Publications by authors named "Luis J V Galietta"

111 Publications

Comprehensive Analysis of Combinatorial Pharmacological Treatments to Correct Nonsense Mutations in the CFTR Gene.

Int J Mol Sci 2021 Nov 4;22(21). Epub 2021 Nov 4.

Telethon Institute of Genetics and Medicine (TIGEM), 80078 Pozzuoli, Italy.

Cystic fibrosis (CF) is caused by loss of function of the CFTR chloride channel. A substantial number of CF patients carry nonsense mutations in the gene. These patients cannot directly benefit from pharmacological correctors and potentiators that have been developed for other types of CFTR mutations. We evaluated the efficacy of combinations of drugs targeting at various levels the effects of nonsense mutations: SMG1i to protect CFTR mRNA from nonsense-mediated decay (NMD), G418 and ELX-02 for readthrough, VX-809 and VX-445 to promote protein maturation and function, PTI-428 to enhance CFTR protein synthesis. We found that the extent of rescue and sensitivity to the various agents is largely dependent on the type of mutation, with W1282X and R553X being the mutations most and least sensitive to pharmacological treatments, respectively. In particular, W1282X-CFTR was highly responsive to NMD suppression by SMG1i but also required treatment with VX-445 corrector to show function. In contrast, G542X-CFTR required treatment with readthrough agents and VX-809. Importantly, we never found cooperativity between the NMD inhibitor and readthrough compounds. Our results indicate that treatment of CF patients with nonsense mutations requires a precision medicine approach with the design of specific drug combinations for each mutation.
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http://dx.doi.org/10.3390/ijms222111972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8584557PMC
November 2021

Chloride transport modulators as drug candidates.

Am J Physiol Cell Physiol 2021 12 13;321(6):C932-C946. Epub 2021 Oct 13.

Telethon Institute of Genetics and Medicine, Pozzuoli, Italy.

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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http://dx.doi.org/10.1152/ajpcell.00334.2021DOI Listing
December 2021

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

Int J Mol Sci 2021 May 17;22(10). Epub 2021 May 17.

U.O.C. Genetica Medica, IRCCS Istituto Giannina Gaslini, 16147 Genova, Italy.

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.
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http://dx.doi.org/10.3390/ijms22105262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8156943PMC
May 2021

Ex vivo model predicted in vivo efficacy of CFTR modulator therapy in a child with rare genotype.

Mol Genet Genomic Med 2021 04 13;9(4):e1656. Epub 2021 Mar 13.

Cystic Fibrosis Regional Reference Center,, Department of Paediatric Medicine, Anna Meyer Children's University, Florence, Italy.

Background: New drugs that target the basic defect in cystic fibrosis (CF) patients may now be used in a large number of patients carrying responsive mutations. Nevertheless, further research is needed to extend the benefit of these treatments to patients with rare mutations that are still uncharacterized in vitro and that are not included in clinical trials. For this purpose, ex vivo models are necessary to preliminary assessing the effect of CFTR modulators in these cases.

Method: We report the clinical effectiveness of lumacaftor/ivacaftor therapy prescribed to a CF child with a rare genetic profile (p.Phe508del/p.Gly970Asp) after testing the drug on nasal epithelial cells. We observed a significant drop of the sweat chloride value, as of the lung clearance index. A longer follow-up period is needed to define the effects of therapy on pancreatic status, although an increase of the fecal elastase values was found.

Conclusion: Drug response obtained on nasal epithelial cells correlates with changes in vivo therapeutic endpoints and can be a predictor of clinical efficacy of novel drugs especially in pediatric patients.
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http://dx.doi.org/10.1002/mgg3.1656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8123755PMC
April 2021

Pharmacological approaches to cystic fibrosis.

Eur J Med Chem 2021 04 30;216:113240. Epub 2021 Jan 30.

Telethon Institute of Genetics and Medicine (TIGEM), Campi Flegrei 34, 80078, Pozzuoli, NA, Italy; Department of Translational Medical Sciences (DISMET), University of Naples, "Federico II", Via Sergio Pansini 5, 80131, Naples, Italy.

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http://dx.doi.org/10.1016/j.ejmech.2021.113240DOI Listing
April 2021

Pharmacoproteomics pinpoints HSP70 interaction for correction of the most frequent Wilson disease-causing mutant of ATP7B.

Proc Natl Acad Sci U S A 2020 12 7;117(51):32453-32463. Epub 2020 Dec 7.

