scholarly journals Deubiquitinase-Targeting Chimeras for Targeted Protein Stabilization

2021 ◽  
Author(s):  
Nathaniel J. Henning ◽  
Lydia Boike ◽  
Jessica N. Spradlin ◽  
Carl C. Ward ◽  
Bridget Belcher ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Protein Stabilization ◽  
Therapeutic Modality ◽  
Dependent Manner ◽  
Bronchial Epithelial ◽  
Therapeutic Modalities ◽  
Ligand Discovery ◽  
Δf508 Cftr ◽  
Specific Proteins ◽  
Cftr Protein

AbstractTargeted protein degradation is a powerful therapeutic modality that uses heterobifunctional small-molecules to induce proximity between E3 ubiquitin ligases and target proteins to ubiquitinate and degrade specific proteins of interest. However, many proteins are ubiquitinated and degraded to drive disease pathology; in these cases targeted protein stabilization (TPS), rather than degradation, of the actively degraded target using a small-molecule would be therapeutically beneficial. Here, we present the Deubiquitinase-Targeting Chimera (DUBTAC) platform for TPS of specific proteins. Using chemoproteomic approaches, we discovered the covalent ligand EN523 that targets a non-catalytic allosteric cysteine C23 in the K48 ubiquitin-specific deubiquitinase OTUB1. We then developed a heterobifunctional DUBTAC consisting of our EN523 OTUB1 recruiter linked to lumacaftor, a drug used to treat cystic fibrosis that binds ΔF508-CFTR. We demonstrated proof-of-concept of TPS by showing that this DUBTAC robustly stabilized ΔF508-CFTR in human cystic fibrosis bronchial epithelial cells in an OTUB1-dependent manner. Our study underscores the utility of chemoproteomics-enabled covalent ligand discovery approaches to develop new induced proximity-based therapeutic modalities and introduces the DUBTAC platform for TPS.Editorial summaryWe have developed the Deubiquitinase Targeting Chimera (DUBTAC) platform for targeted protein stabilization. We have discovered a covalent recruiter against the deubiquitinase OTUB1 that we have linked to the mutant ΔF508-CFTR targeting cystic fibrosis drug Lumacaftor to stabilize mutant CFTR protein in cells.

scholarly journals Characterization of Wild-Type and ΔF508 Cystic Fibrosis Transmembrane Regulator in Human Respiratory Epithelia

2005 ◽  
Vol 16 (5) ◽  
pp. 2154-2167 ◽  
Author(s):  
Silvia M. Kreda ◽  
Marcus Mall ◽  
April Mengos ◽  
Lori Rochelle ◽  
James Yankaskas ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Normal Subjects ◽  
Cell Types ◽  
Cystic Fibrosis Transmembrane Regulator ◽  
Ciliated Cells ◽  
Metabolic Fate ◽  
Specific Expression ◽  
Δf508 Cftr ◽  
Cftr Protein ◽  
Cystic Fibrosis Transmembrane

Previous studies in native tissues have produced conflicting data on the localization and metabolic fate of WT and ΔF508 cystic fibrosis transmembrane regulator (CFTR) in the lung. Combining immunocytochemical and biochemical studies utilizing new high-affinity CFTR mAbs with ion transport assays, we examined both 1) the cell type and region specific expression of CFTR in normal airways and 2) the metabolic fate of ΔF508 CFTR and associated ERM proteins in the cystic fibrosis lung. Studies of lungs from a large number of normal subjects revealed that WT CFTR protein localized to the apical membrane of ciliated cells within the superficial epithelium and gland ducts. In contrast, other cell types in the superficial, gland acinar, and alveolar epithelia expressed little WT CFTR protein. No ΔF508 CFTR mature protein or function could be detected in airway specimens freshly excised from a large number of ΔF508 homozygous subjects, despite an intact ERM complex. In sum, our data demonstrate that WT CFTR is predominantly expressed in ciliated cells, and ΔF508 CFTR pathogenesis in native tissues, like heterologous cells, reflects loss of normal protein processing.

