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1
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Wang D, Spoelstra WK, Lin L, Akkerman N, Krueger D, Dayton T, van Zon JS, Tans SJ, van Es JH, Clevers H. Interferon-responsive intestinal BEST4/CA7 + cells are targets of bacterial diarrheal toxins. Cell Stem Cell 2025; 32:598-612.e5. [PMID: 40010349 DOI: 10.1016/j.stem.2025.02.003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2024] [Revised: 09/25/2024] [Accepted: 02/04/2025] [Indexed: 02/28/2025]
Abstract
BEST4/CA7+ cells of the human intestine were recently identified by single-cell RNA sequencing. While their gene expression profile predicts a role in electrolyte balance, BEST4/CA7+ cell function has not been explored experimentally owing to the absence of BEST4/CA7+ cells in mice and the paucity of human in vitro models. Here, we establish a protocol that allows the emergence of BEST4/CA7+ cells in human intestinal organoids. Differentiation of BEST4/CA7+ cells requires activation of Notch signaling and the transcription factor SPIB. BEST4/CA7+ cell numbers strongly increase in response to the cytokine interferon-γ, supporting a role in immunity. Indeed, we demonstrate that BEST4/CA7+ cells generate robust CFTR-mediated fluid efflux when stimulated with bacterial diarrhea-causing toxins and find the norepinephrine-ADRA2A axis as a potential mechanism in blocking BEST4/CA7+ cell-mediated fluid secretion. Our observations identify a central role of BEST4/CA7+ cells in fluid homeostasis in response to bacterial infections.
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Affiliation(s)
- Daisong Wang
- Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, Utrecht 3584 CT, the Netherlands; Oncode Institute, Hubrecht Institute, Utrecht 3584 CT, the Netherlands
| | | | - Lin Lin
- Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, Utrecht 3584 CT, the Netherlands; Oncode Institute, Hubrecht Institute, Utrecht 3584 CT, the Netherlands; The Princess Máxima Center for Pediatric Oncology, Utrecht 3584 CS, the Netherlands
| | - Ninouk Akkerman
- Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, Utrecht 3584 CT, the Netherlands; Oncode Institute, Hubrecht Institute, Utrecht 3584 CT, the Netherlands
| | - Daniel Krueger
- Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, Utrecht 3584 CT, the Netherlands; Oncode Institute, Hubrecht Institute, Utrecht 3584 CT, the Netherlands
| | - Talya Dayton
- Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, Utrecht 3584 CT, the Netherlands; Oncode Institute, Hubrecht Institute, Utrecht 3584 CT, the Netherlands
| | | | - Sander J Tans
- AMOLF, Amsterdam 1009 DB, the Netherlands; Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Delft 2629 HZ, the Netherlands
| | - Johan H van Es
- Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, Utrecht 3584 CT, the Netherlands; Oncode Institute, Hubrecht Institute, Utrecht 3584 CT, the Netherlands
| | - Hans Clevers
- Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, Utrecht 3584 CT, the Netherlands; Oncode Institute, Hubrecht Institute, Utrecht 3584 CT, the Netherlands; The Princess Máxima Center for Pediatric Oncology, Utrecht 3584 CS, the Netherlands.
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2
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Yibcharoenporn C, Kongkaew T, Worakajit N, Khumjiang R, Saetang P, Satitsri S, Rukachaisirikul V, Muanprasat C. Inhibition of CFTR-mediated intestinal chloride secretion by nornidulin: Cellular mechanisms and anti-secretory efficacy in human intestinal epithelial cells and human colonoids. PLoS One 2024; 19:e0314723. [PMID: 39715175 DOI: 10.1371/journal.pone.0314723] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2024] [Accepted: 11/14/2024] [Indexed: 12/25/2024] Open
Abstract
Secretory diarrhea, a major global health concern, particularly among young children, is often characterized by excessive chloride secretion through the cystic fibrosis transmembrane conductance regulator (CFTR) channel. Nornidulin, a fungus-derived natural product from Aspergillus unguis, has previously been shown to inhibit cAMP-induced Cl- secretion in T84 cells (human intestinal cell lines). However, the cellular mechanism of nornidulin in inhibiting cAMP-induced Cl- secretion and its anti-secretory efficacy is still unknown especially in a human colonoid model, a preclinical model recapitulating intestinal physiology in humans. This research study aimed to examine the mechanism of nornidulin to inhibit cAMP-induced chloride secretion and assess its ability to reduce fluid secretion in both T84 cells and human colonoid models. Apical Cl- current analyses showed that nornidulin inhibited CFTR-mediated Cl- current in T84 cells with IC50 of ~1.5 μM. Nornidulin treatment had no effect on CFTR protein expression. Additionally, the inhibitory effects of nornidulin on CFTR-mediated chloride currents were unaffected by the presence of compounds that inhibit negative regulators of CFTR function, such as protein phosphatases, AMP-activated protein kinases, and phosphodiesterases. Interestingly, nornidulin suppressed the increase in intracellular cAMP levels caused by forskolin, an activator of adenylate cyclases, in T84 cells. Using human colonoid models, we found that nornidulin significantly suppressed the forskolin and cholera toxin-induced fluid secretion, indicating that nornidulin exerted an anti-secretory effect in human intestinal epithelia. Collectively, nornidulin represents a novel class of fungus-derived inhibitors of CFTR-mediated Cl- secretion, potentially making it a promising candidate for the development of anti-secretory treatments.
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Affiliation(s)
- Chamnan Yibcharoenporn
- Chakri Naruebodindra Medical Institute, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bang Phli, Samut Prakarn, Thailand
| | - Thidarat Kongkaew
- Chakri Naruebodindra Medical Institute, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bang Phli, Samut Prakarn, Thailand
| | - Nichakorn Worakajit
- Chakri Naruebodindra Medical Institute, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bang Phli, Samut Prakarn, Thailand
- Translational Medicine, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
| | - Rungtiwa Khumjiang
- Chakri Naruebodindra Medical Institute, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bang Phli, Samut Prakarn, Thailand
| | - Praphatsorn Saetang
- Department of Science, Faculty of Science and Technology, Prince of Songkla University, Pattani, Thailand
- Division of Physical Science and Center of Excellence for Innovation in Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla, Thailand
| | - Saravut Satitsri
- Chakri Naruebodindra Medical Institute, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bang Phli, Samut Prakarn, Thailand
| | - Vatcharin Rukachaisirikul
- Division of Physical Science and Center of Excellence for Innovation in Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla, Thailand
| | - Chatchai Muanprasat
- Chakri Naruebodindra Medical Institute, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bang Phli, Samut Prakarn, Thailand
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3
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Gao X, Yeh HI, Yang Z, Fan C, Jiang F, Howard RJ, Lindahl E, Kappes JC, Hwang TC. Allosteric inhibition of CFTR gating by CFTRinh-172 binding in the pore. Nat Commun 2024; 15:6668. [PMID: 39107303 PMCID: PMC11303713 DOI: 10.1038/s41467-024-50641-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2023] [Accepted: 07/09/2024] [Indexed: 08/10/2024] Open
Abstract
Loss-of-function mutations of the CFTR gene cause the life-shortening genetic disease cystic fibrosis (CF), whereas overactivity of CFTR may lead to secretory diarrhea and polycystic kidney disease. While effective drugs targeting the CFTR protein have been developed for the treatment of CF, little progress has been made for diseases caused by hyper-activated CFTR. Here, we solve the cryo-EM structure of CFTR in complex with CFTRinh-172 (Inh-172), a CFTR gating inhibitor with promising potency and efficacy. We find that Inh-172 binds inside the pore of CFTR, interacting with amino acid residues from transmembrane segments (TMs) 1, 6, 8, 9, and 12 through mostly hydrophobic interactions and a salt bridge. Substitution of these residues lowers the apparent affinity of Inh-172. The inhibitor-bound structure reveals re-orientations of the extracellular segment of TMs 1, 8, and 12, supporting an allosteric modulation mechanism involving post-binding conformational changes. This allosteric inhibitory mechanism readily explains our observations that pig CFTR, which preserves all the amino acid residues involved in Inh-172 binding, exhibits a much-reduced sensitivity to Inh-172 and that the apparent affinity of Inh-172 is altered by the CF drug ivacaftor (i.e., VX-770) which enhances CFTR's activity through binding to a site also comprising TM8.
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Affiliation(s)
- Xiaolong Gao
- Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO, 65211, USA.
| | - Han-I Yeh
- Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO, 65211, USA
- Institute of Pharmacology, National Yang Ming Chiao Tung University, College of Medicine, Taipei, Taiwan
- Membrane Protein Structural Biology Research Center, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Zhengrong Yang
- Heersink School of Medicine, University of Alabama School of Medicine, Birmingham, AL, 35233, USA
| | - Chen Fan
- Department of Applied Physics, Science for Life Laboratory, KTH Royal Institute of Technology, Solna, Sweden
- Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, Solna, Sweden
| | - Fan Jiang
- Heersink School of Medicine, University of Alabama School of Medicine, Birmingham, AL, 35233, USA
| | - Rebecca J Howard
- Department of Applied Physics, Science for Life Laboratory, KTH Royal Institute of Technology, Solna, Sweden
- Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, Solna, Sweden
| | - Erik Lindahl
- Department of Applied Physics, Science for Life Laboratory, KTH Royal Institute of Technology, Solna, Sweden
- Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, Solna, Sweden
| | - John C Kappes
- Heersink School of Medicine, University of Alabama School of Medicine, Birmingham, AL, 35233, USA
- Research Service, Birmingham Veterans Affairs Medical Center, Veterans Health Administration, Birmingham, AL, 35233, USA
| | - Tzyh-Chang Hwang
- Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO, 65211, USA.
- Institute of Pharmacology, National Yang Ming Chiao Tung University, College of Medicine, Taipei, Taiwan.
- Membrane Protein Structural Biology Research Center, National Yang Ming Chiao Tung University, Taipei, Taiwan.
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Liu F, Kaplan AL, Levring J, Einsiedel J, Tiedt S, Distler K, Omattage NS, Kondratov IS, Moroz YS, Pietz HL, Irwin JJ, Gmeiner P, Shoichet BK, Chen J. Structure-based discovery of CFTR potentiators and inhibitors. Cell 2024; 187:3712-3725.e34. [PMID: 38810646 PMCID: PMC11262615 DOI: 10.1016/j.cell.2024.04.046] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2023] [Revised: 03/19/2024] [Accepted: 04/29/2024] [Indexed: 05/31/2024]
Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) is a crucial ion channel whose loss of function leads to cystic fibrosis, whereas its hyperactivation leads to secretory diarrhea. Small molecules that improve CFTR folding (correctors) or function (potentiators) are clinically available. However, the only potentiator, ivacaftor, has suboptimal pharmacokinetics and inhibitors have yet to be clinically developed. Here, we combine molecular docking, electrophysiology, cryo-EM, and medicinal chemistry to identify CFTR modulators. We docked ∼155 million molecules into the potentiator site on CFTR, synthesized 53 test ligands, and used structure-based optimization to identify candidate modulators. This approach uncovered mid-nanomolar potentiators, as well as inhibitors, that bind to the same allosteric site. These molecules represent potential leads for the development of more effective drugs for cystic fibrosis and secretory diarrhea, demonstrating the feasibility of large-scale docking for ion channel drug discovery.
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Affiliation(s)
- Fangyu Liu
- Laboratory of Membrane Biology and Biophysics, The Rockefeller University, New York, NY 10065, USA; Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Anat Levit Kaplan
- Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Jesper Levring
- Laboratory of Membrane Biology and Biophysics, The Rockefeller University, New York, NY 10065, USA
| | - Jürgen Einsiedel
- Department of Chemistry and Pharmacy, Medicinal Chemistry, Friedrich-Alexander University Erlangen-Nürnberg, Nikolaus-Fiebiger-Straße 10, D-91058 Erlangen, Germany
| | - Stephanie Tiedt
- Department of Chemistry and Pharmacy, Medicinal Chemistry, Friedrich-Alexander University Erlangen-Nürnberg, Nikolaus-Fiebiger-Straße 10, D-91058 Erlangen, Germany
| | - Katharina Distler
- Department of Chemistry and Pharmacy, Medicinal Chemistry, Friedrich-Alexander University Erlangen-Nürnberg, Nikolaus-Fiebiger-Straße 10, D-91058 Erlangen, Germany
| | - Natalie S Omattage
- Laboratory of Membrane Biology and Biophysics, The Rockefeller University, New York, NY 10065, USA
| | - Ivan S Kondratov
- Enamine Ltd., Chervonotkatska Street 78, 02094 Kyïv, Ukraine; V.P. Kukhar Institute of Bioorganic Chemistry & Petrochemistry, National Academy of Sciences of Ukraine, Murmanska Street 1, 02660 Kyïv, Ukraine
| | - Yurii S Moroz
- Chemspace, Chervonotkatska Street 85, 02094 Kyïv, Ukraine; Taras Shevchenko National University of Kyïv, Volodymyrska Street 60, 01601 Kyïv, Ukraine
| | - Harlan L Pietz
- Laboratory of Membrane Biology and Biophysics, The Rockefeller University, New York, NY 10065, USA
| | - John J Irwin
- Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Peter Gmeiner
- Department of Chemistry and Pharmacy, Medicinal Chemistry, Friedrich-Alexander University Erlangen-Nürnberg, Nikolaus-Fiebiger-Straße 10, D-91058 Erlangen, Germany.
| | - Brian K Shoichet
- Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94143, USA.
| | - Jue Chen
- Laboratory of Membrane Biology and Biophysics, The Rockefeller University, New York, NY 10065, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA.
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5
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Bae H, Kim BR, Jung S, Le J, van der Heide D, Yu W, Park SH, Hilkin BM, Gansemer ND, Powers LS, Kang T, Meyerholz DK, Schuster VL, Jang C, Welsh MJ. Arteriovenous metabolomics in pigs reveals CFTR regulation of metabolism in multiple organs. J Clin Invest 2024; 134:e174500. [PMID: 38743489 PMCID: PMC11213515 DOI: 10.1172/jci174500] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2023] [Accepted: 05/07/2024] [Indexed: 05/16/2024] Open
Abstract
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), a multiorgan disease that is characterized by diverse metabolic defects. However, other than specific CFTR mutations, the factors that influence disease progression and severity remain poorly understood. Aberrant metabolite levels have been reported, but whether CFTR loss itself or secondary abnormalities (infection, inflammation, malnutrition, and various treatments) drive metabolic defects is uncertain. Here, we implemented comprehensive arteriovenous metabolomics in newborn CF pigs, and the results revealed CFTR as a bona fide regulator of metabolism. CFTR loss impaired metabolite exchange across organs, including disruption of lung uptake of fatty acids, yet enhancement of uptake of arachidonic acid, a precursor of proinflammatory cytokines. CFTR loss also impaired kidney reabsorption of amino acids and lactate and abolished renal glucose homeostasis. These and additional unexpected metabolic defects prior to disease manifestations reveal a fundamental role for CFTR in controlling multiorgan metabolism. Such discovery informs a basic understanding of CF, provides a foundation for future investigation, and has implications for developing therapies targeting only a single tissue.
