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1.
Nat Commun ; 13(1): 4270, 2022 07 29.
Article in English | MEDLINE | ID: mdl-35906215

ABSTRACT

Cystic fibrosis is a monogenic lung disease caused by dysfunction of the cystic fibrosis transmembrane conductance regulator anion channel, resulting in significant morbidity and mortality. The progress in elucidating the role of CFTR using established animal and cell-based models led to the recent discovery of effective modulators for most individuals with CF. However, a subset of individuals with CF do not respond to these modulators and there is an urgent need to develop novel therapeutic strategies. In this study, we generate a panel of airway epithelial cells using induced pluripotent stem cells from individuals with common or rare CFTR variants representative of three distinct classes of CFTR dysfunction. To measure CFTR function we adapt two established in vitro assays for use in induced pluripotent stem cell-derived airway cells. In both a 3-D spheroid assay using forskolin-induced swelling as well as planar cultures composed of polarized mucociliary airway epithelial cells, we detect genotype-specific differences in CFTR baseline function and response to CFTR modulators. These results demonstrate the potential of the human induced pluripotent stem cell platform as a research tool to study CF and in particular accelerate therapeutic development for CF caused by rare variants.


Subject(s)
Cystic Fibrosis , Induced Pluripotent Stem Cells , Animals , Cystic Fibrosis/drug therapy , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Epithelial Cells/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Ion Transport
2.
Nat Commun ; 11(1): 1979, 2020 04 24.
Article in English | MEDLINE | ID: mdl-32332735

ABSTRACT

CRISPR-Cas9-associated base editing is a promising tool to correct pathogenic single nucleotide mutations in research or therapeutic settings. Efficient base editing requires cellular exposure to levels of base editors that can be difficult to attain in hard-to-transfect cells or in vivo. Here we engineer a chemically modified mRNA-encoded adenine base editor that mediates robust editing at various cellular genomic sites together with moderately modified guide RNA, and show its therapeutic potential in correcting pathogenic single nucleotide mutations in cell and animal models of diseases. The optimized chemical modifications of adenine base editor mRNA and guide RNA expand the applicability of CRISPR-associated gene editing tools in vitro and in vivo.


Subject(s)
Adenine/chemistry , CRISPR-Cas Systems , RNA, Guide, Kinetoplastida/chemistry , RNA, Messenger/chemistry , Alleles , Animals , Cell Line , Codon , Codon, Nonsense , Cystic Fibrosis/pathology , Gene Editing , HEK293 Cells , Humans , Mice , Mutation , Nucleotides , Phenotype , Plasmids , Transfection , Uridine/analogs & derivatives , Uridine/chemistry
3.
Bioorg Med Chem Lett ; 22(8): 2877-9, 2012 Apr 15.
Article in English | MEDLINE | ID: mdl-22425452

ABSTRACT

We report the synthesis and biological evaluation of a series of novel α-branched pyrazinoyl quaternary amines for their ability to block ion transport via the epithelial sodium channel (ENaC) in human bronchial epithelial cells (HBECs). Compound 12 g has an IC(50) of 30 nM and is highly efficacious in the Guinea-pig tracheal potential difference (TPD) model of ENaC blockade with an ED(50) of 1 µg kg(-1) at 1h. In addition the SAR results demonstrate for the first time the chiral nature of the binding site of human ENaC. As such, pyrazinoyl quaternary amines represent a promising new class of ENaC blockers for the treatment of cystic fibrosis that are structurally distinct from the pyrazinoyl guanidine chemotype found in prototypical ENaC blockers such as amiloride.


Subject(s)
Amines/chemistry , Epithelial Sodium Channel Blockers , Pyrazines/chemistry , Sodium Channel Blockers/chemistry , Amines/pharmacology , Animals , Binding Sites , Bronchi/drug effects , Epithelial Cells/drug effects , Guinea Pigs , Humans , Inhibitory Concentration 50 , Models, Molecular , Molecular Structure , Pyrazines/pharmacology , Sodium Channel Blockers/pharmacology
4.
Bioorg Med Chem Lett ; 22(2): 929-32, 2012 Jan 15.
Article in English | MEDLINE | ID: mdl-22197144

ABSTRACT

We report the identification of a novel series of human epithelial sodium channel (ENaC) blockers that are structurally distinct from the pyrazinoyl guanidine chemotype found in prototypical ENaC blockers such as amiloride. Following a rational design hypothesis a series of quaternary amines were prepared and evaluated for their ability to block ion transport via ENaC in human bronchial epithelial cells (HBECs). Compound 11 has an IC(50) of 200nM and is efficacious in the Guinea-pig tracheal potential difference (TPD) model of ENaC blockade with an ED(50) of 44µgkg(-1) at 1h. As such, pyrazinoyl quaternary amines represent the first examples of a promising new class of human ENaC blockers.