Telethon Institute of Genetics and Medicine, Pozzuoli, Naples 80078, Italy;

Pathogenic mutations in the copper transporter have been hypothesized to affect its protein interaction landscape contributing to loss of function and, thereby, to hepatic copper toxicosis in Wilson disease. Although targeting mutant interactomes was proposed as a therapeutic strategy, druggable interactors for rescue of ATP7B mutants remain elusive. Using proteomics, we found that the frequent H1069Q substitution promotes ATP7B interaction with HSP70, thus accelerating endoplasmic reticulum (ER) degradation of the mutant protein and consequent copper accumulation in hepatic cells. This prompted us to use an HSP70 inhibitor as bait in a bioinformatics search for structurally similar Food and Drug Administration-approved drugs. Among the hits, domperidone emerged as an effective corrector that recovered trafficking and function of ATP7B-H1069Q by impairing its exposure to the HSP70 proteostatic network. Our findings suggest that HSP70-mediated degradation can be safely targeted with domperidone to rescue ER-retained ATP7B mutants and, hence, to counter the onset of Wilson disease.
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http://dx.doi.org/10.1073/pnas.2006648117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7768786PMC
December 2020

Identification, Structure-Activity Relationship, and Biological Characterization of 2,3,4,5-Tetrahydro-1-pyrido[4,3-]indoles as a Novel Class of CFTR Potentiators.

J Med Chem 2020 10 18;63(19):11169-11194. Epub 2020 Sep 18.

D3-PharmaChemistry, Istituto Italiano di Tecnologia (IIT), 16163 Genova, Italy.

Cystic fibrosis (CF) is a life-threatening autosomal recessive disease, caused by mutations in the CF transmembrane conductance regulator (CFTR) chloride channel. CFTR modulators have been reported to address the basic defects associated with CF-causing mutations, partially restoring the CFTR function in terms of protein processing and/or channel gating. Small-molecule compounds, called potentiators, are known to ameliorate the gating defect. In this study, we describe the identification of the 2,3,4,5-tetrahydro-1-pyrido[4,3-]indole core as a novel chemotype of potentiators. In-depth structure-activity relationship studies led to the discovery of enantiomerically pure endowed with a good efficacy in rescuing the gating defect of F508del- and G551D-CFTR and a promising druglike profile. The characterization of γ-carboline showed considerable exposure levels and good oral bioavailability, with detectable distribution to the lungs after oral administration to rats. Overall, these findings may represent an encouraging starting point to further expand this chemical class, adding a new chemotype to the existing classes of CFTR potentiators.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8011931PMC
October 2020

Ionocytes and CFTR Chloride Channel Expression in Normal and Cystic Fibrosis Nasal and Bronchial Epithelial Cells.

Cells 2020 09 13;9(9). Epub 2020 Sep 13.

Telethon Institute of Genetics and Medicine (TIGEM), 80078 Pozzuoli (NA), Italy.

The airway epithelium contains ionocytes, a rare cell type with high expression of Forkhead Box I1 () transcription factor and Cystic Fibrosis Transmembrane conductance Regulator (), a chloride channel that is defective in cystic fibrosis (CF). Our aim was to verify if ionocyte development is altered in CF and to investigate the relationship between ionocytes and CFTR-dependent chloride secretion. We collected nasal cells by brushing to determine ionocyte abundance. Nasal and bronchial cells were also expanded in vitro and reprogrammed to differentiated epithelia for morphological and functional studies. We found a relatively high (~3%) ionocyte abundance in ex vivo nasal samples, with no difference between CF and control individuals. In bronchi, ionocytes instead appeared very rarely as previously reported, thus suggesting a possible proximal-distal gradient in human airways. The difference between nasal and bronchial epithelial cells was maintained in culture, which suggests an epigenetic control of ionocyte development. In the differentiation phase of the culture procedure, we used two media that resulted in a different pattern of CFTR expression: confined to ionocytes or more broadly expressed. CFTR function was similar in both conditions, thus indicating that chloride secretion equally occurs irrespective of CFTR expression pattern.
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http://dx.doi.org/10.3390/cells9092090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565890PMC
September 2020

Current development of CFTR potentiators in the last decade.

Eur J Med Chem 2020 Oct 15;204:112631. Epub 2020 Jul 15.

Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, Via Archirafi 32, 90123, Palermo, Italy.

Cystic fibrosis (CF) is a genetic disorder produced by the loss of function of CFTR, a main chloride channel involved in transepithelial salt and water transport. CFTR function can be rescued by small molecules called "potentiators" which increase gating activity of CFTR on epithelial surfaces. High throughput screening (HTS) assays allowed the identification of new chemical entities endowed with potentiator properties, further improved through medicinal chemistry optimization. In this review, the most relevant classes of CFTR potentiators developed in the last decade were explored, focusing on structure-activity relationships (SAR) of the different chemical entities, as a useful tool for the improvement of their pharmacological activity.
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http://dx.doi.org/10.1016/j.ejmech.2020.112631DOI Listing
October 2020

Intrinsic Abnormalities of Cystic Fibrosis Airway Connective Tissue Revealed by an In Vitro 3D Stromal Model.

Cells 2020 06 1;9(6). Epub 2020 Jun 1.

Istituto Italiano di Tecnologia (IIT), Largo Barsanti e Matteucci 53, 80125 Napoli, Italy.

