is a G protein-coupled receptor (GPCR) found in the brain and peripheral tissues with neurotensin (NTS) being its endogenous peptide ligand. In the brain, NTS modulates dopamine neuronal activity, induces opioid-independent analgesia, and regulates food intake. Recent studies indicate that biasing NTSR1 toward β-arrestin signaling can attenuate the actions of psychostimulants and other drugs of abuse. Here, we provide the cryoEM structures of NTSR1 ternary complexes with heterotrimeric Gq and GoA with and without the brain-penetrant small-molecule SBI-553. In functional studies, we discovered that SBI-553 displays complex allosteric actions exemplified by negative allosteric modulation for G proteins that are Gα subunit selective and positive allosteric modulation and agonism for β-arrestin translocation at NTSR1. Detailed structural analysis of the allosteric binding site illuminated the structural determinants for biased allosteric modulation of SBI-553 on NTSR1.
- Published
- 2023
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10. Ghrelin receptor signaling in health and disease: a biased view.
- Author
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Gross JD, Zhou Y, Barak LS, and Caron MG
- Subjects
- Humans, Ghrelin metabolism, Receptors, Ghrelin genetics, Receptors, Ghrelin metabolism, Signal Transduction physiology
- Abstract
As allosteric complexes, G-protein-coupled receptors (GPCRs) respond to extracellular stimuli and pleiotropically couple to intracellular transducers to elicit signaling pathway-dependent effects in a process known as biased signaling or functional selectivity. One such GPCR, the ghrelin receptor (GHSR
1a ), has a crucial role in restoring and maintaining metabolic homeostasis during disrupted energy balance. Thus, pharmacological modulation of GHSR1a bias could offer a promising strategy to treat several metabolism-based disorders. Here, we summarize current evidence supporting GHSR1a functional selectivity in vivo and highlight recent structural data. We propose that precise determinations of GHSR1a molecular pharmacology and pathway-specific physiological effects will enable discovery of GHSR1a drugs with tailored signaling profiles, thereby providing safer and more effective treatments for metabolic diseases., Competing Interests: Declaration of interests A provisional patent application has been deposited with the USPTO by Duke University related to the N8279/NCATS-SM8864 drug series. A decision on whether to file a formal application will be decided upon in 2023., (Copyright © 2022 Elsevier Ltd. All rights reserved.)- Published
- 2023
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11. Establishment of multi-stage intravenous self-administration paradigms in mice.
- Author
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Slosky LM, Pires A, Bai Y, Clark NB, Hauser ER, Gross JD, Porkka F, Zhou Y, Chen X, Pogorelov VM, Toth K, Wetsel WC, Barak LS, and Caron MG
- Subjects
- Mice, Animals, Mice, Inbred C57BL, Administration, Intravenous, Self Administration, Models, Animal, Drug-Seeking Behavior
- Abstract
Genetically tractable animal models provide needed strategies to resolve the biological basis of drug addiction. Intravenous self-administration (IVSA) is the gold standard for modeling psychostimulant and opioid addiction in animals, but technical limitations have precluded the widespread use of IVSA in mice. Here, we describe IVSA paradigms for mice that capture the multi-stage nature of the disorder and permit predictive modeling. In these paradigms, C57BL/6J mice with long-standing indwelling jugular catheters engaged in cocaine- or remifentanil-associated lever responding that was fixed ratio-dependent, dose-dependent, extinguished by withholding the drug, and reinstated by the presentation of drug-paired cues. The application of multivariate analysis suggested that drug taking in both paradigms was a function of two latent variables we termed incentive motivation and discriminative control. Machine learning revealed that vulnerability to drug seeking and relapse were predicted by a mouse's a priori response to novelty, sensitivity to drug-induced locomotion, and drug-taking behavior. The application of these behavioral and statistical-analysis approaches to genetically-engineered mice will facilitate the identification of neural circuits driving addiction susceptibility and relapse and focused therapeutic development., (© 2022. The Author(s).)
- Published
- 2022
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12. Discovery of a functionally selective ghrelin receptor (GHSR 1a ) ligand for modulating brain dopamine.
- Author
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Gross JD, Kim DW, Zhou Y, Jansen D, Slosky LM, Clark NB, Ray CR, Hu X, Southall N, Wang A, Xu X, Barnaeva E, Wetsel WC, Ferrer M, Marugan JJ, Caron MG, Barak LS, and Toth K
- Subjects
- Animals, Dopamine genetics, GTP-Binding Protein alpha Subunits, Gq-G11 genetics, Male, Mice, Mice, Knockout, Receptors, Ghrelin genetics, Brain metabolism, Dopamine metabolism, GTP-Binding Protein alpha Subunits, Gq-G11 metabolism, Receptors, Ghrelin metabolism
- Abstract
SignificanceThe modulation of growth hormone secretagogue receptor-1a (GHSR
1a ) signaling is a promising strategy for treating brain conditions of metabolism, aging, and addiction. GHSR1a activation results in pleiotropic physiological outcomes through distinct and pharmacologically separable G protein- and β-arrestin (βarr)-dependent signaling pathways. Thus, pathway-selective modulation can enable improved pharmacotherapeutics that can promote therapeutic efficacy while mitigating side effects. Here, we describe the discovery of a brain-penetrant small molecule, N8279 (NCATS-SM8864), that biases GHSR1a conformations toward Gαq activation and reduces aberrant dopaminergic behavior in mice. N8279 represents a promising chemical scaffold to advance the development of better treatments for GHSR1a -related brain disorders involving the pathological dysregulation of dopamine.- Published
- 2022
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13. HER2 Isoforms Uniquely Program Intratumor Heterogeneity and Predetermine Breast Cancer Trajectories During the Occult Tumorigenic Phase.
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Ginzel JD, Acharya CR, Lubkov V, Mori H, Boone PG, Rochelle LK, Roberts WL, Everitt JI, Hartman ZC, Crosby EJ, Barak LS, Caron MG, Chen JQ, Hubbard NE, Cardiff RD, Borowsky AD, Lyerly HK, and Snyder JC
- Subjects
- Animals, Female, Gene Expression Regulation, Neoplastic genetics, Mice, Mice, Knockout, Tumor Microenvironment genetics, Breast Neoplasms genetics, Carcinogenesis genetics, Protein Isoforms genetics, Receptor, ErbB-2 genetics
- Abstract
HER2-positive breast cancers are among the most heterogeneous breast cancer subtypes. The early amplification of HER2 and its known oncogenic isoforms provide a plausible mechanism in which distinct programs of tumor heterogeneity could be traced to the initial oncogenic event. Here a Cancer rainbow mouse simultaneously expressing fluorescently barcoded wildtype (
WT HER2), exon-16 null (d16 HER2), and N-terminally truncated (p95 HER2) HER2 isoforms is used to trace tumorigenesis from initiation to invasion. Tumorigenesis was visualized using whole-gland fluorescent lineage tracing and single-cell molecular pathology. We demonstrate that within weeks of expression, morphologic aberrations were already present and unique to each HER2 isoform. AlthoughWT HER2 cells were abundant throughout the mammary ducts, detectable lesions were exceptionally rare. In contrast,d16 HER2 andp95 HER2 induced rapid tumor development.d16 HER2 incited homogenous and proliferative luminal-like lesions which infrequently progressed to invasive phenotypes whereasp95 HER2 lesions were heterogenous and invasive at the smallest detectable stage. Distinct cancer trajectories were observed ford16 HER2 andp95 HER2 tumors as evidenced by oncogene-dependent changes in epithelial specification and the tumor microenvironment. These data provide direct experimental evidence that intratumor heterogeneity programs begin very early and well in advance of screen or clinically detectable breast cancer. IMPLICATIONS: Although all HER2 breast cancers are treated equally, we show a mechanism by which clinically undetected HER2 isoforms program heterogenous cancer phenotypes through biased epithelial specification and adaptations within the tumor microenvironment., (©2021 American Association for Cancer Research.)- Published
- 2021
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14. Biased Allosteric Modulators: New Frontiers in GPCR Drug Discovery.
- Author
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Slosky LM, Caron MG, and Barak LS
- Subjects
- Allosteric Regulation, Ligands, Signal Transduction, Drug Discovery, Receptors, G-Protein-Coupled
- Abstract
G protein-coupled receptors (GPCRs) are the largest class of cell surface receptors in the genome and the most successful family of targets of FDA-approved drugs. New frontiers in GPCR drug discovery remain, however, as achieving receptor subtype selectivity and controlling off- and on-target side effects are not always possible with classic agonist and antagonist ligands. These challenges may be overcome by focusing development efforts on allosteric ligands that confer signaling bias. Biased allosteric modulators (BAMs) are an emerging class of GPCR ligands that engage less well-conserved regulatory motifs outside the orthosteric pocket and exert pathway-specific effects on receptor signaling. The unique ways that BAMs texturize receptor signaling present opportunities to fine-tune physiology and develop safer, more selective therapeutics. Here, we provide a conceptual framework for understanding the pharmacology of BAMs, explore their therapeutic potential, and discuss strategies for their discovery., Competing Interests: Declaration of Interests US patents 9 868 707 and 10 118 902 relating to the chemistry of ML314, SBI-553, and their derivatives have been issued to the Sanford Burnham Prebys Medical Research Institute and Duke University (M.G.C. and L.S.B.)., (Copyright © 2021 Elsevier Ltd. All rights reserved.)
- Published
- 2021
- Full Text
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15. β-Arrestin-Biased Allosteric Modulator of NTSR1 Selectively Attenuates Addictive Behaviors.
