scholarly journals Partial Rescue of F508del-CFTR Stability and Trafficking Defects by Double Corrector Treatment

2021 ◽  
Vol 22 (10) ◽  
pp. 5262
Author(s):  
Valeria Capurro ◽  
Valeria Tomati ◽  
Elvira Sondo ◽  
Mario Renda ◽  
Anna Borrelli ◽  
...  
Keyword(s):  
Small Molecules ◽  
Type I ◽  
Frequent Mutation ◽  
Bronchial Epithelial ◽  
Efficacy Analysis ◽  
Structural And Functional Properties ◽  
Cftr Protein ◽  
Trafficking Defects ◽  
The Stability ◽  
Primary Bronchial Epithelial Cells

Deletion of phenylalanine at position 508 (F508del) in the CFTR chloride channel is the most frequent mutation in cystic fibrosis (CF) patients. F508del impairs the stability and folding of the CFTR protein, thus resulting in mistrafficking and premature degradation. F508del-CFTR defects can be overcome with small molecules termed correctors. We investigated the efficacy and properties of VX-445, a newly developed corrector, which is one of the three active principles present in a drug (Trikafta®/Kaftrio®) recently approved for the treatment of CF patients with F508del mutation. We found that VX-445, particularly in combination with type I (VX-809, VX-661) and type II (corr-4a) correctors, elicits a large rescue of F508del-CFTR function. In particular, in primary bronchial epithelial cells of CF patients, the maximal rescue obtained with corrector combinations including VX-445 was close to 60–70% of CFTR function in non-CF cells. Despite this high efficacy, analysis of ubiquitylation, resistance to thermoaggregation, protein half-life, and subcellular localization revealed that corrector combinations did not fully normalize F508del-CFTR behavior. Our study indicates that it is still possible to further improve mutant CFTR rescue with the development of corrector combinations having maximal effects on mutant CFTR structural and functional properties.

scholarly journals Discovery of a picomolar potency pharmacological corrector of the mutant CFTR chloride channel

2020 ◽  
Vol 6 (8) ◽  
pp. eaay9669 ◽  
Author(s):  
Nicoletta Pedemonte ◽  
Fabio Bertozzi ◽  
Emanuela Caci ◽  
Federico Sorana ◽  
Paolo Di Fruscia ◽  
...  
Keyword(s):  
Small Molecules ◽  
Chloride Channel ◽  
Frequent Mutation ◽  
High Potency ◽  
Chemical Library ◽  
Bronchial Epithelial ◽  
High Affinity ◽  
Primary Screening ◽  
Affinity Probe ◽  
Final Compound

F508del, the most frequent mutation causing cystic fibrosis (CF), results in mistrafficking and premature degradation of the CFTR chloride channel. Small molecules named correctors may rescue F508del-CFTR and therefore represent promising drugs to target the basic defect in CF. We screened a carefully designed chemical library to find F508del-CFTR correctors. The initial active compound resulting from the primary screening underwent extensive chemical optimization. The final compound, ARN23765, showed an extremely high potency in bronchial epithelial cells from F508del homozygous patients, with an EC50 of 38 picomolar, which is more than 5000-fold lower compared to presently available corrector drugs. ARN23765 also showed high efficacy, synergy with other types of correctors, and compatibility with chronic VX-770 potentiator. Besides being a promising drug, particularly suited for drug combinations, ARN23765 represents a high-affinity probe for CFTR structure-function studies.

scholarly journals Unravelling the Regions of Mutant F508del-CFTR More Susceptible to the Action of Four Cystic Fibrosis Correctors

2019 ◽  
Vol 20 (21) ◽  
pp. 5463 ◽  
Author(s):  
Amico ◽  
Brandas ◽  
Moran ◽  
Baroni
Keyword(s):  
Cystic Fibrosis ◽  
Bacterial Infections ◽  
Chloride Concentration ◽  
Hek293 Cells ◽  
Transmembrane Conductance Regulator ◽  
Quinazoline Derivative ◽  
Sweat Chloride ◽  
Cftr Protein ◽  
Trafficking Defects ◽  
The Stability