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Affiliation(s)
- Hosung Bae
- Department of Biological Chemistry, University of California – Irvine, Irvine, California, USA
| | - Bo Ram Kim
- Department of Internal Medicine, Pappajohn Biomedical Institute, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA
- Howard Hughes Medical Institute, University of Iowa, Iowa City, Iowa, USA
| | - Sunhee Jung
- Department of Biological Chemistry, University of California – Irvine, Irvine, California, USA
| | - Johnny Le
- Department of Biological Chemistry, University of California – Irvine, Irvine, California, USA
| | | | - Wenjie Yu
- Department of Internal Medicine, Pappajohn Biomedical Institute, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA
- Howard Hughes Medical Institute, University of Iowa, Iowa City, Iowa, USA
| | - Sang Hee Park
- Department of Biological Chemistry, University of California – Irvine, Irvine, California, USA
| | - Brieanna M. Hilkin
- Department of Internal Medicine, Pappajohn Biomedical Institute, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA
| | - Nicholas D. Gansemer
- Department of Internal Medicine, Pappajohn Biomedical Institute, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA
| | - Linda S. Powers
- Department of Internal Medicine, Pappajohn Biomedical Institute, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA
| | - Taekyung Kang
- Department of Biological Chemistry, University of California – Irvine, Irvine, California, USA
| | - David K. Meyerholz
- Department of Pathology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA
| | - Victor L. Schuster
- Department of Internal Medicine, Albert Einstein College of Medicine, Bronx, New York, New York, USA
| | - Cholsoon Jang
- Department of Biological Chemistry, University of California – Irvine, Irvine, California, USA
- Center for Complex Biological Systems and
- Center for Epigenetics and Metabolism, University of California – Irvine, Irvine, California, USA
| | - Michael J. Welsh
- Department of Internal Medicine, Pappajohn Biomedical Institute, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA
- Howard Hughes Medical Institute, University of Iowa, Iowa City, Iowa, USA
- Department of Molecular Physiology and Biophysics, Pappajohn Biomedical Institute, Roy J. and Lucille A. Carver College of Medicine University of Iowa, Iowa City, Iowa, USA
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6
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Han X, Li D, Zhu Y, Schneider-Futschik EK. Recommended Tool Compounds for Modifying the Cystic Fibrosis Transmembrane Conductance Regulator Channel Variants. ACS Pharmacol Transl Sci 2024; 7:933-950. [PMID: 38633590 PMCID: PMC11019735 DOI: 10.1021/acsptsci.3c00362] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2023] [Revised: 02/20/2024] [Accepted: 02/23/2024] [Indexed: 04/19/2024]
Abstract
Cystic fibrosis (CF) is a genetic disorder arising from variations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, leading to multiple organ system defects. CFTR tool compounds are molecules that can modify the activity of the CFTR channel. Especially, patients that are currently not able to benefit from approved CFTR modulators, such as patients with rare CFTR variants, benefit from further research in discovering novel tools to modulate CFTR. This Review explores the development and classification of CFTR tool compounds, including CFTR blockers (CFTRinh-172, GlyH-101), potentiators (VRT-532, Genistein), correctors (VRT-325, Corr-4a), and other approved and unapproved modulators, with detailed descriptions and discussions for each compound. The challenges and future directions in targeting rare variants and optimizing drug delivery, and the potential synergistic effects in combination therapies are outlined. CFTR modulation holds promise not only for CF treatment but also for generating CF models that contribute to CF research and potentially treating other diseases such as secretory diarrhea. Therefore, continued research on CFTR tool compounds is critical.
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Affiliation(s)
- XiaoXuan Han
- Department of Biochemistry & Pharmacology,
School of Biomedical Sciences, Faculty of Medicine, Dentistry and
Health Sciences, The University of Melbourne, Parkville, VIC 3010, Australia
| | - Danni Li
- Department of Biochemistry & Pharmacology,
School of Biomedical Sciences, Faculty of Medicine, Dentistry and
Health Sciences, The University of Melbourne, Parkville, VIC 3010, Australia
| | - Yimin Zhu
- Department of Biochemistry & Pharmacology,
School of Biomedical Sciences, Faculty of Medicine, Dentistry and
Health Sciences, The University of Melbourne, Parkville, VIC 3010, Australia
| | - Elena K. Schneider-Futschik
- Department of Biochemistry & Pharmacology,
School of Biomedical Sciences, Faculty of Medicine, Dentistry and
Health Sciences, The University of Melbourne, Parkville, VIC 3010, Australia
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7
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Young PG, Levring J, Fiedorczuk K, Blanchard SC, Chen J. Structural basis for CFTR inhibition by CFTR inh-172. Proc Natl Acad Sci U S A 2024; 121:e2316675121. [PMID: 38422021 DOI: 10.1073/pnas.2316675121] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2023] [Accepted: 01/12/2024] [Indexed: 03/02/2024] Open
Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that regulates electrolyte and fluid balance in epithelial tissues. While activation of CFTR is vital to treating cystic fibrosis, selective inhibition of CFTR is a potential therapeutic strategy for secretory diarrhea and autosomal dominant polycystic kidney disease. Although several CFTR inhibitors have been developed by high-throughput screening, their modes of action remain elusive. In this study, we determined the structure of CFTR in complex with the inhibitor CFTRinh-172 to an overall resolution of 2.7 Å by cryogenic electron microscopy. We observe that CFTRinh-172 binds inside the pore near transmembrane helix 8, a critical structural element that links adenosine triphosphate hydrolysis with channel gating. Binding of CFTRinh-172 stabilizes a conformation in which the chloride selectivity filter is collapsed, and the pore is blocked from the extracellular side of the membrane. Single-molecule fluorescence resonance energy transfer experiments indicate that CFTRinh-172 inhibits channel gating without compromising nucleotide-binding domain dimerization. Together, these data reconcile previous biophysical observations and provide a molecular basis for the activity of this widely used CFTR inhibitor.
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Affiliation(s)
- Paul G Young
- Laboratory of Membrane Biology and Biophysics, The Rockefeller University, New York, NY 10065
- Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD-PhD Program, New York, NY 10065
| | - Jesper Levring
- Laboratory of Membrane Biology and Biophysics, The Rockefeller University, New York, NY 10065
| | - Karol Fiedorczuk
- Laboratory of Membrane Biology and Biophysics, The Rockefeller University, New York, NY 10065
| | - Scott C Blanchard
- Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38101
| | - Jue Chen
- Laboratory of Membrane Biology and Biophysics, The Rockefeller University, New York, NY 10065
- HHMI, The Rockefeller University, New York, NY 10065
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8
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Jones JT, Morelli KA, Vesely EM, Puerner CTS, Pavuluri CK, Ross BS, van Rhijn N, Bromley MJ, Cramer RA. The cystic fibrosis treatment Trikafta affects the growth, viability, and cell wall of Aspergillus fumigatus biofilms. mBio 2023; 14:e0151623. [PMID: 37830825 PMCID: PMC10653927 DOI: 10.1128/mbio.01516-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2023] [Accepted: 09/01/2023] [Indexed: 10/14/2023] Open
Abstract
IMPORTANCE PwCF commonly test positive for pathogenic fungi, and more than 90% of the cystic fibrosis patient population is approved for the modulator treatment, Trikafta. Therefore, it is critical to understand how fungal communities, specifically A. fumigatus, respond to Trikafta exposure. Therefore, we sought to determine whether Trikafta impacted the biology of A. fumigatus biofilms. Our data demonstrate that Trikafta reduces biomass in several laboratory strains as well as clinical strains isolated from the expectorated sputum of pwCF. Furthermore, Trikafta reduces fungal viability and the capacity of biofilms to recover following treatment. Of particular importance, Trikafta affects how A. fumigatus biofilms respond to cell wall stressors, suggesting that Trikafta modulates components of the cell wall. Since the cell wall directly affects how a host immune system will respond to and effectively neutralize pathogens, our work, demonstrating that Trikafta impacts the A. fumigatus cell wall, is potentially highly relevant to fungal-induced disease pathogenesis.
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Affiliation(s)
- Jane T. Jones
- Department of Microbiology and Immunology, Dartmouth Geisel School of Medicine, Hanover, New Hampshire, USA
| | - Kaesi A. Morelli
- Department of Microbiology and Immunology, Dartmouth Geisel School of Medicine, Hanover, New Hampshire, USA
| | - Elisa M. Vesely
- Department of Microbiology and Immunology, Dartmouth Geisel School of Medicine, Hanover, New Hampshire, USA
| | - Charles T. S. Puerner
- Department of Microbiology and Immunology, Dartmouth Geisel School of Medicine, Hanover, New Hampshire, USA
| | - Chetan K. Pavuluri
- Department of Microbiology and Immunology, Dartmouth Geisel School of Medicine, Hanover, New Hampshire, USA
| | - Brandon S. Ross
- Department of Microbiology and Immunology, Dartmouth Geisel School of Medicine, Hanover, New Hampshire, USA
| | - Norman van Rhijn
- Manchester Fungal Infection Group, Division of Evolution, Infection, and Genomics, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom
- Antimicrobial Resistance Network, University of Manchester, Manchester, United Kingdom
| | - Michael J. Bromley
- Manchester Fungal Infection Group, Division of Evolution, Infection, and Genomics, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom
- Antimicrobial Resistance Network, University of Manchester, Manchester, United Kingdom
| | - Robert A. Cramer
- Department of Microbiology and Immunology, Dartmouth Geisel School of Medicine, Hanover, New Hampshire, USA
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9
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Harras MF, Sabour R, Farghaly TA, Ibrahim MH. Drug Repurposing Approach in Developing New Furosemide Analogs as Antimicrobial Candidates and Anti-PBP: Design, Synthesis, and Molecular Docking. Bioorg Chem 2023; 137:106585. [PMID: 37163813 DOI: 10.1016/j.bioorg.2023.106585] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2022] [Revised: 04/09/2023] [Accepted: 05/01/2023] [Indexed: 05/12/2023]
Abstract
Multidrug-resistant microorganisms have become a global health problem, prompting research into new antimicrobials. Drug repurposing is a new technique in drug discovery used to improve drug development success. As a well-studied medication with a sulfonamide moiety, furosemide was chosen to study its antimicrobial effect on different microbial strains. In addition, a new family of furosemide analogs was investigated for their antimicrobial efficacy. According to the obtained results, the majority of the examined molecules exhibited potential antimicrobial activity. Compounds 3b and 4a had the best anti-MRSA results, with an MIC = 7.81 µg/mL. They also demonstrated potent anti-gram-negative activity against E. coli (MIC = 1.95 µg/mL and 3.91 µg/mL, respectively). A time-killing kinetics study against E. coli and MRSA showed bactericidal actions of 3b and 4a within 120-150 min. Moreover, an anti-PBP activity and an in vitro cytotoxicity evaluation were performed. Furosemide decreased the PBP2a levels in MRSA by 21.5% compared to the control. However, the furosemide analogs 3b and 4a demonstrated superior anti-PBP activity (55.9 and 57.1 % reduction in the expression of PBP2a, respectively). In addition, compound 4a was nearly nontoxic to normal WI-38 cells (IC50 = 248.60 μg /mL) indicating its high safety profile. Finally, the ability of furosemide and compounds 3b and 4a to bind to the target PBP2a enzyme has also been supported by molecular docking research.
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Affiliation(s)
- Marwa F Harras
- Department of Pharmaceutical Medicinal Chemistry and Drug Design, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo, Egypt
| | - Rehab Sabour
- Department of Pharmaceutical Medicinal Chemistry and Drug Design, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo, Egypt
| | - Thoraya A Farghaly
- Department of Chemistry, Faculty of Applied Science, Umm Al-Qura University, Makkah 24230, Saudi Arabia
| | - Mona H Ibrahim
- Department of Pharmaceutical Medicinal Chemistry and Drug Design, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo, Egypt
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10
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Lin J, Gettings SM, Talbi K, Schreiber R, Taggart MJ, Preller M, Kunzelmann K, Althaus M, Gray MA. Pharmacological inhibitors of the cystic fibrosis transmembrane conductance regulator exert off-target effects on epithelial cation channels. Pflugers Arch 2023; 475:167-179. [PMID: 36205782 PMCID: PMC9849171 DOI: 10.1007/s00424-022-02758-9] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2022] [Revised: 09/28/2022] [Accepted: 10/03/2022] [Indexed: 02/01/2023]
Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel and the epithelial Na+ channel (ENaC) play essential roles in transepithelial ion and fluid transport in numerous epithelial tissues. Inhibitors of both channels have been important tools for defining their physiological role in vitro. However, two commonly used CFTR inhibitors, CFTRinh-172 and GlyH-101, also inhibit non-CFTR anion channels, indicating they are not CFTR specific. However, the potential off-target effects of these inhibitors on epithelial cation channels has to date not been addressed. Here, we show that both CFTR blockers, at concentrations routinely employed by many researchers, caused a significant inhibition of store-operated calcium entry (SOCE) that was time-dependent, poorly reversible and independent of CFTR. Patch clamp experiments showed that both CFTRinh-172 and GlyH-101 caused a significant block of Orai1-mediated whole cell currents, establishing that they likely reduce SOCE via modulation of this Ca2+ release-activated Ca2+ (CRAC) channel. In addition to off-target effects on calcium channels, both inhibitors significantly reduced human αβγ-ENaC-mediated currents after heterologous expression in Xenopus oocytes, but had differential effects on δβγ-ENaC function. Molecular docking identified two putative binding sites in the extracellular domain of ENaC for both CFTR blockers. Together, our results indicate that caution is needed when using these two CFTR inhibitors to dissect the role of CFTR, and potentially ENaC, in physiological processes.
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Affiliation(s)
- JinHeng Lin
- grid.1006.70000 0001 0462 7212Biosciences Institute, Newcastle University, Newcastle upon Tyne, NE2 4HH UK ,grid.4991.50000 0004 1936 8948Present Address: Department of Pharmacology, University of Oxford, Oxford, OX1 3QT UK
| | - Sean M. Gettings
- grid.1006.70000 0001 0462 7212School of Natural and Environmental Sciences, Newcastle University, Newcastle upon Tyne, NE1 7RU UK
| | - Khaoula Talbi
- grid.7727.50000 0001 2190 5763Physiological Institute, University of Regensburg, 93053 Regensburg, Germany
| | - Rainer Schreiber
- grid.7727.50000 0001 2190 5763Physiological Institute, University of Regensburg, 93053 Regensburg, Germany
| | - Michael J. Taggart
- grid.1006.70000 0001 0462 7212Biosciences Institute, Newcastle University, Newcastle upon Tyne, NE2 4HH UK
| | - Matthias Preller
- grid.425058.e0000 0004 0473 3519Department of Natural Sciences/Institute for Functional Gene Analytics, Structural Biology Group, Bonn-Rhein-Sieg University of Applied Sciences, 53359 Rheinbach, Germany
| | - Karl Kunzelmann
- grid.7727.50000 0001 2190 5763Physiological Institute, University of Regensburg, 93053 Regensburg, Germany
| | - Mike Althaus
- grid.1006.70000 0001 0462 7212School of Natural and Environmental Sciences, Newcastle University, Newcastle upon Tyne, NE1 7RU UK ,grid.425058.e0000 0004 0473 3519Present Address: Department of Natural Sciences /Institute for Functional Gene Analytics, Ion Transport Physiology Group, Bonn-Rhein-Sieg University of Applied Sciences, 53359 Rheinbach, Germany
| | - Michael A. Gray
- grid.1006.70000 0001 0462 7212Biosciences Institute, Newcastle University, Newcastle upon Tyne, NE2 4HH UK
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11
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Russell T, Gangotia D, Barry G. Assessing the potential of repurposing ion channel inhibitors to treat emerging viral diseases and the role of this host factor in virus replication. Biomed Pharmacother 2022; 156:113850. [DOI: 10.1016/j.biopha.2022.113850] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2022] [Revised: 09/25/2022] [Accepted: 10/06/2022] [Indexed: 12/03/2022] Open
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12
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A Potent Inhibitor of the Cystic Fibrosis Transmembrane Conductance Regulator Blocks Disease and Morbidity Due to Toxigenic Vibrio cholerae. Toxins (Basel) 2022; 14:toxins14030225. [PMID: 35324722 PMCID: PMC8948642 DOI: 10.3390/toxins14030225] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2022] [Revised: 03/09/2022] [Accepted: 03/11/2022] [Indexed: 01/24/2023] Open
Abstract
Vibrio cholerae uses cholera toxin (CT) to cause cholera, a severe diarrheal disease in humans that can lead to death within hours of the onset of symptoms. The catalytic activity of CT in target epithelial cells increases cellular levels of 3',5'-cyclic AMP (cAMP), leading to the activation of the cystic fibrosis transmembrane conductance regulator (CFTR), an apical ion channel that transports chloride out of epithelial cells, resulting in an electrolyte imbalance in the intestinal lumen and massive water loss. Here we report that when administered perorally, benzopyrimido-pyrrolo-oxazinedione, (R)-BPO-27), a potent small molecule inhibitor of CFTR, blocked disease symptoms in a mouse model for acute diarrhea caused by toxigenic V. cholerae. We show that both (R)-BPO-27 and its racemic mixture, (R/S)-BPO-27, are able to protect mice from CT-dependent diarrheal disease and death. Furthermore, we show that, consistent with the ability of the compound to block the secretory diarrhea induced by CT, BPO-27 has a measurable effect on suppressing the gut replication and survival of V. cholerae, including a 2010 isolate from Haiti that is representative of the most predominant 'variant strains' that are causing epidemic and pandemic cholera worldwide. Our results suggest that BPO-27 should advance to human Phase I studies that could further address its safety and efficacy as therapeutic or preventative drug intervention for diarrheal syndromes, including cholera, that are mediated by CFTR channel activation.