- Author
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Slosky LM, Bai Y, Toth K, Ray C, Rochelle LK, Badea A, Chandrasekhar R, Pogorelov VM, Abraham DM, Atluri N, Peddibhotla S, Hedrick MP, Hershberger P, Maloney P, Yuan H, Li Z, Wetsel WC, Pinkerton AB, Barak LS, and Caron MG
- Subjects
- Allosteric Regulation drug effects, Allosteric Regulation physiology, Animals, Behavior, Addictive drug therapy, Cell Line, Female, HEK293 Cells, Humans, Male, Mice, Mice, Inbred C57BL, Models, Animal, Receptors, G-Protein-Coupled metabolism, Signal Transduction drug effects, Signal Transduction physiology, Small Molecule Libraries pharmacology, Behavior, Addictive metabolism, Receptors, Neurotensin metabolism, beta-Arrestins metabolism
- Abstract
Small molecule neurotensin receptor 1 (NTSR1) agonists have been pursued for more than 40 years as potential therapeutics for psychiatric disorders, including drug addiction. Clinical development of NTSR1 agonists has, however, been precluded by their severe side effects. NTSR1, a G protein-coupled receptor (GPCR), signals through the canonical activation of G proteins and engages β-arrestins to mediate distinct cellular signaling events. Here, we characterize the allosteric NTSR1 modulator SBI-553. This small molecule not only acts as a β-arrestin-biased agonist but also extends profound β-arrestin bias to the endogenous ligand by selectively antagonizing G protein signaling. SBI-553 shows efficacy in animal models of psychostimulant abuse, including cocaine self-administration, without the side effects characteristic of balanced NTSR1 agonism. These findings indicate that NTSR1 G protein and β-arrestin activation produce discrete and separable physiological effects, thus providing a strategy to develop safer GPCR-targeting therapeutics with more directed pharmacological action., Competing Interests: Declaration of Interests US Patents 9,868,707 and 10,118,902 relating to the chemistry of ML314, SBI-553, and their derivatives have been issued to the Sanford Burnham Prebys Medical Research Institute and Duke University (A.B.P., M.P.H., P.M., S.P., P.M., L.S.B., and M.G.C.)., (Copyright © 2020 Elsevier Inc. All rights reserved.)
- Published
- 2020
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16. A cancer rainbow mouse for visualizing the functional genomics of oncogenic clonal expansion.
- Author
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Boone PG, Rochelle LK, Ginzel JD, Lubkov V, Roberts WL, Nicholls PJ, Bock C, Flowers ML, von Furstenberg RJ, Stripp BR, Agarwal P, Borowsky AD, Cardiff RD, Barak LS, Caron MG, Lyerly HK, and Snyder JC
- Subjects
- Animals, Carcinogenesis, Cell Proliferation, Colonic Neoplasms metabolism, Colonic Neoplasms physiopathology, Humans, Mice, Mutation, Neoplastic Stem Cells metabolism, Oncogenes, Thrombospondins genetics, Thrombospondins metabolism, Colonic Neoplasms genetics, Disease Models, Animal, Neoplastic Stem Cells cytology
- Abstract
Field cancerization is a premalignant process marked by clones of oncogenic mutations spreading through the epithelium. The timescales of intestinal field cancerization can be variable and the mechanisms driving the rapid spread of oncogenic clones are unknown. Here we use a Cancer rainbow (Crainbow) modelling system for fluorescently barcoding somatic mutations and directly visualizing the clonal expansion and spread of oncogenes. Crainbow shows that mutations of ß-catenin (Ctnnb1) within the intestinal stem cell results in widespread expansion of oncogenes during perinatal development but not in adults. In contrast, mutations that extrinsically disrupt the stem cell microenvironment can spread in adult intestine without delay. We observe the rapid spread of premalignant clones in Crainbow mice expressing oncogenic Rspondin-3 (RSPO3), which occurs by increasing crypt fission and inhibiting crypt fixation. Crainbow modelling provides insight into how somatic mutations rapidly spread and a plausible mechanism for predetermining the intratumor heterogeneity found in colon cancers.
- Published
- 2019
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17. Discovery of β-Arrestin Biased, Orally Bioavailable, and CNS Penetrant Neurotensin Receptor 1 (NTR1) Allosteric Modulators.
- Author
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Pinkerton AB, Peddibhotla S, Yamamoto F, Slosky LM, Bai Y, Maloney P, Hershberger P, Hedrick MP, Falter B, Ardecky RJ, Smith LH, Chung TDY, Jackson MR, Caron MG, and Barak LS
- Subjects
- Administration, Oral, Allosteric Regulation drug effects, Animals, Biological Availability, Central Nervous System Diseases metabolism, Dopamine Plasma Membrane Transport Proteins deficiency, Dopamine Plasma Membrane Transport Proteins metabolism, Dose-Response Relationship, Drug, Female, Locomotion drug effects, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Molecular Structure, Quinazolines administration & dosage, Quinazolines chemistry, Rats, Receptors, Neurotensin metabolism, Structure-Activity Relationship, beta-Arrestins administration & dosage, beta-Arrestins chemistry, Central Nervous System Diseases drug therapy, Drug Discovery, Quinazolines pharmacology, Receptors, Neurotensin antagonists & inhibitors, beta-Arrestins pharmacology
- Abstract
Neurotensin receptor 1 (NTR1) is a G protein coupled receptor that is widely expressed throughout the central nervous system where it acts as a neuromodulator. Neurotensin receptors have been implicated in a wide variety of CNS disorders, but despite extensive efforts to develop small molecule ligands there are few reports of such compounds. Herein we describe the optimization of a quinazoline based lead to give 18 (SBI-553), a potent and brain penetrant NTR1 allosteric modulator.
- Published
- 2019
- Full Text
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18. Encoding the β-Arrestin Trafficking Fate of Ghrelin Receptor GHSR1a: C-Tail-Independent Molecular Determinants in GPCRs.
- Author
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Toth K, Nagi K, Slosky LM, Rochelle L, Ray C, Kaur S, Shenoy SK, Caron MG, and Barak LS
- Abstract
G-protein-coupled receptors (GPCRs) can bias signaling through distinct biochemical pathways that originate from G-protein/receptor and β-arrestin/receptor complexes. Receptor conformations supporting β-arrestin engagement depend on multiple receptor determinants. Using ghrelin receptor GHR1a, we demonstrate by bioluminescence resonance energy transfer and fluorescence microscopy a critical role for its second intracellular loop 2 (ICL2) domain in stabilizing β-arrestin/GHSR1a core interactions and determining receptor trafficking fate. We validate our findings in ICL2 gain- and loss-of-function experiments assessing β-arrestin and ubiquitin-dependent internalization of the CC chemokine receptor, CCR1. Like all CC and CXC subfamily chemokine receptors, CCR1 lacks a critical proline residue found in the ICL2 consensus domain of rhodopsin-family GPCRs. Our study indicates that ICL2, C-tail determinants, and the orthosteric binding pocket that regulates β-arrestin/receptor complex stability are sufficient to encode a broad repertoire of the trafficking fates observed for rhodopsin-family GPCRs, suggesting they provide the essential elements for regulating a large fraction of β-arrestin signaling bias., Competing Interests: The authors declare no competing financial interest., (Copyright © 2019 American Chemical Society.)
- Published
- 2019
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19. A Brief History of the β-Arrestins.
- Author
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Caron MG and Barak LS
- Subjects
- Animals, History, 20th Century, Humans, beta-Arrestins therapeutic use, beta-Arrestins history, beta-Arrestins metabolism
- Abstract
Arrestins have now been implicated in the actions of virtually every G protein-coupled receptor (GPCR) for which they have been examined. Originally discovered for their role in the turnoff of visual perception, their newly discovered pleotropic functions in the cellular and physiological actions of GPCRs not only illuminate new mechanisms of signal transduction but also offer new avenues for therapeutic utility. Below, in this introductory chapter, we provide a short historical description and synopsis of how arrestins conceptually became associated with the function of GPCRs.
- Published
- 2019
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20. Ghrelin receptor antagonism of hyperlocomotion in cocaine-sensitized mice requires βarrestin-2.
- Author
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Toth K, Slosky LM, Pack TF, Urs NM, Boone P, Mao L, Abraham D, Caron MG, and Barak LS
- Subjects
- Animals, Cell Line, Tumor, Central Nervous System Agents, Dopaminergic Neurons drug effects, Dopaminergic Neurons metabolism, Female, Ghrelin metabolism, HEK293 Cells, Humans, Male, Mice, Inbred C57BL, Mice, Knockout, Motor Activity physiology, Piperidines pharmacology, Quinazolinones pharmacology, Receptors, Ghrelin metabolism, beta-Arrestin 2 genetics, Cocaine pharmacology, Cocaine-Related Disorders metabolism, Dopamine Uptake Inhibitors pharmacology, Motor Activity drug effects, Receptors, Ghrelin antagonists & inhibitors, beta-Arrestin 2 metabolism
- Abstract
The "brain-gut" peptide ghrelin, which mediates food-seeking behaviors, is recognized as a very strong endogenous modulator of dopamine (DA) signaling. Ghrelin binds the G protein-coupled receptor GHSR1a, and administration of ghrelin increases the rewarding properties of psychostimulants while ghrelin receptor antagonists decrease them. In addition, the GHSR1a signals through βarrestin-2 to regulate actin/stress fiber rearrangement, suggesting βarrestin-2 participation in the regulation of actin-mediated synaptic plasticity for addictive substances like cocaine. The effects of ghrelin receptor ligands on reward strongly suggest that modulation of ghrelin signaling could provide an effective strategy to ameliorate undesirable behaviors arising from addiction. To investigate this possibility, we tested the effects of ghrelin receptor antagonism in a cocaine behavioral sensitization paradigm using DA neuron-specific βarrestin-2 KO mice. Our results show that these mice sensitize to cocaine as well as wild-type littermates. The βarrestin-2 KO mice, however, no longer respond to the locomotor attenuating effects of the GHSR1a antagonist YIL781. The data presented here suggest that the separate stages of addictive behavior differ in their requirements for βarrestin-2 and show that pharmacological inhibition of βarrestin-2 function through GHSR1a antagonism is not equivalent to the loss of βarrestin-2 function achieved by genetic ablation. These data support targeting GHSR1a signaling in addiction therapy but indicate that using signaling biased compounds that modulate βarrestin-2 activity differentially from G protein activity may be required., (© 2017 Wiley Periodicals, Inc.)
- Published
- 2018
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21. Design, synthesis and biological evaluation of GPR55 agonists.