Cystic fibrosis (CF) is a genetic disease associated with the defective function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein that causes obstructive disease and chronic bacterial infections in airway epithelia. The most prevalent CF-causing mutation, the deletion of phenylalanine at position 508 (F508del), leads to CFTR misfolding, trafficking defects and premature degradation. A number of correctors that are able to partially rescue F508del-CFTR processing defects have been identified. Clinical trials have demonstrated that, unfortunately, mono-therapy with the best correctors identified to date does not ameliorate lung function or sweat chloride concentration in homozygous F508del patients. Understanding the mechanisms exerted by currently available correctors to increase mutant F508del-CFTR expression is essential for the development of new CF-therapeutics. We investigated the activity of correctors on the mutant F508del and wild type (WT) CFTR to identify the protein domains whose expression is mostly affected by the action of correctors, and we investigated their mechanisms of action. We found that the four correctors under study, lumacaftor (VX809), the quinazoline derivative VX325, the bithiazole compound corr4a, and the new molecule tezacaftor (VX661), do not influence either the total expression or the maturation of the WT-CFTR transiently expressed in human embryonic kidney 293 (HEK293) cells. Contrarily, they significantly enhance the expression and the maturation of the full length F508del molecule. Three out of four correctors, VX809, VX661 and VX325, seem to specifically improve the expression and the maturation of the mutant CFTR N-half (M1N1, residues 1–633). By contrast, the CFTR C-half (M2N2, residues 837–1480) appears to be the region mainly affected by corr4a. VX809 was shown to stabilize both the WT- and F508del-CFTR N-half isoforms, while VX661 and VX325 demonstrated the ability to enhance the stability only of the mutant F508del polypeptide.

scholarly journals Haemophilus influenzae causes cellular trans-differentiation in human bronchial epithelia

2021 ◽  
Vol 27 (3) ◽  
pp. 251-259
Author(s):  
Michael Glöckner ◽  
Sebastian Marwitz ◽  
Kristina Rohmann ◽  
Henrik Watz ◽  
Dörte Nitschkowski ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Haemophilus Influenzae ◽  
Bronchial Epithelium ◽  
Bronchial Epithelial Cells ◽  
Respiratory Pathogen ◽  
Chronic Obstructive ◽  
Bronchial Epithelial ◽  
Extracellular Matrix Components ◽  
The Impact ◽  
Primary Bronchial Epithelial Cells

Non-typeable Haemophilus influenzae (NTHi) is the most common respiratory pathogen in patients with chronic obstructive disease. Limited data is available investigating the impact of NTHi infections on cellular re-differentiation processes in the bronchial mucosa. The aim of this study was to assess the effects of stimulation with NTHi on the bronchial epithelium regarding cellular re-differentiation processes using primary bronchial epithelial cells harvested from infection-free patients undergoing bronchoscopy. The cells were then cultivated using an air-liquid interface and stimulated with NTHi and TGF-β. Markers of epithelial and mesenchymal cells were analyzed using immunofluorescence, Western blot and qRT-PCR. Stimulation with both NTHi and TGF-ß led to a marked increase in the expression of the mesenchymal marker vimentin, while E-cadherin as an epithelial marker maintained a stable expression throughout the experiments. Furthermore, expression of collagen 4 and the matrix-metallopeptidases 2 and 9 were increased after stimulation, while the expression of tissue inhibitors of metallopeptidases was not affected by pathogen stimulation. In this study we show a direct pathogen-induced trans-differentiation of primary bronchial epithelial cells resulting in a co-localization of epithelial and mesenchymal markers and an up-regulation of extracellular matrix components.