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13
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Worgall TS. Sphingolipids and Asthma. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2022; 1372:145-155. [DOI: 10.1007/978-981-19-0394-6_10] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
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14
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Abstract
Chloride transport across cell membranes is broadly involved in epithelial fluid transport, cell volume and pH regulation, muscle contraction, membrane excitability, and organellar acidification. The human genome encodes at least 53 chloride-transporting proteins with expression in cell plasma or intracellular membranes, which include chloride channels, exchangers, and cotransporters, some having broad anion specificity. Loss-of-function mutations in chloride transporters cause a wide variety of human diseases, including cystic fibrosis, secretory diarrhea, kidney stones, salt-wasting nephropathy, myotonia, osteopetrosis, hearing loss, and goiter. Although impactful advances have been made in the past decade in drug treatment of cystic fibrosis using small molecule modulators of the defective cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, other chloride channels and solute carrier proteins (SLCs) represent relatively underexplored target classes for drug discovery. New opportunities have emerged for the development of chloride transport modulators as potential therapeutics for secretory diarrheas, constipation, dry eye disorders, kidney stones, polycystic kidney disease, hypertension, and osteoporosis. Approaches to chloride transport-targeted drug discovery are reviewed herein, with focus on chloride channel and exchanger classes in which recent preclinical advances have been made in the identification of small molecule modulators and in proof of concept testing in experimental animal models.
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Affiliation(s)
- Alan S Verkman
- Department of Medicine, University of California, San Francisco, California.,Department of Physiology, University of California, San Francisco, California
| | - Luis J V Galietta
- Telethon Institute of Genetics and Medicine, Pozzuoli, Italy.,Department of Translational Medical Sciences, University of Naples Federico II, Naples, Italy
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15
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Larsen MB, Choi JJ, Wang X, Myerburg MM, Frizzell RA, Bertrand CA. Separating the contributions of SLC26A9 and CFTR to anion secretion in primary human bronchial epithelia. Am J Physiol Lung Cell Mol Physiol 2021; 321:L1147-L1160. [PMID: 34668421 PMCID: PMC8715023 DOI: 10.1152/ajplung.00563.2020] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2020] [Revised: 10/01/2021] [Accepted: 10/15/2021] [Indexed: 11/22/2022] Open
Abstract
Aberrant anion secretion across the bronchial epithelium is associated with airway disease, most notably in cystic fibrosis. Although the cystic fibrosis transmembrane conductance regulator (CFTR) is recognized as the primary source of airway anion secretion, alternative anion transport mechanisms play a contributing role. An alternative anion transporter of growing interest is SLC26A9, a constitutively active chloride channel that has been shown to interact with CFTR and may also contribute to bicarbonate secretion. Interest in SLC26A9 has been fueled by genome-wide association studies that suggest it is a significant modifier of CF disease severity. Despite this growing evidence that SLC26A9 plays an important role in the airway, its presence and function in bronchial epithelia remain poorly understood, in part, because its activity is difficult to separate from the activity of CFTR. Here, we present results using primary human bronchial epithelia (HBE) from multiple patient sources to confirm that SLC26A9 mRNA is present in HBE and that its constitutive channel activity is unaffected by knockdown of CFTR. Furthermore, SLC26A9 and CFTR show differential responses to common inhibitors of anion secretion. Finally, we assess the impact of bicarbonate on the activity of SLC26A9 and CFTR. These results confirm that SLC26A9 is the primary source of constitutive anion secretion across HBE, and should inform future studies focused on activation of SLC26A9 as an alternative anion channel in CF. These results should provide a strong foundation to investigate how single-nucleotide polymorphisms in SLC26A9 modulate airway disease.
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Affiliation(s)
- Mads B Larsen
- Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
| | - Jeannie J Choi
- Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
| | - Xiaohui Wang
- Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
| | - Michael M Myerburg
- Department of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
| | - Raymond A Frizzell
- Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
- Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
| | - Carol A Bertrand
- Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
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16
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Becq F, Mirval S, Carrez T, Lévêque M, Billet A, Coraux C, Sage E, Cantereau A. The rescue of F508del-CFTR by elexacaftor/tezacaftor/ivacaftor (Trikafta) in human airway epithelial cells is underestimated due to the presence of ivacaftor. Eur Respir J 2021; 59:13993003.00671-2021. [PMID: 34266939 DOI: 10.1183/13993003.00671-2021] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2021] [Accepted: 06/25/2021] [Indexed: 11/05/2022]
Abstract
Trikafta, currently the leading therapeutic in Cystic Fibrosis (CF), has demonstrated a real clinical benefit. This treatment is the triple combination therapy of two folding correctors elexacaftor/tezacaftor (VX445/VX661) plus the gating potentiator ivacaftor (VX770). In this study, our aim was to compare the properties of F508del-CFTR in cells treated with either lumacaftor (VX809), tezacaftor, elexacaftor, elexacaftor/tezacaftor with or without ivacaftor. We studied F508del-CFTR function, maturation and membrane localisation by Ussing chamber and whole-cell patch clamp recordings, Western blot and immunolocalization experiments. With human primary airway epithelial cells and the cell lines CFBE and BHK expressing F508del, we found that, whereas the combination elexacaftor/tezacaftor/ivacaftor was efficient in rescuing F508del-CFTR abnormal maturation, apical membrane location and function, the presence of ivacaftor limits these effects. The basal F508del-CFTR short-circuit current was significantly increased by elexacaftor/tezacaftor/ivacaftor and elexacaftor/tezacaftor compared to other correctors and non-treated cells, an effect dependent on ivacaftor and cAMP. These results suggest that the level of the basal F508del-CFTR current might be a marker for correction efficacy in CF cells. When cells were treated with ivacaftor combined to any correctors, the F508del-CFTR current was unresponsive to the subsequently acute addition of ivacaftor unlike the CFTR potentiators genistein and Cact-A1 which increased elexacaftor/tezacaftor/ivacaftor and elexacaftor/tezacaftor-corrected F508del-CFTR currents. These findings show that ivacaftor reduces the correction efficacy of Trikafta. Thus, combining elexacaftor/tezacaftor with a different potentiator might improve the therapeutic efficacy for treating CF patients.
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Affiliation(s)
- Frédéric Becq
- Laboratoire Signalisation et Transports Ioniques Membranaires, Université de Poitiers, France
| | - Sandra Mirval
- Laboratoire Signalisation et Transports Ioniques Membranaires, Université de Poitiers, France
| | - Thomas Carrez
- Laboratoire Signalisation et Transports Ioniques Membranaires, Université de Poitiers, France.,ManRos therapeutics, Presqu'île de Perharidy, Roscoff, France
| | - Manuella Lévêque
- Laboratoire Signalisation et Transports Ioniques Membranaires, Université de Poitiers, France
| | - Arnaud Billet
- Laboratoire Signalisation et Transports Ioniques Membranaires, Université de Poitiers, France
| | - Christelle Coraux
- INSERM UMR-S 1250, Université de Reims-Champagne Ardenne, Reims, France
| | - Edouard Sage
- INRAE UMR 0892, Université Versailles-Saint-Quentin-en-Yvelines, Versailles, France.,Service de chirurgie thoracique et transplantation pulmonaire, Hôpital Foch, Suresnes, France
| | - Anne Cantereau
- Laboratoire Signalisation et Transports Ioniques Membranaires, Université de Poitiers, France
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17
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García R, Falduti C, Clauzure M, Jara R, Massip-Copiz MM, de Los Ángeles Aguilar M, Santa-Coloma TA, Valdivieso ÁG. CFTR chloride channel activity modulates the mitochondrial morphology in cultured epithelial cells. Int J Biochem Cell Biol 2021; 135:105976. [PMID: 33845203 DOI: 10.1016/j.biocel.2021.105976] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2021] [Revised: 04/01/2021] [Accepted: 04/02/2021] [Indexed: 01/10/2023]
Abstract
The impairment of the CFTR channel activity, a cAMP-activated chloride (Cl-) channel responsible for cystic fibrosis (CF), has been associated with a variety of mitochondrial alterations such as modified gene expression, impairment in oxidative phosphorylation, increased reactive oxygen species (ROS), and a disbalance in calcium homeostasis. The mechanisms by which these processes occur in CF are not fully understood. Previously, we demonstrated a reduced MTND4 expression and a failure in the mitochondrial complex I (mCx-I) activity in CF cells. Here we hypothesized that the activity of CFTR might modulate the mitochondrial fission/fusion balance, explaining the decreased mCx-I. The mitochondrial morphology and the levels of mitochondrial dynamic proteins MFN1 and DRP1 were analysed in IB3-1 CF cells, and S9 (IB3-1 expressing wt-CFTR), and C38 (IB3-1 expressing a truncated functional CFTR) cells. The mitochondrial morphology of IB3-1 cells compared to S9 and C38 cells showed that the impaired CFTR activity induced a fragmented mitochondrial network with increased rounded mitochondria and shorter branches. Similar results were obtained by using the CFTR pharmacological inhibitors CFTR(inh)-172 and GlyH101 on C38 cells. These morphological changes were accompanied by modifications in the levels of the mitochondrial dynamic proteins MFN1, DRP1, and p(616)-DRP1. IB3-1 CF cells treated with Mdivi-1, an inhibitor of mitochondrial fission, restored the mCx-I activity to values similar to those seen in S9 and C38 cells. These results suggest that the mitochondrial fission/fusion balance is regulated by the CFTR activity and might be a potential target to treat the impaired mCx-I activity in CF.
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Affiliation(s)
- Rocío García
- Laboratory of Cellular and Molecular Biology, Institute for Biomedical Research (BIOMED), School of Medical Sciences, Pontifical Catholic University of Argentina (UCA), the National Scientific and Technical Research Council of Argentina (CONICET), Buenos Aires, Argentina
| | - Camila Falduti
- Laboratory of Cellular and Molecular Biology, Institute for Biomedical Research (BIOMED), School of Medical Sciences, Pontifical Catholic University of Argentina (UCA), the National Scientific and Technical Research Council of Argentina (CONICET), Buenos Aires, Argentina
| | - Mariángeles Clauzure
- Laboratory of Cellular and Molecular Biology, Institute for Biomedical Research (BIOMED), School of Medical Sciences, Pontifical Catholic University of Argentina (UCA), the National Scientific and Technical Research Council of Argentina (CONICET), Buenos Aires, Argentina
| | - Raquel Jara
- Laboratory of Cellular and Molecular Biology, Institute for Biomedical Research (BIOMED), School of Medical Sciences, Pontifical Catholic University of Argentina (UCA), the National Scientific and Technical Research Council of Argentina (CONICET), Buenos Aires, Argentina
| | - María M Massip-Copiz
- Laboratory of Cellular and Molecular Biology, Institute for Biomedical Research (BIOMED), School of Medical Sciences, Pontifical Catholic University of Argentina (UCA), the National Scientific and Technical Research Council of Argentina (CONICET), Buenos Aires, Argentina
| | - María de Los Ángeles Aguilar
- Laboratory of Cellular and Molecular Biology, Institute for Biomedical Research (BIOMED), School of Medical Sciences, Pontifical Catholic University of Argentina (UCA), the National Scientific and Technical Research Council of Argentina (CONICET), Buenos Aires, Argentina
| | - Tomás A Santa-Coloma
- Laboratory of Cellular and Molecular Biology, Institute for Biomedical Research (BIOMED), School of Medical Sciences, Pontifical Catholic University of Argentina (UCA), the National Scientific and Technical Research Council of Argentina (CONICET), Buenos Aires, Argentina
| | - Ángel G Valdivieso
- Laboratory of Cellular and Molecular Biology, Institute for Biomedical Research (BIOMED), School of Medical Sciences, Pontifical Catholic University of Argentina (UCA), the National Scientific and Technical Research Council of Argentina (CONICET), Buenos Aires, Argentina.
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18
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Leenaars CH, Vries RBD, Reijmer J, Holthaus D, Visser D, Heming A, Elzinga J, Kempkes RW, Beumer W, Punt C, Meijboom FL, Ritskes-Hoitinga M. Animal models for cystic fibrosis: a systematic search and mapping review of the literature. Part 2: nongenetic models. Lab Anim 2021; 55:307-316. [PMID: 33557683 DOI: 10.1177/0023677221990688] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Various animal models are available to study cystic fibrosis (CF). These models may help to enhance our understanding of the pathology and contribute to the development of new treatments. We systematically searched all publications on CF animal models. Because of the large number of models retrieved, we split this mapping review into two parts. Previously, we presented the genetic CF animal models. In this paper we present the nongenetic CF animal models. While genetic animal models may, in theory, be preferable for genetic diseases, the phenotype of a genetic model does not automatically resemble human disease. Depending on the research question, other animal models may thus be more informative.We searched Pubmed and Embase and identified 12,303 unique publications (after duplicate removal). All references were screened for inclusion by two independent reviewers. The genetic animal models for CF (from 636 publications) were previously described. The non-genetic CF models (from 189 publications) are described in this paper, grouped by model type: infection-based, pharmacological, administration of human materials, xenografts and other. As before for the genetic models, an overview of basic model characteristics and outcome measures is provided. This CF animal model overview can be the basis for an objective, evidence-based model choice for specific research questions. Besides, it can help to retrieve relevant background literature on outcome measures of interest.