- Author
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Fakhouri L, Cook CD, Al-Huniti MH, Console-Bram LM, Hurst DP, Spano MBS, Nasrallah DJ, Caron MG, Barak LS, Reggio PH, Abood ME, and Croatt MP
- Subjects
- Dose-Response Relationship, Drug, Humans, Molecular Structure, Receptors, Cannabinoid, Structure-Activity Relationship, Thiourea analogs & derivatives, Thiourea chemical synthesis, Drug Design, Receptors, G-Protein-Coupled agonists, Thiourea pharmacology
- Abstract
GPR55, a G protein-coupled receptor, is an attractive target to alleviate inflammatory and neuropathic pain and treat osteoporosis and cancer. Identifying a potent and selective ligand will aid to further establish the specific physiological roles and pharmacology of the receptor. Towards this goal, a targeted library of 22 compounds was synthesized in a modular fashion to obtain structure-activity relationship information. The general route consisted of coupling a variety of p-aminophenyl sulfonamides to isothiocyanates to form acylthioureas. For the synthesis of a known naphthyl ethyl alcohol motif, route modification led to a shorter and more efficient process. The 22 analogues were analyzed for their ability to serve as agonists at GPR55 and valuable information for both ends of the molecule was ascertained., (Copyright © 2017 Elsevier Ltd. All rights reserved.)
- Published
- 2017
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22. Protamine is an antagonist of apelin receptor, and its activity is reversed by heparin.
- Author
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Le Gonidec S, Chaves-Almagro C, Bai Y, Kang HJ, Smith A, Wanecq E, Huang XP, Prats H, Knibiehler B, Roth BL, Barak LS, Caron MG, Valet P, Audigier Y, and Masri B
- Subjects
- Animals, Apelin Receptors, Cell Line, Tumor, Female, Gene Expression Regulation drug effects, Gene Expression Regulation physiology, HEK293 Cells, Humans, Male, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Receptors, G-Protein-Coupled genetics, Receptors, G-Protein-Coupled metabolism, Heparin pharmacology, Protamines pharmacology, Receptors, G-Protein-Coupled antagonists & inhibitors
- Abstract
Apelin signaling plays an important role during embryo development and regulates angiogenesis, cardiovascular activity, and energy metabolism in adulthood. Overexpression and hyperactivity of this signaling pathway is observed in various pathologic states, such as cardiovascular diseases and cancer, which highlights the importance of inhibiting apelin receptor (APJ); therefore, we developed a cell-based screening assay that uses fluorescence microscopy to identify APJ antagonists. This approach led us to identify the U.S. Food and Drug Administration-approved compound protamine-already used clinically after cardiac surgery-as an agent to bind to heparin and thereby reverse its anticlotting activity. Protamine displays a 390-nM affinity for APJ and behaves as a full antagonist with regard to G protein and β-arrestin-dependent intracellular signaling. Ex vivo and in vivo , protamine abolishes well-known apelin effects, such as angiogenesis, glucose tolerance, and vasodilatation. Remarkably, protamine antagonist activity is fully reversed by heparin treatment both in vitro and in vivo Thus, our results demonstrate a new pharmacologic property of protamine-blockade of APJ-that could explain some adverse effects observed in protamine-treated patients. Moreover, our data reveal that the established antiangiogenic activity of protamine would rely on APJ antagonism.-Le Gonidec, S., Chaves-Almagro, C., Bai, Y., Kang, H. J., Smith, A., Wanecq, E., Huang, X.-P., Prats, H., Knibiehler, B., Roth, B. L., Barak, L. S., Caron, M. G., Valet, P., Audigier, Y., Masri, B. Protamine is an antagonist of apelin receptor, and its activity is reversed by heparin., (© FASEB.)
- Published
- 2017
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23. Inhibiting clathrin-mediated endocytosis of the leucine-rich G protein-coupled receptor-5 diminishes cell fitness.
- Author
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Snyder JC, Rochelle LK, Ray C, Pack TF, Bock CB, Lubkov V, Lyerly HK, Waggoner AS, Barak LS, and Caron MG
- Subjects
- Adenosine Triphosphatases chemistry, Animals, Cell Line, Tumor, Cell Lineage, Cell Proliferation, Dioxolanes chemistry, Epithelium metabolism, Female, Homeostasis, Humans, Lignans chemistry, Mice, Mice, Inbred C57BL, Protein Isoforms, Rats, Stem Cells cytology, Stochastic Processes, Wnt Signaling Pathway, Clathrin chemistry, Endocytosis, Leucine chemistry, Receptors, G-Protein-Coupled chemistry
- Abstract
The leucine-rich G protein-coupled receptor-5 (LGR5) is expressed in adult tissue stem cells of many epithelia, and its overexpression is negatively correlated with cancer prognosis. LGR5 potentiates WNT/β-catenin signaling through its unique constitutive internalization property that clears negative regulators of the WNT-receptor complex from the membrane. However, both the mechanism and physiological relevance of LGR5 internalization are unclear. Therefore, a natural product library was screened to discover LGR5 internalization inhibitors and gain mechanistic insight into LGR5 internalization. The plant lignan justicidin B blocked the constitutive internalization of LGR5. Justicidin B is structurally similar to more potent vacuolar-type H
+ -ATPase inhibitors, which all inhibited LGR5 internalization by blocking clathrin-mediated endocytosis. We then tested the physiological relevance of LGR5 internalization blockade in vivo A LGR5-rainbow (LBOW) mouse line was engineered to express three different LGR5 isoforms along with unique fluorescent protein lineage reporters in the same mouse. In this manner, the effects of each isoform on cell fate can be simultaneously assessed through simple fluorescent imaging for each lineage reporter. LBOW mice express three different forms of LGR5, a wild-type form that constitutively internalizes and two mutant forms whose internalization properties have been compromised by genetic perturbations within the carboxyl-terminal tail. LBOW was activated in the intestinal epithelium, and a year-long lineage-tracing course revealed that genetic blockade of LGR5 internalization diminished cell fitness. Together these data provide proof-of-concept genetic evidence that blocking the clathrin-mediated endocytosis of LGR5 could be used to pharmacologically control cell behavior., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)- Published
- 2017
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24. Probing the Allosteric Role of the α5 Subunit of α3β4α5 Nicotinic Acetylcholine Receptors by Functionally Selective Modulators and Ligands.
- Author
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Ray C, Soderblom EJ, Bai Y, Carroll FI, Caron MG, and Barak LS
- Subjects
- Allosteric Regulation, Calcium metabolism, Electrophoresis, Polyacrylamide Gel, HEK293 Cells, Humans, Ligands, Receptors, Nicotinic metabolism
- Abstract
Nicotinic acetylcholine receptors regulate the nicotine dependence encountered with cigarette smoking, and this has stimulated a search for drugs binding the responsible receptor subtypes. Studies link a gene cluster encoding for α3β4α5-D398N nicotinic acetylcholine receptors to lung cancer risk as well as link a second mutation in this cluster to an increased risk for nicotine dependence. However, there are currently no recognized drugs for discriminating α3β4α5 signaling. In this study, we describe the development of homogeneous HEK-293 cell clones of α3β4 and α3β4α5 receptors appropriate for drug screening and characterizing biochemical and pharmacological properties of incorporated α5 subunits. Clones were assessed for plasma membrane expression of the individual receptor subunits by mass spectrometry and immunochemistry, and their calcium flux was measured in the presence of a library of kinase inhibitors and a focused library of acetylcholine receptor ligands. We demonstrated an incorporation of two α3 subunits in approximately 98% of plasma membrane receptor pentamers, indicating a 2/3 subunit expression ratio of α3 to β4 alone or to coexpressed β4 and α5. With prolonged nicotine exposure, the plasma membrane expression of receptors with and without incorporated α5 increased. Whereas α5 subunit expression decreased the cell calcium response to nicotine and reduced plasma membrane receptor number, it partially protected receptors from nicotine mediated desensitization. Hit compounds from both libraries suggest the α5 and α5-D398N subunits allosterically modify the behavior of nicotine at the parent α3β4 nicotinic acetylcholine receptor. These studies identify pharmacological tools from two distinct classes of drugs, antagonists and modifiers that are α5 and α5-D398N subtype selective that provide a means to characterize the role of the CHRNA5/A3/B4 gene cluster in smoking and cancer.
- Published
- 2017
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25. Benzimidazole inhibitors from the Niclosamide chemotype inhibit Wnt/β-catenin signaling with selectivity over effects on ATP homeostasis.
- Author
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Mook RA Jr, Ren XR, Wang J, Piao H, Barak LS, Kim Lyerly H, and Chen W
- Subjects
- Benzimidazoles chemistry, Cell Line, Tumor, HEK293 Cells, Homeostasis, Humans, Niclosamide chemistry, Structure-Activity Relationship, Adenosine Triphosphate metabolism, Benzimidazoles pharmacology, Niclosamide pharmacology, Signal Transduction drug effects, Wnt Proteins metabolism, beta Catenin metabolism
- Abstract
The Wnt signaling pathway plays a key role in organ and tissue homeostasis, and when dysregulated, can become a major underlying mechanism of disease, particularly cancer. We reported previously that the anthelmintic drug Niclosamide inhibits Wnt/β-catenin signaling and suppresses colon cancer cell growth in vitro and in vivo. To define Niclosamide's mechanism of Wnt/β-catenin inhibition, and to improve its selectivity and pharmacokinetic properties as an anticancer treatment, we designed a novel class of benzimidazole inhibitors of Wnt/β-catenin signaling based on SAR studies of the Niclosamide salicylanilide chemotype. Niclosamide has multiple biological activities. To address selectivity in our design, we interrogated a protonophore SAR model and used the principle of conformational restriction to identify novel Wnt/β-catenin inhibitors with less effect on ATP cellular homeostasis. These studies led to the identification of 4-chloro-2-(5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl) phenol (4) and related derivatives with greater selectivity for Wnt/β-catenin signaling inhibition vs. differential effects on cellular ATP homeostasis. This is the first report that the Wnt signaling inhibitory activity of Niclosamide can be translated into a new chemical class and to show that its effects on ATP homeostasis can be separated from its inhibitory effects on Wnt signaling. These compounds could be useful tools to elucidate the mechanism of Niclosamide's inhibition of Wnt signaling, and aid the discovery of inhibitors with improved pharmacologic properties to treat cancer and diseases in which Niclosamide has important biological activity., (Copyright © 2017 Elsevier Ltd. All rights reserved.)
- Published
- 2017
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26. ML314: A Biased Neurotensin Receptor Ligand for Methamphetamine Abuse.