Subject(s)
Amines/chemistry , Drug Design , Epithelial Cells/drug effects , Epithelial Sodium Channel Blockers , Sodium Channel Blockers/chemical synthesis , Sodium Channel Blockers/pharmacology , Amines/pharmacology , Bronchi/cytology , Dose-Response Relationship, Drug , Epithelial Cells/metabolism , Epithelial Sodium Channels/metabolism , Humans , Sodium Channel Blockers/chemistry , Structure-Activity Relationship
5.
Magn Reson Med ; 62(5): 1164-74, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19780168

ABSTRACT

Dysfunctions in mucociliary clearance are associated with the accelerated loss of lung function in several respiratory diseases. Approaches enabling the in vivo visualization of mucus dynamics in rodents at high resolution and sensitivity would be beneficial for experimental lung research. We describe the synthesis and characterization of two bilabeled amino dextran-based probes binding specifically to mucin. Labeling of secreted mucus and of mucin in goblet cells in the lungs of lipopolysaccharide-challenged rats has been demonstrated in vivo with near-infrared fluorescence and MRI and confirmed by histology. The effects of uridine triphosphate were then studied in lipopolysaccharide-challenged rats by simultaneously administering the imaging probe and the compound. The data suggest that uridine triphosphate increased the mucociliary clearance, but at the same time induced a release of mucin from goblet cells, thus not contributing to the overall reduction of mucus in the lung. The approach outlined here enables one to derive information on mucus clearance, as well as secretion. Such a global view on mucus dynamics may prove invaluable when testing new pharmacological agents aimed at improving mucociliary clearance.


Subject(s)
Gadolinium , Image Enhancement/methods , Lung/metabolism , Lung/pathology , Magnetic Resonance Imaging/methods , Mucins/metabolism , Pneumonia/metabolism , Pneumonia/pathology , Animals , Carbocyanines/pharmacokinetics , Contrast Media , Gadolinium/pharmacokinetics , Lipopolysaccharides , Male , Microscopy, Fluorescence/methods , Pneumonia/chemically induced , Rats , Reproducibility of Results , Sensitivity and Specificity
6.
Am J Physiol Lung Cell Mol Physiol ; 288(3): L514-22, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15516486

ABSTRACT

Repetitive, acute inflammatory insults elicited by cigarette smoke (CS) contribute to the development of chronic obstructive pulmonary disease (COPD), a disorder associated with lung inflammation and mucus hypersecretion. Presently, there is a poor understanding of the acute inflammatory mechanisms involved in this process. The aims of this study were to develop an acute model to investigate temporal inflammatory changes occurring after CS exposure. Rats were exposed to whole body CS (once daily) generated from filtered research cigarettes. Initial studies indicated the generation of a neutrophilic/mucus hypersecreting lung phenotype in <4 days. Subsequent studies demonstrated that just two exposures to CS (15 h apart) elicited a robust inflammatory/mucus hypersecretory phenotype that was used to investigate mechanisms driving this response. Cytokine-induced neutrophil chemoattractants (CINCs) 1-3, the rat growth-related oncogene-alpha family homologs, and IL-1beta demonstrated time-dependent increases in lung tissue or lavage fluid over the 24-h period following CS exposure. The temporal changes in the neutrophil chemokines, CINCs 1-3, mirrored increases in neutrophil infiltration, indicative of a role in neutrophil migration. In addition, a specific CXCR2 antagonist, SB-332235, effectively inhibited CS-induced neutrophilia in a dose-dependent manner, supporting this conclusion. This modeling of the response of the rat airways to acute CS exposure indicates 1) as few as two exposures to CS will induce a phenotype with similarities to COPD and 2) a novel role for CINCs in the generation of this response. These observations represent a paradigm for the study of acute, repetitive lung insults that contribute to the development of chronic disease.


Subject(s)
Lung/metabolism , Mucus/metabolism , Nicotiana/adverse effects , Pneumonia/etiology , Receptors, Interleukin-8B/metabolism , Smoke/adverse effects , Animals , Ligands , Male , Rats , Rats, Sprague-Dawley
7.
Exp Lung Res ; 30(1): 59-71, 2004.
Article in English | MEDLINE | ID: mdl-14967604

ABSTRACT

In this study, the authors describe a new technique enabling the rapid assessment of mucociliary clearance (MCC) in rats and characterize this aspect of innate host defense in 2 animal models of bronchitis. Following instillation into the airways, fluorescent microspheres were rapidly cleared over 24 hours, with 60% to 80% of clearance occurring within 4 hours. On a background of airway neutrophilia and mucus hypersecretion, induced by either lipopolysaccharide or cigarette smoke, MCC was significantly enhanced. This reserve capacity in the MCC system will need to become overwhelmed in order to model the clinically observed impairment of lung mucus clearance in an animal system.


Subject(s)
Bronchitis/physiopathology , Lipopolysaccharides , Mucociliary Clearance , Smoking/adverse effects , Animals , Bronchitis/chemically induced , Cilia/physiology , Disease Models, Animal , Lung/metabolism , Lung/physiopathology , Male , Microspheres , Mucus/metabolism , Rats , Rats, Sprague-Dawley , Rats, Wistar
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