Calcium-pump inhibitors induce functional surface expression of ΔF508-CFTR protein in cystic fibrosis epithelial cells

2002 ◽  
Vol 8 (5) ◽  
pp. 485-492 ◽  
Author(s):  
Marie E. Egan ◽  
Judith Glöckner-Pagel ◽  
Catherine A. Ambrose ◽  
Paula A. Cahill ◽  
Lamiko Pappoe ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Surface Expression ◽  
Calcium Pump ◽  
Functional Surface ◽  
Δf508 Cftr ◽  
Cftr Protein

scholarly journals Vitamin D represses rhinovirus replication in cystic fibrosis cells by inducing LL-37

2015 ◽  
Vol 47 (2) ◽  
pp. 520-530 ◽  
Author(s):  
Aline Schögler ◽  
Ricardo J. Muster ◽  
Elisabeth Kieninger ◽  
Carmen Casaulta ◽  
Caroline Tapparel ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Vitamin D ◽  
Viral Load ◽  
Inverse Correlation ◽  
Dependent Manner ◽  
Bronchial Epithelial ◽  
Immunomodulatory Properties ◽  
Anti Inflammatory ◽  
Dose Dependent ◽  
Primary Bronchial Epithelial Cells

Vitamin D has immunomodulatory properties in the defence against pathogens. Its insufficiency is a widespread feature of cystic fibrosis (CF) patients, which are repeatedly suffering from rhinovirus (RV)-induced pulmonary exacerbations.To investigate whether vitamin D has antiviral activity, primary bronchial epithelial cells from CF children were pre-treated with vitamin D and infected with RV16. Antiviral and anti-inflammatory activity of vitamin D was assessed. RV and LL-37 levels were measured in bronchoalveolar lavage (BAL) of CF children infected with RV.Vitamin D reduced RV16 load in a dose-dependent manner in CF cells (10−7 M, p<0.01). The antiviral response mediated by interferons remained unchanged by vitamin D in CF cells. Vitamin D did not exert anti-inflammatory properties in RV-infected CF cells. Vitamin D increased the expression of the antimicrobial peptide LL-37 up to 17.4-fold (p<0.05). Addition of exogenous LL-37 decreased viral replication by 4.4-fold in CF cells (p<0.05). An inverse correlation between viral load and LL-37 levels in CF BAL (r=−0.48, p<0.05) was observed.RV replication in primary CF bronchial cells was reduced by vitamin D through the induction of LL-37. Clinical studies are needed to determine the importance of an adequate control of vitamin D for prevention of virus-induced pulmonary CF exacerbations.

scholarly journals ΔF508 CFTR protein expression in tissues from patients with cystic fibrosis

1999 ◽  
Vol 103 (10) ◽  
pp. 1379-1389 ◽  
Author(s):  
Nanette Kälin ◽  
Andreas Claaß ◽  
Martin Sommer ◽  
Edith Puchelle ◽  
Burkhard Tümmler
Keyword(s):  
Cystic Fibrosis ◽  
Protein Expression ◽  
Δf508 Cftr ◽  
Cftr Protein

scholarly journals Lumacaftor and Matrine: Possible Therapeutic Combination to Counteract the Inflammatory Process in Cystic Fibrosis

Biomolecules ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 422
Author(s):  
Michela Pecoraro ◽  
Silvia Franceschelli ◽  
Maria Pascale
Keyword(s):  
Cystic Fibrosis ◽  
Inflammatory Process ◽  
Chloride Transport ◽  
Electrolyte Imbalance ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Δf508 Cftr ◽  
Cftr Protein ◽  
Cell Cytosol ◽  
Cystic Fibrosis Transmembrane