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Barak LS, Bai Y, Peterson S, Evron T, Urs NM, Peddibhotla S, Hedrick MP, Hershberger P, Maloney PR, Chung TD, Rodriguiz RM, Wetsel WC, Thomas JB, Hanson GR, Pinkerton AB, and Caron MG
- Subjects
- Allosteric Regulation, Animals, Dopamine Plasma Membrane Transport Proteins genetics, Ligands, Locomotion drug effects, Mice, Mice, Inbred C57BL, Mice, Knockout, Amphetamine-Related Disorders metabolism, Methamphetamine adverse effects, Piperazines metabolism, Quinazolines metabolism, Receptors, Neurotensin metabolism
- Abstract
Pharmacological treatment for methamphetamine addiction will provide important societal benefits. Neurotensin receptor NTR1 and dopamine receptor distributions coincide in brain areas regulating methamphetamine-associated reward, and neurotensin peptides produce behaviors opposing psychostimulants. Therefore, undesirable methamphetamine-associated activities should be treatable with druggable NTR1 agonists, but no such FDA-approved therapeutics exist. We address this limitation with proof-of-concept data for ML314, a small-molecule, brain penetrant, β-arrestin biased, NTR1 agonist. ML314 attenuates amphetamine-like hyperlocomotion in dopamine transporter knockout mice, and in C57BL/6J mice it attenuates methamphetamine-induced hyperlocomotion, potentiates the psychostimulant inhibitory effects of a ghrelin antagonist, and reduces methamphetamine-associated conditioned place preference. In rats, ML314 blocks methamphetamine self-administration. ML314 acts as an allosteric enhancer of endogenous neurotensin, unmasking stoichiometric numbers of hidden NTR1 binding sites in transfected-cell membranes or mouse striatal membranes, while additionally supporting NTR1 endocytosis in cells in the absence of NT peptide. These results indicate ML314 is a viable, preclinical lead for methamphetamine abuse treatment and support an allosteric model of G protein-coupled receptor signaling.
- Published
- 2016
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27. Design, synthesis, and analysis of antagonists of GPR55: Piperidine-substituted 1,3,4-oxadiazol-2-ones.
- Author
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Meza-Aviña ME, Lingerfelt MA, Console-Bram LM, Gamage TF, Sharir H, Gettys KE, Hurst DP, Kotsikorou E, Shore DM, Caron MG, Rao N, Barak LS, Abood ME, Reggio PH, and Croatt MP
- Subjects
- Animals, Arrestins metabolism, CHO Cells, Cricetulus, Humans, Molecular Docking Simulation, Oxadiazoles chemical synthesis, Piperidines chemical synthesis, Receptors, Cannabinoid, Receptors, G-Protein-Coupled metabolism, Structure-Activity Relationship, beta-Arrestins, Drug Design, Oxadiazoles chemistry, Oxadiazoles pharmacology, Piperidines chemistry, Piperidines pharmacology, Receptors, G-Protein-Coupled antagonists & inhibitors
- Abstract
A series of 1,3,4-oxadiazol-2-ones was synthesized and tested for activity as antagonists at GPR55 in cellular beta-arrestin redistribution assays. The synthesis was designed to be modular in nature so that a sufficient number of analogues could be rapidly accessed to explore initial structure-activity relationships. The design of analogues was guided by the docking of potential compounds into a model of the inactive form of GPR55. The results of the assays were used to learn more about the binding pocket of GPR55. With this oxadiazolone scaffold, it was determined that modification of the aryl group adjacent to the oxadiazolone ring was often detrimental and that the distal cyclopropane was beneficial for activity. These results will guide further exploration of this receptor., (Copyright © 2016 Elsevier Ltd. All rights reserved.)
- Published
- 2016
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28. A rapid and affordable screening platform for membrane protein trafficking.
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Snyder JC, Pack TF, Rochelle LK, Chakraborty SK, Zhang M, Eaton AW, Bai Y, Ernst LA, Barak LS, Waggoner AS, and Caron MG
- Subjects
- Drug Discovery economics, HEK293 Cells, High-Throughput Screening Assays economics, Humans, Protein Transport, Reproducibility of Results, Drug Discovery methods, High-Throughput Screening Assays methods, Membrane Proteins metabolism
- Abstract
Background: Membrane proteins regulate a diversity of physiological processes and are the most successful class of targets in drug discovery. However, the number of targets adequately explored in chemical space and the limited resources available for screening are significant problems shared by drug-discovery centers and small laboratories. Therefore, a low-cost and universally applicable screen for membrane protein trafficking was developed., Results: This high-throughput screen (HTS), termed IRFAP-HTS, utilizes the recently described MarsCy1-fluorogen activating protein and the near-infrared and membrane impermeant fluorogen SCi1. The cell surface expression of MarsCy1 epitope-tagged receptors can be visualized by simple addition of SCi1. User-friendly, rapid, and quantitative detection occurs on a standard infrared western-blotting scanner. The reliability and robustness of IRFAP-HTS was validated by confirming human vasopressin-2 receptor and dopamine receptor-2 trafficking in response to agonist or antagonist. The IRFAP-HTS screen was deployed against the leucine-rich G protein-coupled receptor-5 (Lgr5). Lgr5 is expressed in stem cells, modulates Wnt/ß-catenin signaling, and is therefore a promising drug target. However, small molecule modulators have yet to be reported. The constitutive internalization of Lgr5 appears to be one primary mode through which its function is regulated. Therefore, IRFAP-HTS was utilized to screen 11,258 FDA-approved and drug-like small molecules for those that antagonize Lgr5 internalization. Glucocorticoids were found to potently increase Lgr5 expression at the plasma membrane., Conclusion: The IRFAP-HTS platform provides a versatile solution for screening more targets with fewer resources. Using only a standard western-blotting scanner, we were able to screen 5,000 compounds per hour in a robust and quantitative assay. Multi-purposing standardly available laboratory equipment eliminates the need for idiosyncratic and more expensive high-content imaging systems. The modular and user-friendly IRFAP-HTS is a significant departure from current screening platforms. Small laboratories will have unprecedented access to a robust and reliable screening platform and will no longer be limited by the esoteric nature of assay development, data acquisition, and post-screening analysis. The discovery of glucocorticoids as modulators for Lgr5 trafficking confirms that IRFAP-HTS can accelerate drug-discovery and drug-repurposing for even the most obscure targets.
- Published
- 2015
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29. Structure-activity studies of Wnt/β-catenin inhibition in the Niclosamide chemotype: Identification of derivatives with improved drug exposure.
- Author
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Mook RA Jr, Wang J, Ren XR, Chen M, Spasojevic I, Barak LS, Lyerly HK, and Chen W
- Subjects
- Cell Line, Tumor, Cell Proliferation, Humans, Structure-Activity Relationship, Niclosamide therapeutic use, Wnt Proteins antagonists & inhibitors, Wnt Signaling Pathway drug effects, beta Catenin metabolism
- Abstract
The Wnt signaling pathway plays a key role in regulation of organ development and tissue homeostasis. Dysregulated Wnt activity is one of the major underlying mechanisms responsible for many diseases including cancer. We previously reported the FDA-approved anthelmintic drug Niclosamide inhibits Wnt/β-catenin signaling and suppresses colon cancer cell growth in vitro and in vivo. Niclosamide is a multi-functional drug that possesses important biological activity in addition to inhibition of Wnt/β-catenin signaling. Here, we studied the SAR of Wnt signaling inhibition in the anilide and salicylamide region of Niclosamide. We found that the 4'-nitro substituent can be effectively replaced by trifluoromethyl or chlorine and that the potency of inhibition was dependent on the substitution pattern in the anilide ring. Non-anilide, N-methyl amides and reverse amide derivatives lost significant potency, while acylated salicylamide derivatives inhibited signaling with potency similar to non-acyl derivatives. Niclosamide's low systemic exposure when dosed orally may hinder its use to treat systemic disease. To overcome this limitation we identified an acyl derivative of Niclosamide, DK-520 (compound 32), that significantly increased both the plasma concentration and the duration of exposure of Niclosamide when dosed orally. The studies herein provide a medicinal chemical foundation to improve the pharmacokinetic exposure of Niclosamide and Wnt-signaling inhibitors based on the Niclosamide chemotype. The identification of novel derivatives of Niclosamide that metabolize to Niclosamide and increase its drug exposure may provide important research tools for in vivo studies and provide drug candidates for treating cancers with dysregulated Wnt signaling including drug-resistant cancers. Moreover, since Niclosamide is a multi-functional drug, new research tools such as DK520 could directly result in novel treatments against bacterial and viral infection, lupus, and metabolic diseases such as type II diabetes, NASH and NAFLD., (Copyright © 2015 Elsevier Ltd. All rights reserved.)
- Published
- 2015
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30. Lgr4 and Lgr5 drive the formation of long actin-rich cytoneme-like membrane protrusions.
- Author
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Snyder JC, Rochelle LK, Marion S, Lyerly HK, Barak LS, and Caron MG
- Subjects
- Adult, Arrestins metabolism, Biological Transport, Blotting, Western, HEK293 Cells, Humans, Immunoprecipitation, Pseudopodia physiology, Signal Transduction, Stem Cells cytology, beta-Arrestin 2, beta-Arrestins, Actins metabolism, Cell Membrane metabolism, Cell Surface Extensions metabolism, Receptors, G-Protein-Coupled metabolism, Stem Cells metabolism
- Abstract
Embryonic development and adult tissue homeostasis require precise information exchange between cells and their microenvironment to coordinate cell behavior. A specialized class of ultra-long actin-rich filopodia, termed cytonemes, provides one mechanism for this spatiotemporal regulation of extracellular cues. We provide here a mechanism whereby the stem-cell marker Lgr5, and its family member Lgr4, promote the formation of cytonemes. Lgr4- and Lgr5-induced cytonemes exceed lengths of 80 µm, are generated through stabilization of nascent filopodia from an underlying lamellipodial-like network and functionally provide a pipeline for the transit of signaling effectors. As proof-of-principle, we demonstrate that Lgr5-induced cytonemes act as conduits for cell signaling by demonstrating that the actin motor and filopodial cargo carrier protein myosin X (Myo10) and the G-protein-coupled receptor (GPCR) signaling effector β-arrestin-2 (Arrb2) transit into cytonemes. This work delineates a biological function for Lgr4 and Lgr5 and provides the rationale to fully investigate Lgr4 and Lgr5 function and cytonemes in mammalian stem cell and cancer stem cell behavior., (© 2015. Published by The Company of Biologists Ltd.)
- Published
- 2015
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31. Perhexiline promotes HER3 ablation through receptor internalization and inhibits tumor growth.