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Affiliation(s)
- Cathalijn Hc Leenaars
- SYRCLE, Department for Health Evidence, Radboud Institute for Health Sciences, Radboud University Medical Center, The Netherlands.,Faculty of Veterinary Medicine, Department of Animals in Science and Society, Utrecht University, The Netherlands.,Institute for Laboratory Animal Science, Hannover Medical School, Germany
| | - Rob Bm de Vries
- SYRCLE, Department for Health Evidence, Radboud Institute for Health Sciences, Radboud University Medical Center, The Netherlands
| | - Joey Reijmer
- SYRCLE, Department for Health Evidence, Radboud Institute for Health Sciences, Radboud University Medical Center, The Netherlands
| | - David Holthaus
- SYRCLE, Department for Health Evidence, Radboud Institute for Health Sciences, Radboud University Medical Center, The Netherlands
| | - Damian Visser
- SYRCLE, Department for Health Evidence, Radboud Institute for Health Sciences, Radboud University Medical Center, The Netherlands
| | - Anna Heming
- SYRCLE, Department for Health Evidence, Radboud Institute for Health Sciences, Radboud University Medical Center, The Netherlands
| | - Janneke Elzinga
- SYRCLE, Department for Health Evidence, Radboud Institute for Health Sciences, Radboud University Medical Center, The Netherlands
| | - Rosalie Wm Kempkes
- SYRCLE, Department for Health Evidence, Radboud Institute for Health Sciences, Radboud University Medical Center, The Netherlands
| | | | - Carine Punt
- ProQR Therapeutics NV,Leiden, the Netherlands; Present position: BunyaVax BV, Lelystad, The Netherlands
| | - Franck Lb Meijboom
- Faculty of Veterinary Medicine, Department of Animals in Science and Society, Utrecht University, The Netherlands
| | - Merel Ritskes-Hoitinga
- SYRCLE, Department for Health Evidence, Radboud Institute for Health Sciences, Radboud University Medical Center, The Netherlands.,Department of Clinical Medicine, Aarhus University, Denmark
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19
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Heras AF, Veerappan A, Silver RB, Emala CW, Worgall TS, Perez-Zoghbi J, Worgall S. Increasing Sphingolipid Synthesis Alleviates Airway Hyperreactivity. Am J Respir Cell Mol Biol 2020; 63:690-698. [PMID: 32706610 DOI: 10.1165/rcmb.2020-0194oc] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Impaired sphingolipid synthesis is linked genetically to childhood asthma and functionally to airway hyperreactivity (AHR). The objective was to investigate whether sphingolipid synthesis could be a target for asthma therapeutics. The effects of GlyH-101 and fenretinide via modulation of de novo sphingolipid synthesis on AHR was evaluated in mice deficient in SPT (serine palmitoyl-CoA transferase), the rate-limiting enzyme of sphingolipid synthesis. The drugs were also used directly in human airway smooth-muscle and epithelial cells to evaluate changes in de novo sphingolipid metabolites and calcium release. GlyH-101 and fenretinide increased sphinganine and dihydroceramides (de novo sphingolipid metabolites) in lung epithelial and airway smooth-muscle cells, decreased the intracellular calcium concentration in airway smooth-muscle cells, and decreased agonist-induced contraction in proximal and peripheral airways. GlyH-101 also decreased AHR in SPT-deficient mice in vivo. This study identifies the manipulation of sphingolipid synthesis as a novel metabolic therapeutic strategy to alleviate AHR.
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Affiliation(s)
| | | | | | | | - Tilla S Worgall
- Department of Pathology and Cell Biology, Columbia University, New York, New York
| | | | - Stefan Worgall
- Department of Pediatrics.,Department of Genetic Medicine, and.,Drukier Institute for Children's Health, Weill Cornell Medicine, New York, New York; and
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20
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de Jonge HR, Ardelean MC, Bijvelds MJC, Vergani P. Strategies for cystic fibrosis transmembrane conductance regulator inhibition: from molecular mechanisms to treatment for secretory diarrhoeas. FEBS Lett 2020; 594:4085-4108. [PMID: 33113586 PMCID: PMC7756540 DOI: 10.1002/1873-3468.13971] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2020] [Revised: 09/22/2020] [Accepted: 10/15/2020] [Indexed: 02/06/2023]
Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR) is an unusual ABC transporter. It acts as an anion‐selective channel that drives osmotic fluid transport across many epithelia. In the gut, CFTR is crucial for maintaining fluid and acid‐base homeostasis, and its activity is tightly controlled by multiple neuro‐endocrine factors. However, microbial toxins can disrupt this intricate control mechanism and trigger protracted activation of CFTR. This results in the massive faecal water loss, metabolic acidosis and dehydration that characterize secretory diarrhoeas, a major cause of malnutrition and death of children under 5 years of age. Compounds that inhibit CFTR could improve emergency treatment of diarrhoeal disease. Drawing on recent structural and functional insight, we discuss how existing CFTR inhibitors function at the molecular and cellular level. We compare their mechanisms of action to those of inhibitors of related ABC transporters, revealing some unexpected features of drug action on CFTR. Although challenges remain, especially relating to the practical effectiveness of currently available CFTR inhibitors, we discuss how recent technological advances might help develop therapies to better address this important global health need.
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Affiliation(s)
- Hugo R. de Jonge
- Department of Gastroenterology & HepatologyErasmus University Medical CenterRotterdamThe Netherlands
| | - Maria C. Ardelean
- Department of Neuroscience, Physiology and PharmacologyUniversity College LondonUK
- Department of Natural SciencesUniversity College LondonUK
| | - Marcel J. C. Bijvelds
- Department of Gastroenterology & HepatologyErasmus University Medical CenterRotterdamThe Netherlands
| | - Paola Vergani
- Department of Neuroscience, Physiology and PharmacologyUniversity College LondonUK
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21
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Inhibition of CFTR-mediated intestinal chloride secretion by a fungus-derived arthropsolide A: Mechanism of action and anti-diarrheal efficacy. Eur J Pharmacol 2020; 885:173393. [DOI: 10.1016/j.ejphar.2020.173393] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2020] [Revised: 07/10/2020] [Accepted: 07/20/2020] [Indexed: 11/19/2022]
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22
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Abstract
Cerebral edema is a pathological hallmark of various central nervous system (CNS) insults, including traumatic brain injury (TBI) and excitotoxic injury such as stroke. Due to the rigidity of the skull, edema-induced increase of intracranial fluid significantly complicates severe CNS injuries by raising intracranial pressure and compromising perfusion. Mortality due to cerebral edema is high. With mortality rates up to 80% in severe cases of stroke, it is the leading cause of death within the first week. Similarly, cerebral edema is devastating for patients of TBI, accounting for up to 50% mortality. Currently, the available treatments for cerebral edema include hypothermia, osmotherapy, and surgery. However, these treatments only address the symptoms and often elicit adverse side effects, potentially in part due to non-specificity. There is an urgent need to identify effective pharmacological treatments for cerebral edema. Currently, ion channels represent the third-largest target class for drug development, but their roles in cerebral edema remain ill-defined. The present review aims to provide an overview of the proposed roles of ion channels and transporters (including aquaporins, SUR1-TRPM4, chloride channels, glucose transporters, and proton-sensitive channels) in mediating cerebral edema in acute ischemic stroke and TBI. We also focus on the pharmacological inhibitors for each target and potential therapeutic strategies that may be further pursued for the treatment of cerebral edema.
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23
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Rodrat M, Jantarajit W, Ng DRS, Harvey BSJ, Liu J, Wilkinson WJ, Charoenphandhu N, Sheppard DN. Carbon monoxide-releasing molecules inhibit the cystic fibrosis transmembrane conductance regulator Cl - channel. Am J Physiol Lung Cell Mol Physiol 2020; 319:L997-L1009. [PMID: 32936026 DOI: 10.1152/ajplung.00440.2019] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
The gasotransmitter carbon monoxide (CO) regulates fluid and electrolyte movements across epithelial tissues. However, its action on anion channels is incompletely understood. Here, we investigate the direct action of CO on the cystic fibrosis transmembrane conductance regulator (CFTR) by applying CO-releasing molecules (CO-RMs) to the intracellular side of excised inside-out membrane patches from cells heterologously expressing wild-type human CFTR. Addition of increasing concentrations of tricarbonyldichlororuthenium(II) dimer (CORM-2) (1-300 μM) inhibited CFTR channel activity, whereas the control RuCl3 (100 μM) was without effect. CORM-2 predominantly inhibited CFTR by decreasing the frequency of channel openings and, hence, open probability (Po). But, it also reduced current flow through open channels with very fast kinetics, particularly at elevated concentrations. By contrast, the chemically distinct CO-releasing molecule CORM-3 inhibited CFTR by decreasing Po without altering current flow through open channels. Neither depolarizing the membrane voltage nor raising the ATP concentration on the intracellular side of the membrane affected CFTR inhibition by CORM-2. Interestingly, CFTR inhibition by CORM-2, but not by CFTRinh-172, was prevented by prior enhancement of channel activity by the clinically approved CFTR potentiator ivacaftor. Similarly, when added after CORM-2, ivacaftor completely relieved CFTR inhibition. In conclusion, CORM-2 has complex effects on wild-type human CFTR consistent with allosteric inhibition and open-channel blockade. Inhibition of CFTR by CO-releasing molecules suggests that CO regulates CFTR activity and that the gasotransmitter has tissue-specific effects on epithelial ion transport. The action of ivacaftor on CFTR Cl- channels inhibited by CO potentially expands the drug's clinical utility.
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Affiliation(s)
- Mayuree Rodrat
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, United Kingdom.,Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok, Thailand.,Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand
| | - Walailak Jantarajit
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, United Kingdom.,Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok, Thailand.,Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand
| | - Demi R S Ng
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, United Kingdom
| | - Bartholomew S J Harvey
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, United Kingdom
| | - Jia Liu
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, United Kingdom
| | | | - Narattaphol Charoenphandhu
- Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok, Thailand.,Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand.,Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, Thailand.,The Academy of Science, The Royal Society of Thailand, Dusit, Bangkok, Thailand
| | - David N Sheppard
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, United Kingdom
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24
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Laselva O, Bartlett C, Popa A, Ouyang H, Gunawardena TNA, Gonska T, Moraes TJ, Bear CE. Emerging preclinical modulators developed for F508del-CFTR have the potential to be effective for ORKAMBI resistant processing mutants. J Cyst Fibros 2020; 20:106-119. [PMID: 32741662 DOI: 10.1016/j.jcf.2020.07.015] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2020] [Revised: 07/07/2020] [Accepted: 07/22/2020] [Indexed: 10/23/2022]
Abstract
BACKGROUND F508del is prototypical of Class 2 CFTR mutations associated with protein misprocessing and reduced function. Corrector compounds like lumacaftor partially rescue the processing defect of F508del-CFTR whereas potentiators like ivacaftor, enhance its channel activity once trafficked to the cell surface. We asked if emerging modulators developed for F508del-CFTR can rescue Class 2 mutations previously shown to be poorly responsive to lumacaftor and ivacaftor. METHODS Rescue of mutant CFTRs by the correctors: AC1, AC2-1 or AC2-2 and the potentiator, AP2, was studied in HEK-293 cells and in primary human nasal epithelial (HNE) cultures, using a membrane potential assay and Ussing chamber, respectively. RESULTS In HEK-293 cells, we found that a particular combination of corrector molecules (AC1 plus AC2-1) and a potentiator (AP2) was effective in rescuing both the misprocessing and reduced function of M1101K and G85E respectively. These findings were recapitulated in patient-derived nasal cultures, although another corrector combination, AC1 plus AC2-2 also improved misprocessing in these primary tissues. Interestingly, while this corrector combination only led to a modest increase in the abundance of mature N1303K-CFTR it did enable its functional expression in the presence of the potentiator, AP2, in part, because the nominal corrector, AC2-2 also exhibits potentiator activity. CONCLUSIONS Strategic combinations of novel modulators can potentially rescue Class 2 mutants thought to be relatively unresponsive to lumacaftor and ivacaftor.
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Affiliation(s)
- Onofrio Laselva
- Programme in Molecular Medicine, Hospital for Sick Children, Toronto, Canada; Department of Physiology, University of Toronto, Toronto, Canada
| | - Claire Bartlett
- Programme in Translational Medicine, Hospital for Sick Children, Toronto, Canada
| | - Alec Popa
- Programme in Molecular Medicine, Hospital for Sick Children, Toronto, Canada
| | - Hong Ouyang
- Programme in Translational Medicine, Hospital for Sick Children, Toronto, Canada
| | | | - Tanja Gonska
- Programme in Translational Medicine, Hospital for Sick Children, Toronto, Canada; Department of Paediatrics, University of Toronto, Toronto, Canada
| | - Theo J Moraes
- Programme in Translational Medicine, Hospital for Sick Children, Toronto, Canada; Department of Paediatrics, University of Toronto, Toronto, Canada
| | - Christine E Bear
- Programme in Molecular Medicine, Hospital for Sick Children, Toronto, Canada; Department of Physiology, University of Toronto, Toronto, Canada; Department of Biochemistry, University of Toronto, Toronto, Canada.
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25
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Khalaf M, Scott-Ward T, Causer A, Saynor Z, Shepherd A, Górecki D, Lewis A, Laight D, Shute J. Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in Human Lung Microvascular Endothelial Cells Controls Oxidative Stress, Reactive Oxygen-Mediated Cell Signaling and Inflammatory Responses. Front Physiol 2020; 11:879. [PMID: 32848840 PMCID: PMC7403513 DOI: 10.3389/fphys.2020.00879] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2019] [Accepted: 06/29/2020] [Indexed: 12/19/2022] Open
Abstract
Background Perturbation of endothelial function in people with cystic fibrosis (CF) has been reported, which may be associated with endothelial cell expression of the cystic fibrosis transmembrane conductance regulator (CFTR). Previous reports indicate that CFTR activity upregulates endothelial barrier function, endothelial nitric oxide synthase (eNOS) expression and NO release, while limiting interleukin-8 (IL-8) release, in human umbilical vein endothelial cells (HUVECs) in cell culture. In view of reported microvascular dysfunction in people with CF we investigated the role of CFTR expression and activity in the regulation of oxidative stress, cell signaling and inflammation in human lung microvascular endothelial cells (HLMVECs) in cell culture. Methods HLMVECs were cultured in the absence and presence of the CFTR inhibitor GlyH-101 and CFTR siRNA. CFTR expression was analyzed using qRT-PCR, immunocytochemistry (IHC) and western blot, and function by membrane potential assay. IL-8 expression was analyzed using qRT-PCR and ELISA. Nrf2 expression, and NF-κB and AP-1 activation were determined using IHC and western blot. The role of the epidermal growth factor receptor (EGFR) in CFTR signaling was investigated using the EGFR tyrosine kinase inhibitor AG1478. Oxidative stress was measured as intracellular ROS and hydrogen peroxide (H2O2) concentration. VEGF and SOD-2 were measured in culture supernatants by ELISA. Results HLMVECs express low levels of CFTR that increase following inhibition of CFTR activity. Inhibition of CFTR, significantly increased intracellular ROS and H2O2 levels over 30 min and significantly decreased Nrf2 expression by 70% while increasing SOD-2 expression over 24 h. CFTR siRNA significantly increased constitutive expression of IL-8 by HLMVECs. CFTR inhibition activated the AP-1 pathway and increased IL-8 expression, without effect on NF-κB activity. Conversely, TNF-α activated the NF-κB pathway and increased IL-8 expression. The effects of TNF-α and GlyH-101 on IL-8 expression were additive and inhibited by AG1478. Inhibition of both CFTR and EGFR in HLMVECs significantly increased VEGF expression. The antioxidant N-acetyl cysteine significantly reduced ROS production and the increase in IL-8 and VEGF expression following CFTR inhibition. Conclusion Functional endothelial CFTR limits oxidative stress and contributes to the normal anti-inflammatory state of HLMVECs. Therapeutic strategies to restore endothelial CFTR function in CF are warranted.