Cystic fibrosis is a monogenic, autosomal, recessive disease characterized by an alteration of chloride transport caused by mutations in the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene. The loss of Phe residue in position 508 (ΔF508-CFTR) causes an incorrect folding of the protein causing its degradation and electrolyte imbalance. CF patients are extremely predisposed to the development of a chronic inflammatory process of the bronchopulmonary system. When the cells of a tissue are damaged, the immune cells are activated and trigger the production of free radicals, provoking an inflammatory process. In addition to routine therapies, today drugs called correctors are available for mutations such as ΔF508-CFTR as well as for others less frequent ones. These active molecules are supposed to facilitate the maturation of the mutant CFTR protein, allowing it to reach the apical membrane of the epithelial cell. Matrine induces ΔF508-CFTR release from the endoplasmic reticulum to cell cytosol and its localization on the cell membrane. We now have evidence that Matrine and Lumacaftor not only restore the transport of mutant CFTR protein, but probably also counteract the inflammatory process by improving the course of the disease.

scholarly journals Gene-Specific Nonsense-Mediated mRNA Decay Targeting for Cystic Fibrosis Therapy

2021 ◽  
Author(s):  
Young Jin Kim ◽  
Tomoki Nomakuchi ◽  
Foteini Papaleonidopoulou ◽  
Adrian R. Krainer
Keyword(s):  
Cystic Fibrosis ◽  
Mrna Expression ◽  
Antisense Oligonucleotides ◽  
Mrna Decay ◽  
Bronchial Epithelial ◽  
Premature Termination Codons ◽  
Normal Expression ◽  
Allele Specific ◽  
Nonsense Mediated Mrna Decay ◽  
Cftr Protein

Low CFTR mRNA expression due to nonsense-mediated mRNA decay (NMD) is a major hurdle in developing a therapy for cystic fibrosis (CF) caused by the W1282X mutation in the CFTR gene. CFTR-W1282X truncated protein retains partial function, so increasing its levels by inhibiting NMD of its mRNA will likely be beneficial. Because NMD regulates the normal expression of many genes, gene-specific stabilization of CFTR-W1282X mRNA expression is more desirable than general NMD inhibition. Synthetic antisense oligonucleotides (ASOs) designed to prevent binding of exon junction complexes (EJC) downstream of premature termination codons (PTCs) attenuate NMD in a gene-specific manner. We developed a cocktail of three ASOs that specifically increases the expression of CFTR W1282X mRNA and CFTR protein in ASO-transfected human bronchial epithelial cells. This treatment increased the CFTR-mediated chloride current. These results set the stage for clinical development of an allele-specific therapy for CF caused by the W1282X mutation.

scholarly journals Transforming Growth Factor-β1 Selectively Recruits microRNAs to the RNA-Induced Silencing Complex and Degrades CFTR mRNA under Permissive Conditions in Human Bronchial Epithelial Cells

2019 ◽  
Vol 20 (19) ◽  
pp. 4933 ◽  
Author(s):  
Nilay Mitash ◽  
Fangping Mu ◽  
Joshua E. Donovan ◽  
Michael M. Myerburg ◽  
Sarangarajan Ranganathan ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Growth Factor ◽  
Epithelial Cells ◽  
Transforming Growth Factor ◽  
Bronchial Epithelial Cells ◽  
Common Mutation ◽  
Human Bronchial Epithelial Cells ◽  
Bronchial Epithelial ◽  
Cftr Protein