- Author
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Ren XR, Wang J, Osada T, Mook RA Jr, Morse MA, Barak LS, Lyerly HK, and Chen W
- Subjects
- Animals, Breast Neoplasms genetics, Breast Neoplasms pathology, Cell Line, Tumor, Cell Membrane metabolism, Cell Proliferation drug effects, Disease Models, Animal, Female, Humans, Mice, Neuregulins metabolism, Neuregulins pharmacology, Protein Transport drug effects, Proteolysis drug effects, Receptor, ErbB-3 genetics, Signal Transduction drug effects, Tumor Burden drug effects, Ubiquitination drug effects, Xenograft Model Antitumor Assays, Antineoplastic Agents pharmacology, Breast Neoplasms metabolism, Perhexiline pharmacology, Receptor, ErbB-3 metabolism
- Abstract
Introduction: Human epidermal growth factor receptor HER3 has been implicated in promoting the aggressiveness and metastatic potential of breast cancer. Upregulation of HER3 has been found to be a major mechanism underlying drug resistance to EGFR and HER2 tyrosine kinase inhibitors and to endocrine therapy in the treatment of breast cancer. Thus, agents that reduce HER3 expression at the plasma membrane may synergize with current therapies and offer a novel therapeutic strategy to improve treatment., Methods: We devised an image-based screening platform using membrane localized HER3-YFP to identify small molecules that promote HER3 internalization and degradation. In vitro and in vivo tumor models were used to characterize the signaling effects of perhexiline, an anti-anginal drug, identified by the screening platform., Results: We found perhexiline, an anti-anginal drug, selectively internalized HER3, decreased HER3 expression, and subsequently inhibited signaling downstream of HER3. Consistent with these results, perhexiline inhibited breast cancer cell proliferation in vitro and tumor growth in vivo., Conclusions: This is the first demonstration that HER3 can be targeted with small molecules by eliminating it from the cell membrane. The novel approach used here led to the discovery that perhexiline ablates HER3 expression, and offers an opportunity to identify HER3 ablation modulators as innovative therapeutics to improve survival in breast cancer patients.
- Published
- 2015
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32. Phenotypic regulation of the sphingosine 1-phosphate receptor miles apart by G protein-coupled receptor kinase 2.
- Author
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Burczyk M, Burkhalter MD, Blätte T, Matysik S, Caron MG, Barak LS, and Philipp M
- Subjects
- Animals, Arrestins metabolism, Cell Membrane metabolism, Endocytosis, GTP-Binding Proteins metabolism, HEK293 Cells, Heart embryology, Heart Defects, Congenital metabolism, Heart Defects, Congenital pathology, Humans, Mice, Phenotype, Protein Serine-Threonine Kinases metabolism, Serine-Threonine Kinase 3, Signal Transduction, Sphingosine-1-Phosphate Receptors, Zebrafish embryology, beta-Arrestin 2, beta-Arrestins, rhoA GTP-Binding Protein metabolism, G-Protein-Coupled Receptor Kinase 2 metabolism, Mutation genetics, Receptors, Lysosphingolipid metabolism, Zebrafish metabolism, Zebrafish Proteins metabolism
- Abstract
The evolutionarily conserved DRY motif at the end of the third helix of rhodopsin-like, class-A G protein-coupled receptors (GPCRs) is a major regulator of receptor stability, signaling activity, and β-arrestin-mediated internalization. Substitution of the DRY arginine with histidine in the human vasopressin receptor results in a loss-of-function phenotype associated with diabetes insipidus. The analogous R150H substitution of the DRY motif in zebrafish sphingosine-1 phosphate receptor 2 (S1p2) produces a mutation, miles apart m(93) (mil(m93)), that not only disrupts signaling but also impairs heart field migration. We hypothesized that constitutive S1p2 desensitization is the underlying cause of this strong zebrafish developmental defect. We observed in cell assays that the wild-type S1p2 receptor is at the cell surface whereas in distinct contrast the S1p2 R150H receptor is found in intracellular vesicles, blocking G protein but not arrestin signaling activity. Surface S1p2 R150H expression could be restored by inhibition of G protein-coupled receptor kinase 2 (GRK2). Moreover, we observed that β-arrestin 2 and GRK2 colocalize with S1p2 in developing zebrafish embryos and depletion of GRK2 in the S1p2 R150H miles apart zebrafish partially rescued cardia bifida. The ability of reduced GRK2 activity to reverse a developmental phenotype associated with constitutive desensitization supports efforts to genetically or pharmacologically target this kinase in diseases involving biased GPCR signaling.
- Published
- 2015
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33. G Protein and β-arrestin signaling bias at the ghrelin receptor.
- Author
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Evron T, Peterson SM, Urs NM, Bai Y, Rochelle LK, Caron MG, and Barak LS
- Subjects
- Arrestin genetics, GTP-Binding Proteins genetics, HEK293 Cells, Humans, Mitogen-Activated Protein Kinase 1 genetics, Mitogen-Activated Protein Kinase 1 metabolism, Mitogen-Activated Protein Kinase 3 genetics, Mitogen-Activated Protein Kinase 3 metabolism, Neuronal Plasticity physiology, Protein Stability, Protein Structure, Secondary, Protein Transport physiology, Receptors, Ghrelin genetics, Receptors, Vasopressin genetics, Receptors, Vasopressin metabolism, Arrestin metabolism, GTP-Binding Proteins metabolism, MAP Kinase Signaling System physiology, Receptors, Ghrelin metabolism
- Abstract
The G protein-coupled ghrelin receptor GHSR1a is a potential pharmacological target for treating obesity and addiction because of the critical role ghrelin plays in energy homeostasis and dopamine-dependent reward. GHSR1a enhances growth hormone release, appetite, and dopamine signaling through G(q/11), G(i/o), and G(12/13) as well as β-arrestin-based scaffolds. However, the contribution of individual G protein and β-arrestin pathways to the diverse physiological responses mediated by ghrelin remains unknown. To characterize whether a signaling bias occurs for GHSR1a, we investigated ghrelin signaling in a number of cell-based assays, including Ca(2+) mobilization, serum response factor response element, stress fiber formation, ERK1/2 phosphorylation, and β-arrestin translocation, utilizing intracellular second loop and C-tail mutants of GHSR1a. We observed that GHSR1a and β-arrestin rapidly form metastable plasma membrane complexes following exposure to an agonist, but replacement of the GHSR1a C-tail by the tail of the vasopressin 2 receptor greatly stabilizes them, producing complexes observable on the plasma membrane and also in endocytic vesicles. Mutations of the contiguous conserved amino acids Pro-148 and Leu-149 in the GHSR1a intracellular second loop generate receptors with a strong bias to G protein and β-arrestin, respectively, supporting a role for conformation-dependent signaling bias in the wild-type receptor. Our results demonstrate more balance in GHSR1a-mediated ERK signaling from G proteins and β-arrestin but uncover an important role for β-arrestin in RhoA activation and stress fiber formation. These findings suggest an avenue for modulating drug abuse-associated changes in synaptic plasticity via GHSR1a and indicate the development of GHSR1a-biased ligands as a promising strategy for selectively targeting downstream signaling events., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2014
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34. IL-6/STAT3 promotes regeneration of airway ciliated cells from basal stem cells.
- Author
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Tadokoro T, Wang Y, Barak LS, Bai Y, Randell SH, and Hogan BL
- Subjects
- Animals, Bronchi cytology, Cell Differentiation physiology, Cilia physiology, Disease Models, Animal, Epithelial Cells cytology, Epithelial Cells physiology, Green Fluorescent Proteins genetics, Humans, Interleukin-6 genetics, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Phosphorylation physiology, Primary Cell Culture, Regeneration physiology, Respiratory Mucosa physiology, STAT3 Transcription Factor genetics, Signal Transduction physiology, Stem Cells cytology, Stem Cells physiology, Trachea cytology, Interleukin-6 metabolism, Respiratory Mucosa cytology, STAT3 Transcription Factor metabolism
- Abstract
The pseudostratified airway epithelium of the lung contains a balanced proportion of multiciliated and secretory luminal cells that are maintained and regenerated by a population of basal stem cells. However, little is known about how these processes are modulated in vivo, and about the potential role of cytokine signaling between stem and progenitor cells and their niche. Using a clonal 3D organoid assay, we found that IL-6 stimulated, and Stat3 inhibitors reduced, the generation of ciliated vs. secretory cells from basal cells. Gain-of-function and loss-of-function studies with cultured mouse and human basal cells suggest that IL-6/Stat3 signaling promotes ciliogenesis at multiple levels, including increases in multicilin gene and forkhead box protein J1 expression and inhibition of the Notch pathway. To test the role of IL-6 in vivo genetically, we followed the regeneration of mouse tracheal epithelium after ablation of luminal cells by inhaled SO2. Stat3 is activated in basal cells and their daughters early in the repair process, correlating with an increase in Il-6 expression in platelet-derived growth factor receptor alpha(+) mesenchymal cells in the stroma. Conditional deletion in basal cells of suppressor of cytokine signaling 3, encoding a negative regulator of the Stat3 pathway, results in an increase in multiciliated cells at the expense of secretory and basal cells. By contrast, Il-6 null mice regenerate fewer ciliated cells and an increased number of secretory cells after injury. The results support a model in which IL-6, produced in the reparative niche, functions to enhance the differentiation of basal cells, and thereby acts as a "friend" to promote airway repair rather than a "foe."
- Published
- 2014
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35. Imidazole-derived agonists for the neurotensin 1 receptor.
- Author
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Hershberger PM, Hedrick MP, Peddibhotla S, Mangravita-Novo A, Gosalia P, Li Y, Gray W, Vicchiarelli M, Smith LH, Chung TD, Thomas JB, Caron MG, Pinkerton AB, Barak LS, and Roth GP
- Subjects
- Animals, Dose-Response Relationship, Drug, Humans, Imidazoles chemical synthesis, Imidazoles chemistry, Mice, Molecular Structure, Structure-Activity Relationship, Imidazoles pharmacology, Receptors, Neurotensin agonists
- Abstract
A scaffold-hop program seeking full agonists of the neurotensin-1 (NTR1) receptor identified the probe molecule ML301 (1) and associated analogs, including its naphthyl analog (14) which exhibited similar properties. Compound 1 showed full agonist behavior (79-93%) with an EC50 of 2.0-4.1μM against NTR1. Compound 1 also showed good activity in a Ca mobilization FLIPR assay (93% efficacy at 298nM), consistent with it functioning via the Gq coupled pathway, and good selectivity relative to NTR2 and GPR35. In further profiling, 1 showed low potential for promiscuity and good overall pharmacological data. This report describes the discovery, synthesis, and SAR of 1 and associated analogs. Initial in vitro pharmacologic characterization is also presented., (Copyright © 2013 Elsevier Ltd. All rights reserved.)