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Affiliation(s)
- Maha Khalaf
- School of Pharmacy and Biomedical Sciences, Institute of Biological and Biomedical Sciences, University of Portsmouth, Portsmouth, United Kingdom
| | - Toby Scott-Ward
- School of Pharmacy and Biomedical Sciences, Institute of Biological and Biomedical Sciences, University of Portsmouth, Portsmouth, United Kingdom
| | - Adam Causer
- Department of Sport and Exercise Science, University of Portsmouth, Portsmouth, United Kingdom
| | - Zoe Saynor
- Department of Sport and Exercise Science, University of Portsmouth, Portsmouth, United Kingdom
| | - Anthony Shepherd
- Department of Sport and Exercise Science, University of Portsmouth, Portsmouth, United Kingdom
| | - Dariusz Górecki
- School of Pharmacy and Biomedical Sciences, Institute of Biological and Biomedical Sciences, University of Portsmouth, Portsmouth, United Kingdom
| | - Anthony Lewis
- School of Pharmacy and Biomedical Sciences, Institute of Biological and Biomedical Sciences, University of Portsmouth, Portsmouth, United Kingdom
| | - David Laight
- School of Pharmacy and Biomedical Sciences, Institute of Biological and Biomedical Sciences, University of Portsmouth, Portsmouth, United Kingdom
| | - Janis Shute
- School of Pharmacy and Biomedical Sciences, Institute of Biological and Biomedical Sciences, University of Portsmouth, Portsmouth, United Kingdom
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26
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Rehman T, Thornell IM, Pezzulo AA, Thurman AL, Romano Ibarra GS, Karp PH, Tan P, Duffey ME, Welsh MJ. TNFα and IL-17 alkalinize airway surface liquid through CFTR and pendrin. Am J Physiol Cell Physiol 2020; 319:C331-C344. [PMID: 32432926 PMCID: PMC7500220 DOI: 10.1152/ajpcell.00112.2020] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
The pH of airway surface liquid (ASL) is a key factor that determines respiratory host defense; ASL acidification impairs and alkalinization enhances key defense mechanisms. Under healthy conditions, airway epithelia secrete base ([Formula: see text]) and acid (H+) to control ASL pH (pHASL). Neutrophil-predominant inflammation is a hallmark of several airway diseases, and TNFα and IL-17 are key drivers. However, how these cytokines perturb pHASL regulation is uncertain. In primary cultures of differentiated human airway epithelia, TNFα decreased and IL-17 did not change pHASL. However, the combination (TNFα+IL-17) markedly increased pHASL by increasing [Formula: see text] secretion. TNFα+IL-17 increased expression and function of two apical [Formula: see text] transporters, CFTR anion channels and pendrin Cl-/[Formula: see text] exchangers. Both were required for maximal alkalinization. TNFα+IL-17 induced pendrin expression primarily in secretory cells where it was coexpressed with CFTR. Interestingly, significant pendrin expression was not detected in CFTR-rich ionocytes. These results indicate that TNFα+IL-17 stimulate [Formula: see text] secretion via CFTR and pendrin to alkalinize ASL, which may represent an important defense mechanism in inflamed airways.
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Affiliation(s)
- Tayyab Rehman
- Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa
| | - Ian M Thornell
- Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa
| | - Alejandro A Pezzulo
- Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa
| | - Andrew L Thurman
- Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa
| | - Guillermo S Romano Ibarra
- Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa
| | - Philip H Karp
- Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa.,Howard Hughes Medical Institute, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa
| | - Ping Tan
- Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa
| | - Michael E Duffey
- Department of Physiology and Biophysics, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, New York
| | - Michael J Welsh
- Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa.,Howard Hughes Medical Institute, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa.,Department of Molecular Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa
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27
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Tarique AA, Evron T, Zhang G, Tepper MA, Morshed MM, Andersen ISG, Begum N, Sly PD, Fantino E. Anti-inflammatory effects of lenabasum, a cannabinoid receptor type 2 agonist, on macrophages from cystic fibrosis. J Cyst Fibros 2020; 19:823-829. [PMID: 32387042 DOI: 10.1016/j.jcf.2020.03.015] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2019] [Revised: 02/12/2020] [Accepted: 03/24/2020] [Indexed: 12/24/2022]
Abstract
BACKGROUND Lenabasum is an oral synthetic cannabinoid receptor type 2 agonist previously shown to reduce the production of key airway pro-inflammatory cytokines known to play a role in cystic fibrosis (CF). In a double-blinded, randomized, placebo-control phase 2 study, lenabasum lowered the rate of pulmonary exacerbation among patients with CF. The present study was undertaken to investigate anti-inflammatory mechanisms of lenabasum exhibits in CF macrophages. METHODS We used monocyte-derived macrophages (MDMs) from healthy donors (n = 15), MDMs with CFTR inhibited with C-172 (n = 5) and MDMs from patients with CF (n = 4). Monocytes were differentiated to macrophages and polarized into classically activated (M1) macrophages by LPS or alternatively activated (M2) macrophages by IL-13 in presence or absence of lenabasum. RESULTS Lenabasum had no effect on differentiation, polarization and function of macrophages from healthy individuals. However, in CF macrophages lenabasum downregulated macrophage polarization into the pro-inflammatory M1 phenotype and secretion of the pro-inflammatory cytokines IL-8 and TNF-α in a dose-dependent manner. An improvement in phagocytic activity was also observed following lenabasum treatment. Although lenabasum did not restore the impaired polarization of anti-inflammatory M2 macrophage, it reduced the levels of IL-13 and enhanced the endocytic function of CF MDMs. The effects of lenabasum on MDMs with CFTR inhibited by C-172 were not as obvious. CONCLUSION In CF macrophages lenabasum modulates macrophage polarization and function in vitro in a way that would reduce inflammation in vivo. Further studies are warranted to determine the link between activating the CBR2 receptor and CFTR.
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Affiliation(s)
- Abdullah A Tarique
- Child Health Research Centre (CHRC), The University of Queensland, Brisbane, Australia
| | - Tama Evron
- Corbus Pharmaceuticals, Inc., Norwood, MA, USA
| | | | | | - Mohammed M Morshed
- Child Health Research Centre (CHRC), The University of Queensland, Brisbane, Australia
| | - Isabella S G Andersen
- Child Health Research Centre (CHRC), The University of Queensland, Brisbane, Australia
| | - Nelufa Begum
- Child Health Research Centre (CHRC), The University of Queensland, Brisbane, Australia
| | - Peter D Sly
- Child Health Research Centre (CHRC), The University of Queensland, Brisbane, Australia.
| | - Emmanuelle Fantino
- Child Health Research Centre (CHRC), The University of Queensland, Brisbane, Australia
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28
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Akita H, Yoshie S, Ishida T, Takeishi Y, Hazama A. Negative chronotropic and inotropic effects of lubiprostone on iPS cell-derived cardiomyocytes via activation of CFTR. BMC Complement Med Ther 2020; 20:118. [PMID: 32306956 PMCID: PMC7169008 DOI: 10.1186/s12906-020-02923-6] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2020] [Accepted: 04/07/2020] [Indexed: 11/10/2022] Open
Abstract
Background Lubiprostone (LBP) is a novel chloride channel opener that has been reported to activate chloride channel protein 2 (ClC-2) and cystic fibrosis transmembrane conductance regulator (CFTR). LBP facilitates fluid secretion by activating CFTR in the intestine and is used as a drug for treating chronic constipation. While ClC-2 and CFTR expression has been confirmed in cardiomyocytes (CMs), the effect of LBP on CMs has not yet been investigated. Thus, the present study aimed to investigate the effect of LBP on CMs using mouse-induced pluripotent stem (iPS) cell-derived CMs (iPS-CMs). Methods We induced mouse iPS cells into CMs through embryoid body (EB) formation. We compared the differentiated cells to CMs isolated from adult and fetal mice using gene expression, spontaneous beating rate, and contraction ratio analyses. Results Gene expression analysis revealed that, in the iPS-CMs, the mRNA expression of the undifferentiated cell markers Rex1 and Nanog decreased, whereas the expression of the unique cardiomyocyte markers cardiac troponin I (cTnI) and cardiac troponin T (cTNT), increased. Immunostaining showed that the localization of cTnI and connexin-43 in the iPS-CMs was similar to that in the primary fetal CMs (FCMs) and adult CMs (ACMs). LBP decreased the spontaneous beating rate of the iPS-CMs and FCMs, and decreased the contraction ratio of the iPS-CMs and ACMs. The reduction in the beating rate and contraction ratio caused by LBP was inhibited by glycine hydrazide (GlyH), which is a CFTR inhibitor. Conclusion These results suggest that LBP stimulates CFTR in CMs and that LBP has negative chronotropic and inotropic effects on CMs. LBP may be useful for treating cardiac diseases such as heart failure, ischemia, and arrhythmia.
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Affiliation(s)
- Hiraku Akita
- Department of Cardiovascular Medicine, Fukushima Medical University, Fukushima, Japan
| | - Susumu Yoshie
- Department of Cellular and Integrative Physiology, School of Medicine, Fukushima Medical University, 1 Hikarigaoka, Fukushima City, 960-1295, Japan
| | - Takafumi Ishida
- Department of Cardiovascular Medicine, Fukushima Medical University, Fukushima, Japan
| | - Yasuchika Takeishi
- Department of Cardiovascular Medicine, Fukushima Medical University, Fukushima, Japan
| | - Akihiro Hazama
- Department of Cellular and Integrative Physiology, School of Medicine, Fukushima Medical University, 1 Hikarigaoka, Fukushima City, 960-1295, Japan.
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29
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Lim DJ, McCormick J, Skinner D, Zhang S, Elder JB, McLemore JG, Allen M, West JM, Grayson JW, Rowe SM, Woodworth BA, Cho DY. Controlled delivery of ciprofloxacin and ivacaftor via sinus stent in a preclinical model of Pseudomonas sinusitis. Int Forum Allergy Rhinol 2019; 10:481-488. [PMID: 31872532 DOI: 10.1002/alr.22514] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2019] [Revised: 11/09/2019] [Accepted: 12/03/2019] [Indexed: 12/14/2022]
Abstract
BACKGROUND Pseudomonas aeruginosa is common in chronic rhinosinusitus (CRS) and frequently resistant to antibiotic treatment. We recently described the ciprofloxacin and ivacaftor-releasing biodegradable sinus stent (CISS)-a drug-delivery system that administers ciprofloxacin and the mucociliary activator (ivacaftor) at high local concentrations with prolonged mucosal contact time and sustained delivery. The objective of this study is to evaluate the efficacy of the CISS in a rabbit model of P aeruginosa (PAO1 strain) sinusitis. METHODS Ciprofloxacin/ivacaftor (double layer) was coated on biodegradable poly-D/L-lactic acid (PLLA). A total of 10 sinus stents (5 bare PLLA stent controls, 5 CISSs) were placed unilaterally in rabbit maxillary sinuses via dorsal sinusotomy after inducing infection for 1 week with PAO1. Animals were assessed 3 weeks after stent insertion with sinus culture, nasal endoscopy, computed tomography scan, histopathology, and in-vivo sinus potential difference (SPD) assay. RESULTS Rabbits treated with CISS had significant reductions in computed tomography (∆ Kerschner scale: control, 0.55 ± 0.92; CISS, -5.92 ± 1.69; p = 0.024) and endoscopy (control, 4.0 ± 0.0; CISS, 1.875 ± 0.74; p = 0.003) scores. A 2-log reduction of PAO1 was observed (control, -2.14 ± 0.77; CISS, 1.84 ± 1.52; p = 0.047). SPD revealed significantly increased Cl- transport in the CISS group compared with the control group (Cl- -free + forskolin ΔPD: control, -4.23 ± 1.04 mV; CISS, -18.36 ± 6.31 mV; p = 0.026). Finally, marked improvements were noted in the histology of the mucosa and submucosa in treated animals. CONCLUSION The CISS had robust clinical efficacy in treating P aeruginosa rabbit sinusitis. The innovative design of double-layered drug coating on the surface of the biodegradable stent may provide therapeutic advantages over current treatment strategies for P aeruginosa sinusitis.
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Affiliation(s)
- Dong-Jin Lim
- Department of Otolaryngology-Head & Neck Surgery, University of Alabama at Birmingham, Birmingham, AL
| | - Justin McCormick
- Department of Otolaryngology-Head & Neck Surgery, University of Alabama at Birmingham, Birmingham, AL
| | - Daniel Skinner
- Department of Otolaryngology-Head & Neck Surgery, University of Alabama at Birmingham, Birmingham, AL
| | - Shaoyan Zhang
- Department of Otolaryngology-Head & Neck Surgery, University of Alabama at Birmingham, Birmingham, AL
| | - Jeffrey B Elder
- Department of Otolaryngology-Head & Neck Surgery, University of Alabama at Birmingham, Birmingham, AL
| | - John G McLemore
- Department of Otolaryngology-Head & Neck Surgery, University of Alabama at Birmingham, Birmingham, AL
| | - Mark Allen
- Department of Otolaryngology-Head & Neck Surgery, University of Alabama at Birmingham, Birmingham, AL
| | - John Martin West
- Department of Otolaryngology-Head & Neck Surgery, University of Alabama at Birmingham, Birmingham, AL
| | - Jessica W Grayson
- Department of Otolaryngology-Head & Neck Surgery, University of Alabama at Birmingham, Birmingham, AL
| | - Steven M Rowe
- Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL.,Department of Medicine, University of Alabama at Birmingham, Birmingham, AL.,Department of Pediatrics, University of Alabama at Birmingham, Birmingham, AL.,Department of Cell Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL
| | - Bradford A Woodworth
- Department of Otolaryngology-Head & Neck Surgery, University of Alabama at Birmingham, Birmingham, AL.,Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL
| | - Do-Yeon Cho
- Department of Otolaryngology-Head & Neck Surgery, University of Alabama at Birmingham, Birmingham, AL.,Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL
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30
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Shamsuddin AKM, Quinton PM. Concurrent absorption and secretion of airway surface liquids and bicarbonate secretion in human bronchioles. Am J Physiol Lung Cell Mol Physiol 2019; 316:L953-L960. [PMID: 30838869 PMCID: PMC6589593 DOI: 10.1152/ajplung.00545.2018] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2018] [Revised: 03/01/2019] [Accepted: 03/01/2019] [Indexed: 01/15/2023] Open
Abstract
Although small airways account for the largest fraction of the total conducting airway surfaces, the epithelial fluid and electrolyte transport in small, native airway epithelia has not been well characterized. Investigations have been limited, no doubt, by the complex tissue architecture as well as by its inaccessibility, small dimensions, and lack of applicable assays, especially in human tissues. To better understand how the critically thin layer of airway surface liquid (ASL) is maintained, we applied a "capillary"-Ussing chamber (area ≈1 mm2) to measure ion transport properties of bronchioles with diameters of ~2 mm isolated from resected specimens of excised human lungs. We found that the small human airway, constitutively and concurrently, secretes and absorbs fluid as observed in porcine small airways (50). We found that the human bronchiolar epithelium is also highly anion selective and constitutively secretes bicarbonate ( HCO 3 - ), which can be enhanced pharmacologically by cAMP as well as Ca2+-mediated agonists. Concurrent secretion and absorption of surface liquid along with HCO 3 - secretion help explain how the delicate volume of the fluid lining the human small airway is physiologically buffered and maintained in a steady state that avoids desiccating or flooding the small airway with ASL.
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Affiliation(s)
- A K M Shamsuddin
- Department of Pediatrics, University of California , San Diego, California
| | - Paul M Quinton
- Department of Pediatrics, University of California , San Diego, California
- Division of Biomedical Sciences, University of California , Riverside, California
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31
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Csanády L, Vergani P, Gadsby DC. STRUCTURE, GATING, AND REGULATION OF THE CFTR ANION CHANNEL. Physiol Rev 2019; 99:707-738. [PMID: 30516439 DOI: 10.1152/physrev.00007.2018] [Citation(s) in RCA: 164] [Impact Index Per Article: 27.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) belongs to the ATP binding cassette (ABC) transporter superfamily but functions as an anion channel crucial for salt and water transport across epithelial cells. CFTR dysfunction, because of mutations, causes cystic fibrosis (CF). The anion-selective pore of the CFTR protein is formed by its two transmembrane domains (TMDs) and regulated by its cytosolic domains: two nucleotide binding domains (NBDs) and a regulatory (R) domain. Channel activation requires phosphorylation of the R domain by cAMP-dependent protein kinase (PKA), and pore opening and closing (gating) of phosphorylated channels is driven by ATP binding and hydrolysis at the NBDs. This review summarizes available information on structure and mechanism of the CFTR protein, with a particular focus on atomic-level insight gained from recent cryo-electron microscopic structures and on the molecular mechanisms of channel gating and its regulation. The pharmacological mechanisms of small molecules targeting CFTR's ion channel function, aimed at treating patients suffering from CF and other diseases, are briefly discussed.