<p>Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (<italic>CFTR</italic>) gene lead to cystic fibrosis (CF). The most common mutation F508del inhibits folding and processing of CFTR protein. FDA-approved correctors rescue the biosynthetic processing of F508del-CFTR protein, while potentiators improve the rescued CFTR channel function. Transforming growth factor (TGF-&#x3B2;1), overexpressed in many CF patients, blocks corrector/potentiator rescue by inhibiting CFTR mRNA in vitro. Increased TGF-&#x3B2;1 signaling and acquired CFTR dysfunction are present in other lung diseases. To study the mechanism of TGF-&#x3B2;1 repression of CFTR, we used molecular, biochemical, and functional approaches in primary human bronchial epithelial cells from over 50 donors. TGF-&#x3B2;1 destabilized CFTR mRNA in cells from lungs with chronic disease, including CF, and impaired F508del-CFTR rescue by new-generation correctors. TGF-&#x3B2;1 increased the active pool of selected micro(mi)RNAs validated as CFTR inhibitors, recruiting them to the RNA-induced silencing complex (RISC). Expression of F508del-CFTR globally modulated TGF-&#x3B2;1-induced changes in the miRNA landscape, creating a permissive environment required for degradation of F508del-CFTR mRNA. In conclusion, TGF-&#x3B2;1 may impede the full benefit of corrector/potentiator therapy in CF patients. Studying miRNA recruitment to RISC under disease-specific conditions may help to better characterize the miRNAs utilized by TGF-&#x3B2;1 to destabilize CFTR mRNA.

Increased functional cell surface expression of CFTR and ΔF508-CFTR by the anthracycline doxorubicin

2001 ◽  
Vol 280 (5) ◽  
pp. C1031-C1037 ◽  
Author(s):  
Rangan Maitra ◽  
Collin M. Shaw ◽  
Bruce A. Stanton ◽  
Joshua W. Hamilton
Keyword(s):  
Cystic Fibrosis ◽  
Cell Surface ◽  
Protein Expression ◽  
Surface Expression ◽  
Chloride Secretion ◽  
Cell Surface Expression ◽  
Common Mutation ◽  
Transmembrane Conductance Regulator ◽  
Δf508 Cftr ◽  
Cftr Protein

Cystic fibrosis (CF) is a disease that is caused by mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most common mutation, ΔF508, accounts for 70% of all CF alleles and results in a protein that is defective in folding and trafficking to the cell surface. However, ΔF508-CFTR is functional when properly localized. We report that a single, noncytotoxic dose of the anthracycline doxorubicin (Dox, 0.25 μM) significantly increased total cellular CFTR protein expression, cell surface CFTR protein expression, and CFTR-associated chloride secretion in cultured T84 epithelial cells. Dox treatment also increased ΔF508-CFTR cell surface expression and ΔF508-CFTR-associated chloride secretion in stably transfected Madin-Darby canine kidney cells. These results suggest that anthracycline analogs may be useful for the clinical treatment of CF.

PRO–CF–MED, Clinical Proof of concept for a RNA–targeting Oligonucleotide for a Cystic fibrosis–F508del MEDication, Horizon2020

Impact ◽  
2018 ◽  
Vol 2018 (3) ◽  
pp. 52-54
Author(s):  
Nicolas Lamontagne
Keyword(s):  
Cystic Fibrosis ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Rna Targeting ◽  
Threatening Condition ◽  
Life Threatening ◽  
Cftr Protein ◽  
Disease Modifying ◽  
Cystic Fibrosis Transmembrane ◽  
Specific Challenge

Cystic fibrosis (CF) is a progressive life–shortening disease caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene leading to a dysfunctional CFTR protein. The disease affects over 70,000 patients worldwide and while many mutations are known, the F508del mutation affects 90% of all patients. The absence of CFTR in the plasma membrane leads to a dramatic decrease in chloride efflux, resulting in viscous mucus that causes severe symptoms in vital organs like the lungs and intestines. For CF patients that suffer from the life threatening F508del mutation only palliative treatment exist. PRO–CF–MED addresses the specific challenge of this call by introducing the first disease modifying medication for the treatment of the CF patients with F508del mutation. The PRO–CF–MED project has been designed to assess the potential clinical efficacy of QR–010, an innovative disease modifying oligonucleotide–based treatment for F508del patients. Partners within PRO–CF–MED have generated very promising preclinical evidence for QR–010 which allows for further clinical assessment of QR–010 in clinical trials. PRO–CF–MED will enable the fast translation of QR–010 towards clinical practice and market authorisation. PRO–CF–MED has the potential to transform this life–threatening condition into a manageable one.

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