- Published
- 2014
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36. Identification of the GPR55 antagonist binding site using a novel set of high-potency GPR55 selective ligands.
- Author
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Kotsikorou E, Sharir H, Shore DM, Hurst DP, Lynch DL, Madrigal KE, Heynen-Genel S, Milan LB, Chung TD, Seltzman HH, Bai Y, Caron MG, Barak LS, Croatt MP, Abood ME, and Reggio PH
- Subjects
- Binding Sites, Humans, Inhibitory Concentration 50, Ligands, Models, Molecular, Protein Binding, Receptors, Cannabinoid, Receptors, G-Protein-Coupled metabolism, Drug Evaluation, Preclinical, Receptors, G-Protein-Coupled antagonists & inhibitors, Receptors, G-Protein-Coupled chemistry
- Abstract
GPR55 is a class A G protein-coupled receptor (GPCR) that has been implicated in inflammatory pain, neuropathic pain, metabolic disorder, bone development, and cancer. Initially deorphanized as a cannabinoid receptor, GPR55 has been shown to be activated by non-cannabinoid ligands such as l-α-lysophosphatidylinositol (LPI). While there is a growing body of evidence of physiological and pathophysiological roles for GPR55, the paucity of specific antagonists has limited its study. In collaboration with the Molecular Libraries Probe Production Centers Network initiative, we identified a series of GPR55 antagonists using a β-arrestin, high-throughput, high-content screen of ~300000 compounds. This screen yielded novel, GPR55 antagonist chemotypes with IC50 values in the range of 0.16-2.72 μM [Heynen-Genel, S., et al. (2010) Screening for Selective Ligands for GPR55: Antagonists (ML191, ML192, ML193) (Bookshelf ID NBK66153; PMID entry 22091481)]. Importantly, many of the GPR55 antagonists were completely selective, with no agonism or antagonism against GPR35, CB1, or CB2 up to 20 μM. Using a model of the GPR55 inactive state, we studied the binding of an antagonist series that emerged from this screen. These studies suggest that GPR55 antagonists possess a head region that occupies a horizontal binding pocket extending into the extracellular loop region, a central ligand portion that fits vertically in the receptor binding pocket and terminates with a pendant aromatic or heterocyclic ring that juts out. Both the region that extends extracellularly and the pendant ring are features associated with antagonism. Taken together, our results provide a set of design rules for the development of second-generation GPR55 selective antagonists.
- Published
- 2013
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37. The stem cell-expressed receptor Lgr5 possesses canonical and functionally active molecular determinants critical to β-arrestin-2 recruitment.
- Author
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Snyder JC, Rochelle LK, Barak LS, and Caron MG
- Subjects
- Amino Acid Motifs, Arrestins genetics, HEK293 Cells, Humans, Protein Structure, Tertiary, Receptors, G-Protein-Coupled genetics, beta-Arrestin 2, beta-Arrestins, Arrestins metabolism, Receptors, G-Protein-Coupled metabolism, Signal Transduction physiology
- Abstract
Lgr5 is a membrane protein related to G protein-coupled receptors (GPCR)s whose expression identifies stem cells in multiple tissues and is strongly correlated with cancer. Despite the recent identification of endogenous ligands for Lgr5, its mode of signaling remains enigmatic. The ability to couple to G proteins and βarrestins are classical molecular behaviors of GPCRs that have yet to be observed for Lgr5. Therefore, the goal of this study was to determine if Lgr5 can engage a classical GPCR behavior and elucidate the molecular determinants of this process. Structural analysis of Lgr5 revealed several motifs consistent with its ability to recruit βarr2. Among them, a "SSS" serine cluster located at amino acid position 873-875 within the C-terminal tail (C-tail), is in a region consistent with other GPCRs that bind βarr2 with high-affinity. To test its functionality, a ligand-independent βarr2 translocation assay was implemented. We show that Lgr5 recruits βarr2 and that the "SSS" amino acids (873-875) are absolutely critical to this process. We also demonstrate that for full efficacy, this cluster requires other Lgr5 C-tail serines that were previously shown to be important for constitutive and βarr2 independent internalization of Lgr5. These data are proof of principle that a classical GPCR behavior can be manifested by Lgr5. The existence of alternative ligands or missing effectors of Lgr5 that scaffold this classical GPCR behavior and the downstream signaling pathways engaged should be considered. Characterizing Lgr5 signaling will be invaluable for assessing its role in tissue maintenance, repair, and disease.
- Published
- 2013
- Full Text
- View/download PDF
38. Triphenylmethane dye activation of beta-arrestin.
- Author
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Barak LS, Bai Y, Snyder JC, Wang J, Chen W, and Caron MG
- Subjects
- Cell Line, Cell Line, Tumor, Coloring Agents, Endocytosis, GTP-Binding Proteins metabolism, HEK293 Cells, Humans, Quaternary Ammonium Compounds chemistry, Receptors, Neurotensin chemistry, Receptors, Neurotensin metabolism, Rosaniline Dyes chemistry, Signal Transduction, beta-Arrestins, Arrestins metabolism, Quaternary Ammonium Compounds metabolism, Rosaniline Dyes metabolism
- Abstract
β-Arrestins regulate G protein-coupled receptor signaling as competitive inhibitors and protein adaptors. Low molecular weight biased ligands that bind receptors and discriminate between the G protein dependent arm and β-arrestin, clathrin-associated arm of receptor signaling are considered therapeutically valuable as a result of this distinctive pharmacological behavior. Other than receptor agonists, compounds that activate β-arrestins are not available. We show that within minutes of exposure to the cationic triphenylmethane dyes malachite green and brilliant green, tissue culture cells recruit β-arrestins to clathrin scaffolds in a receptor-activation independent manner. In the presence of these compounds, G protein signaling is inhibited, ERK and GSK3β signaling are preserved, and the recruitment of the beta2-adaptin, AP2 adaptor complex to clathrin as well as transferrin internalization is reduced. Moreover, malachite green binds β-arrestin2-GFP coated immunotrap beads relative to GFP only coated beads. Triphenylmethane dyes are FDA approved for topical use on newborns as components of triple-dye preparations and are not approved but used effectively as aqueous antibiotics in fish husbandry. As possible carcinogens, their chronic ingestion in food preparations, particularly through farmed fish, is discouraged in the U.S. and Europe. Our results indicate triphenylmethane dyes as a result of novel pharmacology may have additional roles as β-arrestin/clathrin pathway signaling modulators in both pharmacology research and clinical therapy.
- Published
- 2013
- Full Text
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39. Discovery of ML314, a Brain Penetrant Non-Peptidic β-Arrestin Biased Agonist of the Neurotensin NTR1 Receptor.
- Author
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Peddibhotla S, Hedrick MP, Hershberger P, Maloney PR, Li Y, Milewski M, Gosalia P, Gray W, Mehta A, Sugarman E, Hood B, Suyama E, Nguyen K, Heynen-Genel S, Vasile S, Salaniwal S, Stonich D, Su Y, Mangravita-Novo A, Vicchiarelli M, Roth GP, Smith LH, Chung TD, Hanson GR, Thomas JB, Caron MG, Barak LS, and Pinkerton AB
- Abstract
The neurotensin 1 receptor (NTR1) is an important therapeutic target for a range of disease states including addiction. A high throughput screening campaign, followed by medicinal chemistry optimization, led to the discovery of a non-peptidic β-arrestin biased agonist for NTR1. The lead compound, 2-cyclopropyl-6,7-dimethoxy-4-(4-(2-methoxyphenyl)- piperazin-1-yl)quinazoline, 32 ( ML314 ), exhibits full agonist behavior against NTR1 (EC
50 = 2.0 μM) in the primary assay and selectivity against NTR2. The effect of 32 is blocked by the NTR1 antagonist SR142948A in a dose dependent manner. Unlike peptide based NTR1 agonists, compound 32 has no significant response in a Ca2+ mobilization assay and is thus a biased agonist that activates the β-arrestin pathway rather than the traditional Gq coupled pathway. This bias has distinct biochemical and functional consequences that may lead to physiological advantages. Compound 32 displays good brain penetration in rodents, and studies examining its in vivo properties are underway.- Published
- 2013
- Full Text
- View/download PDF
40. Novel adamantyl cannabinoids as CB1 receptor probes.
- Author
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Thakur GA, Bajaj S, Paronis C, Peng Y, Bowman AL, Barak LS, Caron MG, Parrish D, Deschamps JR, and Makriyannis A
- Subjects
- Analgesics chemical synthesis, Analgesics pharmacology, Animals, Arrestin metabolism, Body Temperature drug effects, Crystallography, X-Ray, Cyclic AMP metabolism, Dose-Response Relationship, Drug, Female, Hypothermia chemically induced, Models, Molecular, Pain Measurement drug effects, Rats, Rats, Sprague-Dawley, Receptor, Cannabinoid, CB2 drug effects, Structure-Activity Relationship, Adamantane chemical synthesis, Adamantane pharmacology, Cannabinoid Receptor Agonists chemical synthesis, Cannabinoids chemical synthesis, Cannabinoids pharmacology, Receptor, Cannabinoid, CB1 drug effects
- Abstract
In previous studies, compound 1 (AM411), a 3-(1-adamantyl) analogue of the phytocannabinoid (-)-Δ(8)-tetrahydrocannabinol (Δ(8)-THC), was shown to have improved affinity and selectivity for the CB1 receptor. In this work, we further explored the role of the 1-adamantyl group at the C-3 position in a series of tricyclic cannabinoid analogues modified at the 9-northern aliphatic hydroxyl (NAH) position. Of these, 9-hydroxymethyl hexahydrocannabinol 11 (AM4054) exhibited high CB1 affinity and full agonist profile. In the cAMP assay, the 9-hydroxymethyl cannabinol analogue 24 (AM4089) had a partial agonist profile, with high affinity and moderate selectivity for rCB1 over hCB2. In vivo results in rat models of hypothermia and analgesia were congruent with in vitro data. Our in vivo data indicate that 3-(1-adamantyl) substitution, within NAH cannabinergics, imparts improved pharmacological profiles when compared to the corresponding, traditionally used 3-dimethylheptyl analogues and identifies 11 and 24 as potentially useful in vivo CB1 cannabinergic probes.