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Affiliation(s)
- László Csanády
- Department of Medical Biochemistry, Semmelweis University , Budapest , Hungary ; MTA-SE Ion Channel Research Group, Budapest , Hungary ; Department of Neuroscience, Physiology and Pharmacology, University College London , London , United Kingdom ; and Laboratory of Cardiac/Membrane Physiology, The Rockefeller University , New York, New York
| | - Paola Vergani
- Department of Medical Biochemistry, Semmelweis University , Budapest , Hungary ; MTA-SE Ion Channel Research Group, Budapest , Hungary ; Department of Neuroscience, Physiology and Pharmacology, University College London , London , United Kingdom ; and Laboratory of Cardiac/Membrane Physiology, The Rockefeller University , New York, New York
| | - David C Gadsby
- Department of Medical Biochemistry, Semmelweis University , Budapest , Hungary ; MTA-SE Ion Channel Research Group, Budapest , Hungary ; Department of Neuroscience, Physiology and Pharmacology, University College London , London , United Kingdom ; and Laboratory of Cardiac/Membrane Physiology, The Rockefeller University , New York, New York
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Okada Y, Okada T, Sato-Numata K, Islam MR, Ando-Akatsuka Y, Numata T, Kubo M, Shimizu T, Kurbannazarova RS, Marunaka Y, Sabirov RZ. Cell Volume-Activated and Volume-Correlated Anion Channels in Mammalian Cells: Their Biophysical, Molecular, and Pharmacological Properties. Pharmacol Rev 2019; 71:49-88. [PMID: 30573636 DOI: 10.1124/pr.118.015917] [Citation(s) in RCA: 60] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
There are a number of mammalian anion channel types associated with cell volume changes. These channel types are classified into two groups: volume-activated anion channels (VAACs) and volume-correlated anion channels (VCACs). VAACs can be directly activated by cell swelling and include the volume-sensitive outwardly rectifying anion channel (VSOR), which is also called the volume-regulated anion channel; the maxi-anion channel (MAC or Maxi-Cl); and the voltage-gated anion channel, chloride channel (ClC)-2. VCACs can be facultatively implicated in, although not directly activated by, cell volume changes and include the cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, the Ca2+-activated Cl- channel (CaCC), and the acid-sensitive (or acid-stimulated) outwardly rectifying anion channel. This article describes the phenotypical properties and activation mechanisms of both groups of anion channels, including accumulating pieces of information on the basis of recent molecular understanding. To that end, this review also highlights the molecular identities of both anion channel groups; in addition to the molecular identities of ClC-2 and CFTR, those of CaCC, VSOR, and Maxi-Cl were recently identified by applying genome-wide approaches. In the last section of this review, the most up-to-date information on the pharmacological properties of both anion channel groups, especially their half-maximal inhibitory concentrations (IC50 values) and voltage-dependent blocking, is summarized particularly from the standpoint of pharmacological distinctions among them. Future physiologic and pharmacological studies are definitely warranted for therapeutic targeting of dysfunction of VAACs and VCACs.
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Affiliation(s)
- Yasunobu Okada
- Departments of Physiology and Systems Bioscience (Y.O.) and Molecular Cell Physiology (Y.M.), Kyoto Prefectural University of Medicine, Kyoto, Japan; Division of Cell Signaling, National Institute for Physiological Sciences, Okazaki, Japan (Y.O., T.O., M.R.I., M.K., R.Z.S.); Department of Physiology, School of Medicine, Fukuoka University, Fukuoka, Japan (K.S.-N., T.N.); Department of Cell Physiology, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Japan (Y.A.-A.); Department of Pharmaceutical Physiology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan (T.S.); Laboratory of Molecular Physiology, Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan, Tashkent, Uzbekistan (R.S.K., R.Z.S.); and Research Institute for Clinical Physiology, Kyoto Industrial Health Association, Kyoto, Japan (Y.M.)
| | - Toshiaki Okada
- Departments of Physiology and Systems Bioscience (Y.O.) and Molecular Cell Physiology (Y.M.), Kyoto Prefectural University of Medicine, Kyoto, Japan; Division of Cell Signaling, National Institute for Physiological Sciences, Okazaki, Japan (Y.O., T.O., M.R.I., M.K., R.Z.S.); Department of Physiology, School of Medicine, Fukuoka University, Fukuoka, Japan (K.S.-N., T.N.); Department of Cell Physiology, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Japan (Y.A.-A.); Department of Pharmaceutical Physiology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan (T.S.); Laboratory of Molecular Physiology, Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan, Tashkent, Uzbekistan (R.S.K., R.Z.S.); and Research Institute for Clinical Physiology, Kyoto Industrial Health Association, Kyoto, Japan (Y.M.)
| | - Kaori Sato-Numata
- Departments of Physiology and Systems Bioscience (Y.O.) and Molecular Cell Physiology (Y.M.), Kyoto Prefectural University of Medicine, Kyoto, Japan; Division of Cell Signaling, National Institute for Physiological Sciences, Okazaki, Japan (Y.O., T.O., M.R.I., M.K., R.Z.S.); Department of Physiology, School of Medicine, Fukuoka University, Fukuoka, Japan (K.S.-N., T.N.); Department of Cell Physiology, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Japan (Y.A.-A.); Department of Pharmaceutical Physiology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan (T.S.); Laboratory of Molecular Physiology, Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan, Tashkent, Uzbekistan (R.S.K., R.Z.S.); and Research Institute for Clinical Physiology, Kyoto Industrial Health Association, Kyoto, Japan (Y.M.)
| | - Md Rafiqul Islam
- Departments of Physiology and Systems Bioscience (Y.O.) and Molecular Cell Physiology (Y.M.), Kyoto Prefectural University of Medicine, Kyoto, Japan; Division of Cell Signaling, National Institute for Physiological Sciences, Okazaki, Japan (Y.O., T.O., M.R.I., M.K., R.Z.S.); Department of Physiology, School of Medicine, Fukuoka University, Fukuoka, Japan (K.S.-N., T.N.); Department of Cell Physiology, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Japan (Y.A.-A.); Department of Pharmaceutical Physiology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan (T.S.); Laboratory of Molecular Physiology, Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan, Tashkent, Uzbekistan (R.S.K., R.Z.S.); and Research Institute for Clinical Physiology, Kyoto Industrial Health Association, Kyoto, Japan (Y.M.)
| | - Yuhko Ando-Akatsuka
- Departments of Physiology and Systems Bioscience (Y.O.) and Molecular Cell Physiology (Y.M.), Kyoto Prefectural University of Medicine, Kyoto, Japan; Division of Cell Signaling, National Institute for Physiological Sciences, Okazaki, Japan (Y.O., T.O., M.R.I., M.K., R.Z.S.); Department of Physiology, School of Medicine, Fukuoka University, Fukuoka, Japan (K.S.-N., T.N.); Department of Cell Physiology, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Japan (Y.A.-A.); Department of Pharmaceutical Physiology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan (T.S.); Laboratory of Molecular Physiology, Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan, Tashkent, Uzbekistan (R.S.K., R.Z.S.); and Research Institute for Clinical Physiology, Kyoto Industrial Health Association, Kyoto, Japan (Y.M.)
| | - Tomohiro Numata
- Departments of Physiology and Systems Bioscience (Y.O.) and Molecular Cell Physiology (Y.M.), Kyoto Prefectural University of Medicine, Kyoto, Japan; Division of Cell Signaling, National Institute for Physiological Sciences, Okazaki, Japan (Y.O., T.O., M.R.I., M.K., R.Z.S.); Department of Physiology, School of Medicine, Fukuoka University, Fukuoka, Japan (K.S.-N., T.N.); Department of Cell Physiology, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Japan (Y.A.-A.); Department of Pharmaceutical Physiology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan (T.S.); Laboratory of Molecular Physiology, Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan, Tashkent, Uzbekistan (R.S.K., R.Z.S.); and Research Institute for Clinical Physiology, Kyoto Industrial Health Association, Kyoto, Japan (Y.M.)
| | - Machiko Kubo
- Departments of Physiology and Systems Bioscience (Y.O.) and Molecular Cell Physiology (Y.M.), Kyoto Prefectural University of Medicine, Kyoto, Japan; Division of Cell Signaling, National Institute for Physiological Sciences, Okazaki, Japan (Y.O., T.O., M.R.I., M.K., R.Z.S.); Department of Physiology, School of Medicine, Fukuoka University, Fukuoka, Japan (K.S.-N., T.N.); Department of Cell Physiology, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Japan (Y.A.-A.); Department of Pharmaceutical Physiology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan (T.S.); Laboratory of Molecular Physiology, Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan, Tashkent, Uzbekistan (R.S.K., R.Z.S.); and Research Institute for Clinical Physiology, Kyoto Industrial Health Association, Kyoto, Japan (Y.M.)
| | - Takahiro Shimizu
- Departments of Physiology and Systems Bioscience (Y.O.) and Molecular Cell Physiology (Y.M.), Kyoto Prefectural University of Medicine, Kyoto, Japan; Division of Cell Signaling, National Institute for Physiological Sciences, Okazaki, Japan (Y.O., T.O., M.R.I., M.K., R.Z.S.); Department of Physiology, School of Medicine, Fukuoka University, Fukuoka, Japan (K.S.-N., T.N.); Department of Cell Physiology, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Japan (Y.A.-A.); Department of Pharmaceutical Physiology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan (T.S.); Laboratory of Molecular Physiology, Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan, Tashkent, Uzbekistan (R.S.K., R.Z.S.); and Research Institute for Clinical Physiology, Kyoto Industrial Health Association, Kyoto, Japan (Y.M.)
| | - Ranohon S Kurbannazarova
- Departments of Physiology and Systems Bioscience (Y.O.) and Molecular Cell Physiology (Y.M.), Kyoto Prefectural University of Medicine, Kyoto, Japan; Division of Cell Signaling, National Institute for Physiological Sciences, Okazaki, Japan (Y.O., T.O., M.R.I., M.K., R.Z.S.); Department of Physiology, School of Medicine, Fukuoka University, Fukuoka, Japan (K.S.-N., T.N.); Department of Cell Physiology, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Japan (Y.A.-A.); Department of Pharmaceutical Physiology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan (T.S.); Laboratory of Molecular Physiology, Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan, Tashkent, Uzbekistan (R.S.K., R.Z.S.); and Research Institute for Clinical Physiology, Kyoto Industrial Health Association, Kyoto, Japan (Y.M.)
| | - Yoshinori Marunaka
- Departments of Physiology and Systems Bioscience (Y.O.) and Molecular Cell Physiology (Y.M.), Kyoto Prefectural University of Medicine, Kyoto, Japan; Division of Cell Signaling, National Institute for Physiological Sciences, Okazaki, Japan (Y.O., T.O., M.R.I., M.K., R.Z.S.); Department of Physiology, School of Medicine, Fukuoka University, Fukuoka, Japan (K.S.-N., T.N.); Department of Cell Physiology, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Japan (Y.A.-A.); Department of Pharmaceutical Physiology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan (T.S.); Laboratory of Molecular Physiology, Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan, Tashkent, Uzbekistan (R.S.K., R.Z.S.); and Research Institute for Clinical Physiology, Kyoto Industrial Health Association, Kyoto, Japan (Y.M.)
| | - Ravshan Z Sabirov
- Departments of Physiology and Systems Bioscience (Y.O.) and Molecular Cell Physiology (Y.M.), Kyoto Prefectural University of Medicine, Kyoto, Japan; Division of Cell Signaling, National Institute for Physiological Sciences, Okazaki, Japan (Y.O., T.O., M.R.I., M.K., R.Z.S.); Department of Physiology, School of Medicine, Fukuoka University, Fukuoka, Japan (K.S.-N., T.N.); Department of Cell Physiology, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Japan (Y.A.-A.); Department of Pharmaceutical Physiology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan (T.S.); Laboratory of Molecular Physiology, Institute of Bioorganic Chemistry, Academy of Sciences of Uzbekistan, Tashkent, Uzbekistan (R.S.K., R.Z.S.); and Research Institute for Clinical Physiology, Kyoto Industrial Health Association, Kyoto, Japan (Y.M.)
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Gong X, Liao Y, Ahner A, Larsen MB, Wang X, Bertrand CA, Frizzell RA. Different SUMO paralogues determine the fate of wild-type and mutant CFTRs: biogenesis versus degradation. Mol Biol Cell 2018; 30:4-16. [PMID: 30403549 PMCID: PMC6337916 DOI: 10.1091/mbc.e18-04-0252] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
A pathway for cystic fibrosis transmembrane conductance regulator (CFTR) degradation is initiated by Hsp27, which cooperates with Ubc9 and binds to the common F508del mutant to modify it with SUMO-2/3. These SUMO paralogues form polychains, which are recognized by the ubiquitin ligase, RNF4, for proteosomal degradation. Here, protein array analysis identified the SUMO E3, protein inhibitor of activated STAT 4 (PIAS4), which increased wild-type (WT) and F508del CFTR biogenesis in CFBE airway cells. PIAS4 increased immature CFTR threefold and doubled expression of mature CFTR, detected by biochemical and functional assays. In cycloheximide chase assays, PIAS4 slowed immature F508del degradation threefold and stabilized mature WT CFTR at the plasma membrance. PIAS4 knockdown reduced WT and F508del CFTR expression by 40–50%, suggesting a physiological role in CFTR biogenesis. PIAS4 modified F508del CFTR with SUMO-1 in vivo and reduced its conjugation to SUMO-2/3. These SUMO paralogue-specific effects of PIAS4 were reproduced in vitro using purified F508del nucleotide-binding domain 1 and SUMOylation reaction components. PIAS4 reduced endogenous ubiquitin conjugation to F508del CFTR by ∼50% and blocked the impact of RNF4 on mutant CFTR disposal. These findings indicate that different SUMO paralogues determine the fates of WT and mutant CFTRs, and they suggest that a paralogue switch during biogenesis can direct these proteins to different outcomes: biogenesis versus degradation.
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Affiliation(s)
- Xiaoyan Gong
- Departments of Pediatrics and Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15224
| | - Yong Liao
- Departments of Pediatrics and Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15224
| | - Annette Ahner
- Departments of Pediatrics and Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15224
| | - Mads Breum Larsen
- Departments of Pediatrics and Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15224
| | - Xiaohui Wang
- Departments of Pediatrics and Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15224
| | - Carol A Bertrand
- Departments of Pediatrics and Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15224
| | - Raymond A Frizzell
- Departments of Pediatrics and Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15224
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Dey I, Bradbury NA. Physiology of the Gut: Experimental Models for Investigating Intestinal Fluid and Electrolyte Transport. CURRENT TOPICS IN MEMBRANES 2018; 81:337-381. [PMID: 30243437 DOI: 10.1016/bs.ctm.2018.08.003] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Once thought to be exclusively an absorptive tissue, the intestine is now recognized as an important secretory tissue, playing a key role in body ion and fluid homeostasis. Given the intestine's role in fluid homeostasis, it is not surprising that important clinical pathologies arise from imbalances in fluid absorption and secretion. Perhaps the most important examples of this can be seen in enterotoxigenic secretory diarrheas with extreme fluid secretion, and Cystic Fibrosis with little or no fluid secretion. A mechanistic understanding of the cellular pathways regulating ion and fluid transport has been obtained from a variety of approaches and model systems. These have ranged from the intact intestine to a single intestinal epithelial cell type. Although for many years a reductionist approach has held sway for investigating intestinal transport, the growing realization that physiologic processes should really be examined within a physiological context has seen a marked increase in studies using models that are essentially mini-intestines in a dish. The aim of this chapter is to provide a historical context for our understanding of intestinal ion and fluid transport, and to highlight the model systems that have been used to acquire this knowledge.