Updated outcomes for patients with completely resected pulmonary recurrent osteosarcoma: A report from the Children’s Oncology Group.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 10023-10023
Author(s):  
Alexander J. Chou ◽  
Mark D. Krailo ◽  
Ruxu Han ◽  
Allen Buxton ◽  
Damon R. Reed ◽  
...  
Keyword(s):  
Disease Control ◽  
Oncology Group ◽  
Specific Patient ◽  
Efficacy Analysis ◽  
Investigational Agent ◽  
Phase 2 Trial ◽  
Select Group ◽  
Using Data ◽  
The Stability ◽  
Histological Confirmation

10023 Background: Amongst patients with recurrent osteosarcoma (OS), those with resectable pulmonary-only relapse appear to have the best outcomes. Prior analysis of patients with completely resected recurrent OS enrolled on a Children’s Oncology Group (COG) Phase 2 trial AOST0221, which studied the efficacy of inhaled GMCSF, showed a 12- month disease control rate (DCR12) of only 20% (95% CI, 10 – 34%). DCR12 based on this analysis was used as the historical benchmark for efficacy analysis in two recently completed trials, AOST1321 (completely resected cohort only) and AOST1421. We analyzed the stability of the DCR12 benchmark using data from these contemporary studies. Methods: Patients were eligible for AOST1321 if they had undergone resection of all sites of recurrent or refractory OS within 30 days of enrollment and for AOST1421 if they had lung only recurrent OS completely resected within 4 weeks of enrollment. AOST1321 evaluated denosumab while AOST1421 evaluated dinutuximab. Patients with refractory disease, extrapulmonary recurrence or without histological confirmation of relapse were excluded from this analysis. DCR12 was defined as having at least stable disease 12 months after the start of protocol therapy. We report the DCR12 observed on AOST1321, AOST1421 and AOST0221. Results: One hundred and twenty-eight evaluable patients with completely resected recurrent OS were enrolled on AOST0221 (N=49), AOST1321 (N=38) and AOST1421 (N=41). One hundred and one patients were included in this analysis (AOST0221: 37, AOST 1321: 25, AOST1421:39). DCR12 was 14 % (95% CI, 5% – 26%) for AOST0221, 24% (95%CI 10-42%) for AOST1321, and 31% (95% CI, 17% - 45%) for AOST1421. Risk for disease progression did not differ across the 3 included studies. DCR12 for all three studies combined was 23% (95% CI, 15% – 31%). Conclusions: Prognosis for recurrent OS remains dismal, even for those with resectable pulmonary-only disease. Although not statistically significant, dinutuximab may have activity in a select group of relapsed OS patients; combination studies using dinutuximab are planned. The previously described benchmark of disease control at 12 months remained relatively consistent in recent studies for those patients who have resectable pulmonary-only relapse. Therefore, DCR12 remains a useful outcome measure in fully resected OS with lung only metastases. Analyses are ongoing to better define the appropriate threshold to define investigational agent activity in this specific patient population.

scholarly journals 8-azaadenosine and 8-chloroadenosine are not selective inhibitors of ADAR

2021 ◽  
Author(s):  
Kyle A Cottrell ◽  
Luisangely Soto Torres ◽  
Jason D Weber
Keyword(s):  
Small Molecules ◽  
Cell Lines ◽  
Cancer Cell ◽  
Selective Inhibition ◽  
Cancer Cell Lines ◽  
Type I ◽  
Inhibition Of Proliferation ◽  
Translational Repressor ◽  
Attractive Therapeutic Target ◽  
Interferon Pathway

The RNA editing enzyme ADAR, is an attractive therapeutic target for multiple cancers. Through its deaminase activity, ADAR edits adenosine to inosine in dsRNAs. Loss of ADAR in some cancer cell lines causes activation of the type I interferon pathway and the PKR translational repressor, leading to inhibition of proliferation and stimulation of cell death. As such, inhibition of ADAR function is a viable therapeutic strategy for many cancers. However, there are no FDA approved inhibitors of ADAR. Two small molecules have been previously described as inhibitors of ADAR: 8-azaadenosine and 8-chloroadenosine. Here we show that neither molecule is a selective inhibitor of ADAR. Both 8-azaadenosine and 8-chloroadenosine show similar toxicity to ADAR-dependent and independent cancer cell lines. Furthermore, the toxicity of both small molecules is comparable between cell lines with knockdown of ADAR and cells with unperturbed ADAR expression. Treatment with neither molecule causes activation of PKR. Finally, treatment with either molecule has no effect on A-to-I editing of an ADAR substrate. Together these data show that 8-azaadenosine and 8-chloroadenosine are not suitable small molecules for therapies that require selective inhibition of ADAR, and neither should be used in preclinical studies as ADAR inhibitors.

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