- Published
- 2013
- Full Text
- View/download PDF
41. Constitutive internalization of the leucine-rich G protein-coupled receptor-5 (LGR5) to the trans-Golgi network.
- Author
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Snyder JC, Rochelle LK, Lyerly HK, Caron MG, and Barak LS
- Subjects
- Endosomes genetics, Endosomes metabolism, HEK293 Cells, Humans, Protein Transport physiology, Receptors, G-Protein-Coupled genetics, Time Factors, Vesicular Transport Proteins genetics, Vesicular Transport Proteins metabolism, rab GTP-Binding Proteins genetics, rab GTP-Binding Proteins metabolism, rab5 GTP-Binding Proteins genetics, rab5 GTP-Binding Proteins metabolism, rab7 GTP-Binding Proteins, trans-Golgi Network genetics, Receptors, G-Protein-Coupled metabolism, Wnt Signaling Pathway physiology, trans-Golgi Network metabolism
- Abstract
LGR5 is a Wnt pathway associated G protein-coupled receptor (GPCR) that serves as a molecular determinant of stem cells in numerous tissues including the intestine, stomach, hair follicle, eye, and mammary gland. Despite its importance as a marker for this critical niche, little is known about LGR5 signaling nor the biochemical mechanisms and receptor determinants that regulate LGR5 membrane expression and intracellular trafficking. Most importantly, in cells LGR5 is predominantly intracellular, yet the mechanisms underlying this behavior have not been determined. In this work we elucidate a precise trafficking program for LGR5 and identify the motif at its C terminus that is responsible for the observed constitutive internalization. We show that this process is dependent upon dynamin GTPase activity and find that wild-type full-length LGR5 rapidly internalizes into EEA1- and Rab5-positive endosomes. However, LGR5 fails to rapidly recycle to the plasmid membrane through Rab4-positive vesicles, as is common for other GPCRs. Rather, internalized LGR5 transits through Rab7- and Rab9-positive vesicles, co-localizes in vesicles with Vps26, a retromer complex component that regulates retrograde trafficking to the trans-Golgi network (TGN) and reaches a steady-state distribution in the TGN within 2 h. Using mutagenesis, particularly of putative phosphorylation sites, we show that the amino acid pair, serine 861 and 864, is the principal C-tail determinant that mediates LGR5 constitutive internalization. The constitutive internalization of LGR5 to the TGN suggests the existence of novel biochemical roles for its Wnt pathway related, but ill defined signaling program.
- Published
- 2013
- Full Text
- View/download PDF
42. The intestinal glucose-apelin cycle controls carbohydrate absorption in mice.
- Author
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Dray C, Sakar Y, Vinel C, Daviaud D, Masri B, Garrigues L, Wanecq E, Galvani S, Negre-Salvayre A, Barak LS, Monsarrat B, Burlet-Schiltz O, Valet P, Castan-Laurell I, and Ducroc R
- Subjects
- Analysis of Variance, Animals, Biological Transport drug effects, Biological Transport physiology, Blotting, Western, Chromatography, Liquid methods, Disease Models, Animal, Glucose pharmacology, Glucose Transporter Type 2 metabolism, Immunohistochemistry, Male, Mass Spectrometry, Mice, Mice, Inbred C57BL, Microscopy, Confocal, Random Allocation, Reference Values, Sodium-Glucose Transporter 1 metabolism, Carbohydrates pharmacokinetics, Glucose metabolism, Intercellular Signaling Peptides and Proteins metabolism, Intestinal Absorption drug effects, Intestinal Absorption physiology
- Abstract
Background & Aims: Glucose is absorbed into intestine cells via the sodium glucose transporter 1 (SGLT-1) and glucose transporter 2 (GLUT2); various peptides and hormones control this process. Apelin is a peptide that regulates glucose homeostasis and is produced by proximal digestive cells; we studied whether glucose modulates apelin secretion by enterocytes and the effects of apelin on intestinal glucose absorption., Methods: We characterized glucose-related luminal apelin secretion in vivo and ex vivo by mass spectroscopy and immunologic techniques. The effects of apelin on (14)C-labeled glucose transport were determined in jejunal loops and in mice following apelin gavage. We determined levels of GLUT2 and SGLT-1 proteins and phosphorylation of AMPKα2 by immunoblotting. The net effect of apelin on intestinal glucose transepithelial transport was determined in mice., Results: Glucose stimulated luminal secretion of the pyroglutaminated apelin-13 isoform ([Pyr-1]-apelin-13) in the small intestine of mice. Apelin increased specific glucose flux through the gastric epithelial barrier in jejunal loops and in vivo following oral glucose administration. Conversely, pharmacologic apelin blockade in the intestine reduced the increased glycemia that occurs following oral glucose administration. Apelin activity was associated with phosphorylation of AMPKα2 and a rapid increase of the GLUT2/SGLT-1 protein ratio in the brush border membrane., Conclusions: Glucose amplifies its own transport from the intestinal lumen to the bloodstream by increasing luminal apelin secretion. In the lumen, active apelin regulates carbohydrate flux through enterocytes by promoting AMPKα2 phosphorylation and modifying the ratio of SGLT-1:GLUT2. The glucose-apelin cycle might be pharmacologically handled to regulate glucose absorption and assess better control of glucose homeostasis., (Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
43. Small molecule modulators of Wnt/β-catenin signaling.
- Author
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Mook RA Jr, Chen M, Lu J, Barak LS, Lyerly HK, and Chen W
- Subjects
- Cell Line, Tumor, Dose-Response Relationship, Drug, HEK293 Cells, Humans, Molecular Structure, Niclosamide chemistry, Small Molecule Libraries chemistry, Structure-Activity Relationship, beta Catenin metabolism, Niclosamide pharmacology, Small Molecule Libraries pharmacology, Wnt Signaling Pathway drug effects, beta Catenin antagonists & inhibitors
- Abstract
The Wnt signal transduction pathway is dysregulated in many highly prevalent diseases, including cancer. Unfortunately, drug discovery efforts have been hampered by the paucity of targets and drug-like lead molecules amenable to drug discovery. Recently, we reported the FDA-approved anthelmintic drug Niclosamide inhibits Wnt/β-catenin signaling by a unique mechanism, though the target responsible remains unknown. We interrogated the mechanism and structure-activity relationships to understand drivers of potency and to assist target identification efforts. We found inhibition of Wnt signaling by Niclosamide appears unique among the structurally-related anthelmintic agents tested and found the potency and functional response was dependent on small changes in the chemical structure of Niclosamide. Overall, these findings support efforts to identify the target of Niclosamide inhibition of Wnt/β-catenin signaling and the discovery of potent and selective modulators to treat human disease., (Copyright © 2013 Elsevier Ltd. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
44. Identification of a novel Smoothened antagonist that potently suppresses Hedgehog signaling.
- Author
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Wang J, Mook RA Jr, Lu J, Gooden DM, Ribeiro A, Guo A, Barak LS, Lyerly HK, and Chen W
- Subjects
- Animals, Benzamides chemical synthesis, Benzamides pharmacology, Cell Proliferation drug effects, Cells, Cultured, Female, Hair drug effects, Hair growth & development, Humans, Mice, Mice, Inbred C57BL, Mutation, Neurons cytology, Pyridines chemical synthesis, Pyridines pharmacology, Receptors, G-Protein-Coupled genetics, Receptors, G-Protein-Coupled metabolism, Signal Transduction, Smoothened Receptor, Benzamides chemistry, Hedgehog Proteins metabolism, Pyridines chemistry, Receptors, G-Protein-Coupled antagonists & inhibitors
- Abstract
The Hedgehog signaling pathway plays an essential role in embryo development and adult tissue homeostasis, in regulating stem cells and is abnormally activated in many cancers. Given the importance of this signaling pathway, we developed a novel and versatile high-throughput, cell-based screening platform using confocal imaging, based on the role of β-arrestin in Hedgehog signal transduction, that can identify agonists or antagonist of the pathway by a simple change to the screening protocol. Here we report the use of this assay in the antagonist mode to identify novel antagonists of Smoothened, including a compound (A8) with low nanomolar activity against wild-type Smo also capable of binding the Smo point mutant D473H associated with clinical resistance in medulloblastoma. Our data validate this novel screening approach in the further development of A8 and related congeners to treat Hedgehog related diseases, including the treatment of basal cell carcinoma and medulloblastoma., (Copyright © 2012 Elsevier Ltd. All rights reserved.)
- Published
- 2012
- Full Text
- View/download PDF
45. BRET biosensors to study GPCR biology, pharmacology, and signal transduction.
- Author
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Salahpour A, Espinoza S, Masri B, Lam V, Barak LS, and Gainetdinov RR
- Abstract
Bioluminescence resonance energy transfer (BRET)-based biosensors have been extensively used over the last decade to study protein-protein interactions and intracellular signal transduction in living cells. In this review, we discuss the various BRET biosensors that have been developed to investigate biology, pharmacology, and signaling of G protein-coupled receptors (GPCRs). GPCRs form two distinct types of multiprotein signal transduction complexes based upon their inclusion of G proteins or β-arrestins that can be differentially affected by drugs that exhibit functional selectivity toward G protein or β-arrestin signaling. BRET has been especially adept at illuminating the dynamics of protein-protein interactions between receptors, G proteins, β-arrestins, and their many binding partners in living cells; as well as measuring the formation and accumulation of second messengers following receptor activation. Specifically, we discuss in detail the application of BRET to study dopamine and trace amine receptors signaling, presenting examples of an exchange protein activated by cAMP biosensor to measure cAMP, β-arrestin biosensors to determine β-arrestin recruitment to the receptor, and dopamine D2 receptor and trace amine-associated receptor 1 biosensors to investigate heterodimerization between them. As the biochemical spectrum of BRET biosensors expands, the number of signaling pathways that can be measured will concomitantly increase. This will be particularly useful for the evaluation of functional selectivity in which the real-time BRET capability to measure distinct signaling modalities will dramatically shorten the time to characterize new generation of biased drugs. These emerging approaches will further expand the growing application of BRET in the screening for novel pharmacologically active compounds.