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Affiliation(s)
- Isha Dey
- Department of Physiology and Biophysics, Chicago Medical School, North Chicago, IL, United States
| | - Neil A Bradbury
- Department of Physiology and Biophysics, Chicago Medical School, North Chicago, IL, United States
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35
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Gera A, Mohan C, Arora S. Synthesis of Phthaloylglycyl Hydrazide Derivatives: Selective Protection of Phthalimide Group from Hydrazinolysis. Curr Org Synth 2018. [DOI: 10.2174/1570179415666180601083256] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Background:
N-phthalimide amino acid hydrazide is a class of compounds that have the potential
therapeutic use. In general, hydrazinolysis of N-substituted amino acid(s) ester removes the ester group and
yields the corresponding hydrazide. However, in case if N-substitution group is phthalimide, phthalimide
group is cleaved and not the ester group. The resulted compound, therefore, is amino acid ester rather than Nphthalimide
amino acid hydrazide. The above class of compounds, because of susceptibility of phthalimide
group to hydrazinolysis, has previously been synthesized by a lengthy three-step procedure.
Objective:
N-phthaloylglycyl hydrazide was synthesized by using new efficient, simplified, one step process.
Hydrazone derivatives from substituted benzaldehydes, and substituted furaldehyde were also synthesized.
Method:
N-phthaloylglycyl hydrazide was synthesized from the corresponding carboxylic acid using 1-Ethyl-
3-(3-dimethylaminopropyl)carbodiimide (EDC) as a coupling agent and hydroxybenzotriazole (HOBt) as an
activator. Hydrazone derivatives were synthesized by condensation of N-phthaloylglycyl hydrazide with substituted
benzaldehyde/substituted furaldehydes. All the compounds were characterized by IR, 1H-NMR, 13CNMR,
mass spectroscopy and elemental analysis.
Results:
The presence of EDC/HOBt resulted in hydrazinolysis of the carboxylic acid group and not the
phthalimide group. N-phthaloylglycyl hydrazide was synthesized in good yield.
Conclusion:
We report the improved process of the synthesis of N-phthaloylglycyl hydrazide. This is the first
report where stability of phthaloyl amino acid compound to hydrazine is demonstrated. The reaction may be
explored for the reaction schemes where stability of phthalimide group to hydrazinolysis is required.
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Affiliation(s)
- Ankur Gera
- Chitkara College of Pharmacy, Chitkara University, Chandigarh-Patiala National Highway (NH-64), Punjab 140401, India
| | - Chander Mohan
- Rayat-Bahra Institute of Pharmacy, VPO-Bohan, Hoshiarpur, Punjab 146104, India
| | - Sandeep Arora
- Chitkara College of Pharmacy, Chitkara University, Chandigarh-Patiala National Highway (NH-64), Punjab 140401, India
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Adam D, Bilodeau C, Sognigbé L, Maillé É, Ruffin M, Brochiero E. CFTR rescue with VX-809 and VX-770 favors the repair of primary airway epithelial cell cultures from patients with class II mutations in the presence of Pseudomonas aeruginosa exoproducts. J Cyst Fibros 2018; 17:705-714. [PMID: 29661510 DOI: 10.1016/j.jcf.2018.03.010] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2017] [Revised: 03/08/2018] [Accepted: 03/25/2018] [Indexed: 12/18/2022]
Abstract
BACKGROUND Progressive airway damage due to bacterial infections, especially with Pseudomonas aeruginosa remains the first cause of morbidity and mortality in CF patients. Our previous work revealed a repair delay in CF airway epithelia compared to non-CF. This delay was partially prevented after CFTR correction (with VRT-325) in the absence of infection. Our goals were now to evaluate the effect of the Orkambi combination (CFTR VX-809 corrector + VX-770 potentiator) on the repair of CF primary airway epithelia, in infectious conditions. METHODS Primary airway epithelial cell cultures from patients with class II mutations were mechanically injured and wound healing rates and transepithelial resistances were monitored after CFTR rescue, in the absence and presence of P. aeruginosa exoproducts. RESULTS Our data revealed that combined treatment with VX-809 and VX-770 elicited a greater beneficial impact on airway epithelial repair than VX-809 alone, in the absence of infection. The treatment with Orkambi was effective not only in airway epithelial cell cultures from patients homozygous for the F508del mutation but also from heterozygous patients carrying F508del and another class II mutation (N1303 K, I507del). The stimulatory effect of the Orkambi treatment was prevented by CFTR inhibition with GlyH101. Finally, Orkambi combination elicited a slight but significant improvement in airway epithelial repair and transepithelial resistance, despite the presence of P. aeruginosa exoproducts. CONCLUSIONS Our findings indicate that Orkambi may favor airway epithelial integrity in CF patients with class II mutations. Complementary approaches would however be needed to further improve CFTR rescue and airway epithelial repair.
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Affiliation(s)
- Damien Adam
- Centre de recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), Montréal, Québec, Canada; Département de médecine, Université de Montréal, Montréal, Québec, Canada.
| | - Claudia Bilodeau
- Centre de recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), Montréal, Québec, Canada; Département de médecine, Université de Montréal, Montréal, Québec, Canada.
| | - Laura Sognigbé
- Centre de recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), Montréal, Québec, Canada; Département de médecine, Université de Montréal, Montréal, Québec, Canada.
| | - Émilie Maillé
- Centre de recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), Montréal, Québec, Canada.
| | - Manon Ruffin
- Centre de recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), Montréal, Québec, Canada; Département de médecine, Université de Montréal, Montréal, Québec, Canada.
| | - Emmanuelle Brochiero
- Centre de recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), Montréal, Québec, Canada; Département de médecine, Université de Montréal, Montréal, Québec, Canada.
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Hwang TC, Yeh JT, Zhang J, Yu YC, Yeh HI, Destefano S. Structural mechanisms of CFTR function and dysfunction. J Gen Physiol 2018; 150:539-570. [PMID: 29581173 PMCID: PMC5881446 DOI: 10.1085/jgp.201711946] [Citation(s) in RCA: 85] [Impact Index Per Article: 12.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2017] [Accepted: 03/05/2018] [Indexed: 12/18/2022] Open
Abstract
Hwang et al. integrate new structural insights with prior functional studies to reveal the functional anatomy of CFTR chloride channels. Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) chloride channel plays a critical role in regulating transepithelial movement of water and electrolyte in exocrine tissues. Malfunction of the channel because of mutations of the cftr gene results in CF, the most prevalent lethal genetic disease among Caucasians. Recently, the publication of atomic structures of CFTR in two distinct conformations provides, for the first time, a clear overview of the protein. However, given the highly dynamic nature of the interactions among CFTR’s various domains, better understanding of the functional significance of these structures requires an integration of these new structural insights with previously established biochemical/biophysical studies, which is the goal of this review.
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Affiliation(s)
- Tzyh-Chang Hwang
- Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO .,Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, MO.,Department of Biological Engineering, University of Missouri, Columbia, MO
| | - Jiunn-Tyng Yeh
- Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO
| | - Jingyao Zhang
- Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO.,Department of Biological Engineering, University of Missouri, Columbia, MO
| | - Ying-Chun Yu
- Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO.,Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, MO
| | - Han-I Yeh
- Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO.,Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, MO
| | - Samantha Destefano
- Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO.,Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, MO
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38
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Ruffin M, Roussel L, Maillé É, Rousseau S, Brochiero E. Vx-809/Vx-770 treatment reduces inflammatory response to Pseudomonas aeruginosa in primary differentiated cystic fibrosis bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol 2017; 314:L635-L641. [PMID: 29351441 DOI: 10.1152/ajplung.00198.2017] [Citation(s) in RCA: 34] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Cystic fibrosis patients exhibit chronic Pseudomonas aeruginosa respiratory infections and sustained proinflammatory state favoring lung tissue damage and remodeling, ultimately leading to respiratory failure. Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function is associated with MAPK hyperactivation and increased cytokines expression, such as interleukin-8 [chemoattractant chemokine (C-X-C motif) ligand 8 (CXCL8)]. Recently, new therapeutic strategies directly targeting the basic CFTR defect have been developed, and ORKAMBI (Vx-809/Vx-770 combination) is the only Food and Drug Administration-approved treatment for CF patients homozygous for the F508del mutation. Here we aimed to determine the effect of the Vx-809/Vx-770 combination on the induction of the inflammatory response by fully differentiated primary bronchial epithelial cell cultures from CF patients carrying F508del mutations, following exposure to P. aeruginosa exoproducts. Our data unveiled that CFTR functional rescue with Vx-809/Vx-770 drastically reduces CXCL8 (as well as CXCL1 and CXCL2) transcripts and p38 MAPK phosphorylation in response to P. aeruginosa exposure through a CFTR-dependent mechanism. These results suggest that ORKAMBI has anti-inflammatory properties that could decrease lung inflammation and contribute to the observed beneficial impact of this treatment in CF patients.
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Affiliation(s)
- Manon Ruffin
- Centre de recherche du centre hospitalier de l'université de Montréal (CRCHUM) , Montréal, Québec , Canada.,Département de Médecine, Université de Montréal , Montréal, Québec , Canada
| | - Lucie Roussel
- The Meakins-Christie Laboratories at the Research Institute of the McGill University Health Center, McGill University , Montréal, Québec , Canada.,Department of Medicine, McGill University , Montréal, Québec , Canada
| | - Émilie Maillé
- Centre de recherche du centre hospitalier de l'université de Montréal (CRCHUM) , Montréal, Québec , Canada
| | - Simon Rousseau
- The Meakins-Christie Laboratories at the Research Institute of the McGill University Health Center, McGill University , Montréal, Québec , Canada.,Department of Medicine, McGill University , Montréal, Québec , Canada
| | - Emmanuelle Brochiero
- Centre de recherche du centre hospitalier de l'université de Montréal (CRCHUM) , Montréal, Québec , Canada.,Département de Médecine, Université de Montréal , Montréal, Québec , Canada
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39
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Yu B, Jiang Y, Zhang B, Yang H, Ma T. Resveratrol dimer trans-ε-viniferin prevents rotaviral diarrhea in mice by inhibition of the intestinal calcium-activated chloride channel. Pharmacol Res 2017; 129:453-461. [PMID: 29155014 DOI: 10.1016/j.phrs.2017.11.016] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/26/2017] [Revised: 10/27/2017] [Accepted: 11/13/2017] [Indexed: 12/11/2022]
Abstract
We previously identified, by a natural-product screen, resveratrol oligomers as inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Here, we report the resveratrol dimer trans-ε-viniferin (TV) and tetramer r-2-viniferin (RV) as inhibitors of the intestinal calcium-activated chloride channel (CaCC) and demonstrate their antisecretory efficacy in a neonatal mouse model of rotaviral diarrhea. Short-circuit measurements show inhibition of CaCC current in the human colonic cell line HT-29 by TV and RV with IC50∼1 and 20μM, respectively. TV primarily inhibited the physiologically relevant, long-term CaCC current following agonist stimulation, without effect on cytoplasmic Ca2+ signaling. TV and RV inhibited short-circuit current in mouse colon as well. In a neonatal mouse model of rotaviral secretory diarrhea produced by oral inoculation with rotavirus, 2μg TV or 11μg RV inhibited secretory diarrhea by >50%, without effect on the rotaviral infection. Our results support the antisecretory efficacy of non-toxic, natural-product resveratrol oligomers for diarrheas produced by CaCC activation. Because these compounds also inhibit the CFTR chloride channel, they may be useful for antisecretory therapy of a wide range of diarrheas.
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Affiliation(s)
- Bo Yu
- School of Life Sciences, Liaoning Provincial Key Laboratory of Biotechnology and Drug Discovery, Liaoning Normal University, Dalian, PR China
| | - Yu Jiang
- School of Life Sciences, Liaoning Provincial Key Laboratory of Biotechnology and Drug Discovery, Liaoning Normal University, Dalian, PR China; College of Life Science, Jilin Agricultural University, Changchun, PR China
| | - Bo Zhang
- Institute of Traditional Chinese Medicine, Nanjing University of Chinese Medicine, Nanjing, PR China
| | - Hong Yang
- School of Life Sciences, Liaoning Provincial Key Laboratory of Biotechnology and Drug Discovery, Liaoning Normal University, Dalian, PR China.
| | - Tonghui Ma
- Institute of Traditional Chinese Medicine, Nanjing University of Chinese Medicine, Nanjing, PR China.
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40
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Valdivieso ÁG, Mori C, Clauzure M, Massip-Copiz M, Santa-Coloma TA. CFTR modulates RPS27 gene expression using chloride anion as signaling effector. Arch Biochem Biophys 2017; 633:103-109. [DOI: 10.1016/j.abb.2017.09.014] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2017] [Revised: 09/13/2017] [Accepted: 09/20/2017] [Indexed: 12/13/2022]
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41
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Abstract
JGP hosts key papers that shaped the epithelial transport field. Epithelia define the boundaries of the body and often transfer solutes and water from outside to inside (absorption) or from inside to outside (secretion). Those processes involve dual plasma membranes with different transport components that interact with each other. Understanding those functions has entailed breaking down the problem to analyze properties of individual membranes (apical vs. basolateral) and individual transport proteins. It also requires understanding of how those components interact and how they are regulated. This article outlines the modern history of this research as reflected by publications in The Journal of General Physiology.
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Affiliation(s)
- Lawrence G Palmer
- Department of Physiology and Biophysics, Weill-Cornell Medical College, New York, NY
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42
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Gonçalves C, Gomez JP, Même W, Rasolonjatovo B, Gosset D, Nedellec S, Hulin P, Huin C, Le Gall T, Montier T, Lehn P, Pichon C, Guégan P, Cheradame H, Midoux P. Curcumin/poly(2-methyl-2-oxazoline-b-tetrahydrofuran-b-2-methyl-2-oxazoline) formulation: An improved penetration and biological effect of curcumin in F508del-CFTR cell lines. Eur J Pharm Biopharm 2017; 117:168-181. [DOI: 10.1016/j.ejpb.2017.04.015] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2016] [Revised: 03/14/2017] [Accepted: 04/13/2017] [Indexed: 01/29/2023]
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43
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CFTR-dependent defect in alternatively-activated macrophages in cystic fibrosis. J Cyst Fibros 2017; 16:475-482. [DOI: 10.1016/j.jcf.2017.03.011] [Citation(s) in RCA: 41] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2016] [Revised: 02/28/2017] [Accepted: 03/24/2017] [Indexed: 12/31/2022]
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44
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Stauffer BB, Cui G, Cottrill KA, Infield DT, McCarty NA. Bacterial Sphingomyelinase is a State-Dependent Inhibitor of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR). Sci Rep 2017; 7:2931. [PMID: 28592822 PMCID: PMC5462758 DOI: 10.1038/s41598-017-03103-2] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2016] [Accepted: 04/24/2017] [Indexed: 02/07/2023] Open
Abstract
Sphingomyelinase C (SMase) inhibits CFTR chloride channel activity in multiple cell systems, an effect that could exacerbate disease in CF and COPD patients. The mechanism by which sphingomyelin catalysis inhibits CFTR is not known but evidence suggests that it occurs independently of CFTR's regulatory "R" domain. In this study we utilized the Xenopus oocyte expression system to shed light on how CFTR channel activity is reduced by SMase. We found that the pathway leading to inhibition is not membrane delimited and that inhibited CFTR channels remain at the cell membrane, indicative of a novel silencing mechanism. Consistent with an effect on CFTR gating behavior, we found that altering gating kinetics influenced the sensitivity to inhibition by SMase. Specifically, increasing channel activity by introducing the mutation K1250A or pretreating with the CFTR potentiator VX-770 (Ivacaftor) imparted resistance to inhibition. In primary bronchial epithelial cells, we found that basolateral, but not apical, application of SMase leads to a redistribution of sphingomyelin and a reduction in forskolin- and VX-770-stimulated currents. Taken together, these data suggest that SMase inhibits CFTR channel function by locking channels into a closed state and that endogenous CFTR in HBEs is affected by SMase activity.