- Published
- 2012
- Full Text
- View/download PDF
46. The insecticide synergist piperonyl butoxide inhibits hedgehog signaling: assessing chemical risks.
- Author
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Wang J, Lu J, Mook RA Jr, Zhang M, Zhao S, Barak LS, Freedman JH, Lyerly HK, and Chen W
- Subjects
- Animals, Embryonic Development drug effects, Genes, Reporter, Luciferases genetics, Risk Assessment, Veratrum Alkaloids metabolism, Zebrafish embryology, Hedgehog Proteins metabolism, Pesticide Synergists toxicity, Piperonyl Butoxide toxicity, Signal Transduction drug effects
- Abstract
The spread of chemicals, including insecticides, into the environment often raises public health concerns, as exemplified by a recent epidemiologic study associating in utero piperonyl butoxide (PBO) exposure with delayed mental development. The insecticide synergist PBO is listed among the top 10 chemicals detected in indoor dust; a systematic assessment of risks from PBO exposure, as for many toxicants unfortunately, may be underdeveloped when important biological targets that can cause toxicity are unknown. Hedgehog/Smoothened signaling is critical in neurological development. This study was designed to use novel high-throughput in vitro drug screening technology to identify modulators of Hedgehog signaling in environmental chemicals to assist the assessment of their potential risks. A directed library of 1408 environmental toxicants was screened for Hedgehog/Smoothened antagonist activity using a high-content assay that evaluated the interaction between Smoothened and βarrestin2 green fluorescent protein. PBO was identified as a Hedgehog/Smoothened antagonist capable of inhibiting Hedgehog signaling. We found that PBO bound Smoothened and blocked Smoothened overexpression-induced Gli-luciferase reporter activity but had no effect on Gli-1 downstream transcriptional factor-induced Gli activity. PBO inhibited Sonic Hedgehog ligand-induced Gli signaling and mouse cerebellar granular precursor cell proliferation. Moreover, PBO disrupted zebrafish development. Our findings demonstrate the value of high-throughput target-based screening strategies that can successfully evaluate large numbers of environmental toxicants and identify key targets and unknown biological activity that is helpful in properly assessing potential risks.
- Published
- 2012
- Full Text
- View/download PDF
47. Polyclonal HER2-specific antibodies induced by vaccination mediate receptor internalization and degradation in tumor cells.
- Author
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Ren XR, Wei J, Lei G, Wang J, Lu J, Xia W, Spector N, Barak LS, Clay TM, Osada T, Hamilton E, Blackwell K, Hobeika AC, Morse MA, Lyerly HK, and Chen W
- Subjects
- Animals, Cell Line, Tumor, Cell Membrane metabolism, Cell Proliferation, Clathrin-Coated Vesicles metabolism, Endocytosis immunology, Extracellular Signal-Regulated MAP Kinases metabolism, Female, HEK293 Cells, Humans, Mice, Mice, Inbred C57BL, Mice, Inbred NOD, Neoplasm Transplantation, Phosphorylation, Proto-Oncogene Proteins c-akt metabolism, Receptor, ErbB-2 metabolism, Transplantation, Heterologous, Ubiquitination, Vaccination, Antibodies immunology, Breast Neoplasms immunology, Breast Neoplasms therapy, Cancer Vaccines immunology, Cancer Vaccines therapeutic use, Receptor, ErbB-2 immunology
- Abstract
Introduction: Sustained HER2 signaling at the cell surface is an oncogenic mechanism in a significant proportion of breast cancers. While clinically effective therapies targeting HER2 such as mAbs and tyrosine kinase inhibitors exist, tumors overexpressing HER2 eventually progress despite treatment. Thus, abrogation of persistent HER2 expression at the plasma membrane to synergize with current approaches may represent a novel therapeutic strategy., Methods: We generated polyclonal anti-HER2 antibodies (HER2-VIA) by vaccinating mice with an adenovirus expressing human HER2, and assessed their signaling effects in vitro and anti-tumor effects in a xenograft model. In addition, we studied the signaling effects of human HER2-specific antibodies induced by vaccinating breast cancer patients with a HER2 protein vaccine., Results: HER2-VIA bound HER2 at the plasma membrane, initially activating the downstream kinases extracellular signal-regulated protein kinase 1/2 and Akt, but subsequently inducing receptor internalization in clathrin-coated pits in a HER2 kinase-independent manner, followed by ubiquitination and degradation of HER2 into a 130 kDa fragment phosphorylated at tyrosine residues 1,221/1,222 and 1,248. Following vaccination of breast cancer patients with the HER2 protein vaccine, HER2-specific antibodies were detectable and these antibodies bound to cell surface-expressed HER2 and inhibited HER2 signaling through blocking tyrosine 877 phosphorylation of HER2. In contrast to the murine antibodies, human anti-HER2 antibodies induced by protein vaccination did not mediate receptor internalization and degradation., Conclusion: These data provide new insight into HER2 trafficking at the plasma membrane and the changes induced by polyclonal HER2-specific antibodies. The reduction of HER2 membrane expression and HER2 signaling by polyclonal antibodies induced by adenoviral HER2 vaccines supports human clinical trials with this strategy for those breast cancer patients with HER2 therapy-resistant disease.
- Published
- 2012
- Full Text
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48. Discovery of Small Molecule Kappa Opioid Receptor Agonist and Antagonist Chemotypes through a HTS and Hit Refinement Strategy.
- Author
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Frankowski KJ, Hedrick MP, Gosalia P, Li K, Shi S, Whipple D, Ghosh P, Prisinzano TE, Schoenen FJ, Su Y, Vasile S, Sergienko E, Gray W, Hariharan S, Milan L, Heynen-Genel S, Mangravita-Novo A, Vicchiarelli M, Smith LH, Streicher JM, Caron MG, Barak LS, Bohn LM, Chung TD, and Aubé J
- Abstract
Herein we present the outcome of a high throughput screening (HTS) campaign-based strategy for the rapid identification and optimization of selective and general chemotypes for both kappa (κ) opioid receptor (KOR) activation and inhibition. In this program, we have developed potent antagonists (IC(50) < 120 nM) or agonists of high binding affinity (K(i) < 3 nM). In contrast to many important KOR ligands, the compounds presented here are highly modular, readily synthesized and, in most cases, achiral. The four new chemotypes hold promise for further development into chemical tools for studying the KOR or as potential therapeutic lead candidates.
- Published
- 2012
- Full Text
- View/download PDF
49. Modeling of bias for the analysis of receptor signaling in biochemical systems.
- Author
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Barak LS and Peterson S
- Subjects
- Animals, Arrestins chemistry, Arrestins physiology, Bias, Dopamine D2 Receptor Antagonists, HEK293 Cells, Humans, Mice, Probability, Protein Conformation, Quinpirole pharmacology, Receptors, Dopamine D2 physiology, Receptors, G-Protein-Coupled physiology, Signal Transduction, beta-Arrestins, Models, Biological, Receptors, Dopamine D2 chemistry, Receptors, G-Protein-Coupled chemistry
- Abstract
Ligand bias is a recently introduced concept in the receptor signaling field that underlies innovative strategies for targeted drug design. Ligands, as a consequence of conformational selectivity, produce signaling bias in which some downstream biochemical pathways are favored over others, and this contributes to variability in physiological responsiveness. Though the concept of bias and its implications for receptor signaling have become more important, its working definition in biochemical signaling is sufficiently imprecise as to impede the use of bias as an analytical tool. In this work, we provide a precise mathematical definition for receptor signaling bias using a formalism expressly applied to logistic response functions, models of most physiological behaviors. We show that signaling-response bias of biological processes may be represented by hyperbolae, or more generally as families of bias coordinates that index hyperbolae. Furthermore, we show bias is a property of a parametric mapping of these indexes into vertical strings that reside within a cylinder of stacked Poincare disks and that bias factors representing signaling probabilities are the radial distance of the strings from the cylinder axis. The utility of the formalism is demonstrated with logistic hyperbolic plots, by transducer ratio modeling, and with novel examples of Poincare disk plots of Gi and β-arrestin biased dopamine 2 receptor signaling. Our results provide a platform for categorizing compounds using distance relationships in the Poincare disk, indicate that signaling bias is a relatively common phenomenon at low ligand concentrations, and suggest that potent partial agonists and signaling pathway modulators may be preferred leads for signal bias-based therapies.
- Published
- 2012
- Full Text
- View/download PDF
50. Functional interaction between trace amine-associated receptor 1 and dopamine D2 receptor.
- Author
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Espinoza S, Salahpour A, Masri B, Sotnikova TD, Messa M, Barak LS, Caron MG, and Gainetdinov RR
- Subjects
- Animals, Cell Line, Dimerization, Fluorescent Antibody Technique, Haloperidol pharmacology, Humans, Ligands, Mice, Mice, Inbred C57BL, Protein Binding, Receptors, Dopamine D2 metabolism, Receptors, G-Protein-Coupled metabolism
- Abstract
The ability of dopamine receptors to interact with other receptor subtypes may provide mechanisms for modulating dopamine-related functions and behaviors. In particular, there is evidence suggesting that the trace amine-associated receptor 1 (TAAR1) affects the dopaminergic system by regulating the firing rate of dopaminergic neurons or by altering dopamine D2 receptor (D2R) responsiveness to ligands. TAAR1 is a Gα(s) protein-coupled receptor that is activated by biogenic amines, "trace amines," such as β-phenylethylamine (β-PEA) and tyramine that are normally found at low concentrations in the mammalian brain. In the present study, we investigated the biochemical mechanism of interaction between TAAR1 and D2R and the role this interaction plays in D2R-related signaling and behaviors. Using a bioluminescence resonance energy transfer biosensor for cAMP, we demonstrated that the D2R antagonists haloperidol, raclopride, and amisulpride were able to enhance selectively a TAAR1-mediated β-PEA increase of cAMP. Moreover, TAAR1 and D2R were able to form heterodimers when coexpressed in human embryonic kidney 293 cells, and this direct interaction was disrupted in the presence of haloperidol. In addition, in mice lacking TAAR1, haloperidol-induced striatal c-Fos expression and catalepsy were significantly reduced. Taken together, these data suggest that TAAR1 and D2R have functional and physical interactions that could be critical for the modulation of the dopaminergic system by TAAR1 in vivo.
- Published
- 2011
- Full Text
- View/download PDF
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