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Affiliation(s)
- B B Stauffer
- Division of Pulmonology, Allergy/Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Emory + Children's Center for Cystic Fibrosis and Airways Disease Research, Emory University School of Medicine and Children's Healthcare of Atlanta, 2015 Uppergate Drive, Atlanta, GA, 30322, USA
- Molecular and Systems Pharmacology program, Emory University, 201 Dowman Drive, Atlanta, GA, 20322, USA
| | - G Cui
- Division of Pulmonology, Allergy/Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Emory + Children's Center for Cystic Fibrosis and Airways Disease Research, Emory University School of Medicine and Children's Healthcare of Atlanta, 2015 Uppergate Drive, Atlanta, GA, 30322, USA
| | - K A Cottrill
- Molecular and Systems Pharmacology program, Emory University, 201 Dowman Drive, Atlanta, GA, 20322, USA
| | - D T Infield
- Division of Pulmonology, Allergy/Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Emory + Children's Center for Cystic Fibrosis and Airways Disease Research, Emory University School of Medicine and Children's Healthcare of Atlanta, 2015 Uppergate Drive, Atlanta, GA, 30322, USA
| | - N A McCarty
- Division of Pulmonology, Allergy/Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Emory + Children's Center for Cystic Fibrosis and Airways Disease Research, Emory University School of Medicine and Children's Healthcare of Atlanta, 2015 Uppergate Drive, Atlanta, GA, 30322, USA.
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45
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Friard J, Tauc M, Cougnon M, Compan V, Duranton C, Rubera I. Comparative Effects of Chloride Channel Inhibitors on LRRC8/VRAC-Mediated Chloride Conductance. Front Pharmacol 2017; 8:328. [PMID: 28620305 PMCID: PMC5449500 DOI: 10.3389/fphar.2017.00328] [Citation(s) in RCA: 46] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2017] [Accepted: 05/16/2017] [Indexed: 01/08/2023] Open
Abstract
Chloride channels play an essential role in a variety of physiological functions and in human diseases. Historically, the field of chloride channels has long been neglected owing to the lack of powerful selective pharmacological agents that are needed to overcome the technical challenge of characterizing the molecular identities of these channels. Recently, members of the LRRC8 family have been shown to be essential for generating the volume-regulated anion channel (VRAC) current, a chloride conductance that governs the regulatory volume decrease (RVD) process. The inhibitory effects of six commonly used chloride channel inhibitors on VRAC/LRRC8-mediated chloride transport were tested in wild-type HEK-293 cells expressing LRRC8 proteins and devoid of other types of chloride channels (CFTR and ANO1/2). We explored the effectiveness of the inhibitors using the patch-clamp whole-cell approach and fluorescence-based quantification of cellular volume changes during hypotonic challenge. Both DCPIB and NFA inhibited VRAC current in a whole-cell configuration, with IC50 values of 5 ± 1 μM and 55 ± 2 μM, respectively. Surprisingly, GlyH-101 and PPQ-102, two CFTR inhibitors, also inhibited VRAC conductance at concentrations in the range of their current use, with IC50 values of 10 ± 1 μM and 20 ± 1 μM, respectively. T16Ainh-A01, a so-called specific inhibitor of calcium-activated Cl- conductance, blocked the chloride current triggered by hypo-osmotic challenge, with an IC50 of 6 ± 1 μM. Moreover, RVD following hypotonic challenge was dramatically reduced by these inhibitors. CFTRinh-172 was the only inhibitor that had almost no effect on VRAC/LRRC8-mediated chloride conductance. All inhibitors tested except CFTRinh-172 inhibited VRAC/LRRC8-mediated chloride conductance and cellular volume changes during hypotonic challenge. These results shed light on the apparent lack of chloride channel inhibitors specificity and raise the question of how these inhibitors actually block chloride conductances.
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Affiliation(s)
- Jonas Friard
- LP2M CNRS-UMR7370, LabEx ICST, Medical Faculty, Université Côte d'AzurNice, France
| | - Michel Tauc
- LP2M CNRS-UMR7370, LabEx ICST, Medical Faculty, Université Côte d'AzurNice, France
| | - Marc Cougnon
- LP2M CNRS-UMR7370, LabEx ICST, Medical Faculty, Université Côte d'AzurNice, France
| | - Vincent Compan
- Institut de Génomique Fonctionnelle, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Université de MontpellierMontpellier, France
| | - Christophe Duranton
- LP2M CNRS-UMR7370, LabEx ICST, Medical Faculty, Université Côte d'AzurNice, France
| | - Isabelle Rubera
- LP2M CNRS-UMR7370, LabEx ICST, Medical Faculty, Université Côte d'AzurNice, France
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46
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Abstract
CFTR protein is an ion channel regulated by cAMP-dependent phosphorylation and expressed in many types of epithelial cells. CFTR-mediated chloride and bicarbonate secretion play an important role in the respiratory and gastrointestinal systems. Pharmacological modulators of CFTR represent promising drugs for a variety of diseases. In particular, correctors and potentiators may restore the activity of CFTR in cystic fibrosis patients. Potentiators are also potentially useful to improve mucociliary clearance in patients with chronic obstructive pulmonary disease. On the other hand, CFTR inhibitors may be useful to block fluid and electrolyte loss in secretory diarrhea and slow down the progression of polycystic kidney disease.
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Affiliation(s)
- Olga Zegarra-Moran
- U.O.C. Genetica Medica, Istituto Giannina Gaslini, Via Gerolamo Gaslini 5, 16147, Genoa, Italy
| | - Luis J V Galietta
- U.O.C. Genetica Medica, Istituto Giannina Gaslini, Via Gerolamo Gaslini 5, 16147, Genoa, Italy.
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47
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Linsdell P. Architecture and functional properties of the CFTR channel pore. Cell Mol Life Sci 2017; 74:67-83. [PMID: 27699452 PMCID: PMC11107662 DOI: 10.1007/s00018-016-2389-5] [Citation(s) in RCA: 35] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2016] [Accepted: 09/28/2016] [Indexed: 12/12/2022]
Abstract
The main function of the cystic fibrosis transmembrane conductance regulator (CFTR) is as an ion channel for the movement of small anions across epithelial cell membranes. As an ion channel, CFTR must form a continuous pathway across the cell membrane-referred to as the channel pore-for the rapid electrodiffusional movement of ions. This review summarizes our current understanding of the architecture of the channel pore, as defined by electrophysiological analysis and molecular modeling studies. This includes consideration of the characteristic functional properties of the pore, definition of the overall shape of the entire extent of the pore, and discussion of how the molecular structure of distinct regions of the pore might control different facets of pore function. Comparisons are drawn with closely related proteins that are not ion channels, and also with structurally unrelated proteins with anion channel function. A simple model of pore function is also described.
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Affiliation(s)
- Paul Linsdell
- Department of Physiology and Biophysics, Dalhousie University, PO Box 15000, Halifax, NS, B3H 4R2, Canada.
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48
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Cil O, Phuan PW, Gillespie AM, Lee S, Tradtrantip L, Yin J, Tse M, Zachos NC, Lin R, Donowitz M, Verkman AS. Benzopyrimido-pyrrolo-oxazine-dione CFTR inhibitor (R)-BPO-27 for antisecretory therapy of diarrheas caused by bacterial enterotoxins. FASEB J 2016; 31:751-760. [PMID: 27871064 DOI: 10.1096/fj.201600891r] [Citation(s) in RCA: 40] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2016] [Accepted: 10/24/2016] [Indexed: 12/15/2022]
Abstract
Secretory diarrheas caused by bacterial enterotoxins, including cholera and traveler's diarrhea, remain a major global health problem. Inappropriate activation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel occurs in these diarrheas. We previously reported that the benzopyrimido-pyrrolo-oxazinedione (R)-BPO-27 inhibits CFTR chloride conductance with low-nanomolar potency. Here, we demonstrate using experimental mouse models and human enterocyte cultures the potential utility of (R)-BPO-27 for treatment of secretory diarrheas caused by cholera and Escherichia coli enterotoxins. (R)-BPO-27 fully blocked CFTR chloride conductance in epithelial cell cultures and intestine after cAMP agonists, cholera toxin, or heat-stable enterotoxin of E. coli (STa toxin), with IC50 down to ∼5 nM. (R)-BPO-27 prevented cholera toxin and STa toxin-induced fluid accumulation in small intestinal loops, with IC50 down to 0.1 mg/kg. (R)-BPO-27 did not impair intestinal fluid absorption or inhibit other major intestinal transporters. Pharmacokinetics in mice showed >90% oral bioavailability with sustained therapeutic serum levels for >4 h without the significant toxicity seen with 7-d administration at 5 mg/kg/d. As evidence to support efficacy in human diarrheas, (R)-BPO-27 blocked fluid secretion in primary cultures of enteroids from human small intestine and anion current in enteroid monolayers. These studies support the potential utility of (R)-BPO-27 for therapy of CFTR-mediated secretory diarrheas.-Cil, O., Phuan, P.-W., Gillespie, A. M., Lee, S., Tradtrantip, L., Yin, J., Tse, M., Zachos, N. C., Lin, R., Donowitz, M., Verkman, A. S. Benzopyrimido-pyrrolo-oxazine-dione CFTR inhibitor (R)-BPO-27 for antisecretory therapy of diarrheas caused by bacterial enterotoxins.
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Affiliation(s)
- Onur Cil
- Department of Medicine, University of California San Francisco, San Francisco, California, USA.,Department of Physiology, University of California San Francisco, San Francisco, California, USA
| | - Puay-Wah Phuan
- Department of Medicine, University of California San Francisco, San Francisco, California, USA.,Department of Physiology, University of California San Francisco, San Francisco, California, USA
| | - Anne Marie Gillespie
- Department of Medicine, University of California San Francisco, San Francisco, California, USA.,Department of Physiology, University of California San Francisco, San Francisco, California, USA
| | - Sujin Lee
- Department of Medicine, University of California San Francisco, San Francisco, California, USA.,Department of Physiology, University of California San Francisco, San Francisco, California, USA
| | - Lukmanee Tradtrantip
- Department of Medicine, University of California San Francisco, San Francisco, California, USA.,Department of Physiology, University of California San Francisco, San Francisco, California, USA
| | - Jianyi Yin
- Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; and.,Gastroenterology Division, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - Ming Tse
- Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; and.,Gastroenterology Division, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - Nicholas C Zachos
- Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; and.,Gastroenterology Division, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - Ruxian Lin
- Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; and.,Gastroenterology Division, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - Mark Donowitz
- Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; and.,Gastroenterology Division, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - Alan S Verkman
- Department of Medicine, University of California San Francisco, San Francisco, California, USA; .,Department of Physiology, University of California San Francisco, San Francisco, California, USA
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49
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Puga Molina LC, Pinto NA, Torres Rodríguez P, Romarowski A, Vicens Sanchez A, Visconti PE, Darszon A, Treviño CL, Buffone MG. Essential Role of CFTR in PKA-Dependent Phosphorylation, Alkalinization, and Hyperpolarization During Human Sperm Capacitation. J Cell Physiol 2016; 232:1404-1414. [PMID: 27714810 DOI: 10.1002/jcp.25634] [Citation(s) in RCA: 48] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2016] [Accepted: 10/05/2016] [Indexed: 12/17/2022]
Abstract
Mammalian sperm require to spend a limited period of time in the female reproductive tract to become competent to fertilize in a process called capacitation. It is well established that HCO3- is essential for capacitation because it activates the atypical soluble adenylate cyclase ADCY10 leading to cAMP production, and promotes alkalinization of cytoplasm, and membrane hyperpolarization. However, how HCO3- is transported into the sperm is not well understood. There is evidence that CFTR activity is involved in the human sperm capacitation but how this channel is integrated in the complex signaling cascades associated with this process remains largely unknown. In the present work, we have analyzed the extent to which CFTR regulates different events in human sperm capacitation. We observed that inhibition of CFTR affects HCO3- -entrance dependent events resulting in lower PKA activity. CFTR inhibition also affected cAMP/PKA-downstream events such as the increase in tyrosine phosphorylation, hyperactivated motility, and acrosome reaction. In addition, we demonstrated for the first time, that CFTR and PKA activity are essential for the regulation of intracellular pH, and membrane potential in human sperm. Addition of permeable cAMP partially recovered all the PKA-dependent events altered in the presence of inh-172 which is consistent with a role of CFTR upstream of PKA activation. J. Cell. Physiol. 232: 1404-1414, 2017. © 2016 Wiley Periodicals, Inc.
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Affiliation(s)
- Lis C Puga Molina
- Instituto de Biología y Medicina Experimental (IBYME), Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), Buenos Aires, Argentina
| | - Nicolás A Pinto
- Instituto de Biología y Medicina Experimental (IBYME), Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), Buenos Aires, Argentina
| | - Paulina Torres Rodríguez
- Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Cuernavaca, Morelos, México
| | - Ana Romarowski
- Instituto de Biología y Medicina Experimental (IBYME), Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), Buenos Aires, Argentina
| | - Alberto Vicens Sanchez
- Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Cuernavaca, Morelos, México
| | - Pablo E Visconti
- Department of Veterinary and Animal Science, Paige Labs, University of Massachusetts, Amherst, Massachusetts
| | - Alberto Darszon
- Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Cuernavaca, Morelos, México
| | - Claudia L Treviño
- Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Cuernavaca, Morelos, México
| | - Mariano G Buffone
- Instituto de Biología y Medicina Experimental (IBYME), Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), Buenos Aires, Argentina
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50
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Sharma A, Xu Y, Sung B, Vincent CT, Worgall T, Worgall S. Regulation of the Coxsackie and adenovirus receptor expression is dependent on cystic fibrosis transmembrane regulator in airway epithelial cells. Cell Microbiol 2016; 19. [PMID: 27527752 DOI: 10.1111/cmi.12654] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2016] [Revised: 08/08/2016] [Accepted: 08/10/2016] [Indexed: 12/21/2022]
Abstract
The coxsackievirus and adenovirus receptor (CAR), in addition to serving as viral receptor, is a component of tight junctions and plays an important role in tissue homeostasis. Defects in the cystic fibrosis transmembrane regulator (CFTR) in lung epithelial cells are linked to inflammation and susceptibility for respiratory tract infections. Here, we demonstrate that CAR expression and infectivity with adenovirus (Ad) are increased in cystic fibrosis airway epithelial cells. Inhibition of CFTR or histone deacetylase (HDAC) enhanced CAR expression while CFTR overexpression or restoration of the diminished HDAC activity in cystic fibrosis cells reduced CAR expression. This connects the CFTR to CAR expression and infectivity with adenovirus through HDAC.
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Affiliation(s)
- Anurag Sharma
- Department of Pediatrics, Weill Cornell Medicine, New York, New York, USA
| | - Yaqin Xu
- Department of Pediatrics, Weill Cornell Medicine, New York, New York, USA
| | - Biin Sung
- Department of Genetic Medicine, Weill Cornell Medicine, New York, New York, USA
| | - C Theresa Vincent
- Department of Physiology and Biophysics, Weill Cornell Medicine, New York, New York, USA.,Department of Pharmacology and Physiology, Karolinska Institute, Stockholm, Sweden
| | - Tilla Worgall
- Department of Pathology, Columbia University, New York, New York, USA
| | - Stefan Worgall
- Department of Pediatrics, Weill Cornell Medicine, New York, New York, USA.,Department of Genetic Medicine, Weill Cornell Medicine, New York, New York, USA
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