Cystic fibrosis is characterized by lung dysfunction involving mucus hypersecretion, bacterial infections, and inflammatory response. Inflammation triggers pro-fibrotic signals that compromise lung structure and function. At present, several in vitro cystic fibrosis models have been developed to study epithelial dysfunction but none of these focuses on stromal alterations. Here we show a new cystic fibrosis 3D stromal lung model made up of primary fibroblasts embedded in their own extracellular matrix and investigate its morphological and transcriptomic features. Cystic fibrosis fibroblasts showed a high proliferation rate and produced an abundant and chaotic matrix with increased protein content and elastic modulus. More interesting, they had enhanced pro-fibrotic markers and genes involved in epithelial function and inflammatory response. In conclusion, our study reveals that cystic fibrosis fibroblasts maintain in vitro an activated pro-fibrotic state. This abnormality may play in vivo a role in the modulation of epithelial and inflammatory cell behavior and lung remodeling. We argue that the proposed bioengineered model may provide new insights on epithelial/stromal/inflammatory cells crosstalk in cystic fibrosis, paving the way for novel therapeutic strategies.
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http://dx.doi.org/10.3390/cells9061371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7348935PMC
June 2020

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

Sci Adv 2020 02 21;6(8):eaay9669. Epub 2020 Feb 21.

Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy.

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 EC 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.
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http://dx.doi.org/10.1126/sciadv.aay9669DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7034990PMC
February 2020

TRPV4 and purinergic receptor signalling pathways are separately linked in airway epithelia to CFTR and TMEM16A chloride channels.

J Physiol 2019 12 12;597(24):5859-5878. Epub 2019 Nov 12.

Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy.

Key Points: Eact is a putative pharmacological activator of TMEM16A. Eact is strongly effective in recombinant Fischer rat thyroid (FRT) cells but not in airway epithelial cells with endogenous TMEM16A expression. Transcriptomic analysis, gene silencing and functional studies in FRT cells reveal that Eact is actually an activator of the Ca -permeable TRPV4 channel. In airway epithelial cells TRPV4 and TMEM16A are expressed in separate cell types. Intracellular Ca elevation by TRPV4 stimulation leads to CFTR channel activation.

Abstract: TMEM16A is a Ca -activated Cl channel expressed in airway epithelial cells, particularly under conditions of mucus hypersecretion. To investigate the role of TMEM16A, we used Eact, a putative TMEM16A pharmacological activator. However, in contrast to purinergic stimulation, we found little effect of Eact on bronchial epithelial cells under conditions of high TMEM16A expression. We hypothesized that Eact is an indirect activator of TMEM16A. By a combination of approaches, including short-circuit current recordings, bulk and single cell RNA sequencing, intracellular Ca imaging and RNA interference, we found that Eact is actually an activator of the Ca -permeable TRPV4 channel and that the modest effect of this compound in bronchial epithelial cells is due to a separate expression of TMEM16A and TRPV4 in different cell types. Importantly, we found that TRPV4 stimulation induced activation of the CFTR Cl channel. Our study reveals the existence of separate Ca signalling pathways linked to different Cl secretory processes.
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http://dx.doi.org/10.1113/JP278784DOI Listing
December 2019

ANO4 (Anoctamin 4) Is a Novel Marker of Zona Glomerulosa That Regulates Stimulated Aldosterone Secretion.

Hypertension 2019 11 30;74(5):1152-1159. Epub 2019 Sep 30.

From the Centre for Clinical Pharmacology, William Harvey Research Institute, Barts and the London School of Medicine & Dentistry, Queen Mary University of London, United Kingdom Clinical Pharmacology Unit (C.M., L.H.S., M.J.B.).

Microarray comparison of the transcriptomes of human adrenal zona glomerulosa (ZG) and zona fasciculata found several ZG-specific genes that negatively regulate aldosterone secretion. The third and most significantly upregulated ZG-gene (19.9-fold compared with zona fasciculata, =6.58×10) was , a putative Ca-activated chloride channel. We have investigated the role of in human adrenal, and whether it functions like the prototype anoctamin, . We evaluated mRNA and protein expression in human adrenal by qPCR and immunohistochemistry, compared the effects of and overexpression on baseline and stimulated aldosterone secretion and cell proliferation in H295R cells, and analyzed ANO4 activity as a Ca-activated chloride channel in comparison with other anoctamins by a fluorescence-based functional assay. The expression of in ZG was confirmed by qPCR as 23.21-fold upregulated compared with zona fasciculata (n=18; =4.93×10). Immunohistochemistry found cytoplasmic, ZG-selective expression of ANO4 (anoctamin 4) protein. overexpression in H295R cells attenuated calcium-mediated aldosterone secretion and cell proliferation in comparison to controls. The latter effects were in a different direction to those of ANO1. The functional assay showed that, in contrast to ANO1, ANO4 expression results in low levels of calcium-dependent anion transport. In conclusion, ANO4 is one of the most highly expressed genes in ZG. It attenuates stimulated aldosterone secretion and cell proliferation. Although belonging to a family of Ca-activated chloride channels, it does not generate significant plasma membrane chloride channel activity.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.119.13287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6791498PMC
November 2019

An overview on chemical structures as ΔF508-CFTR correctors.

Eur J Med Chem 2019 Oct 15;180:430-448. Epub 2019 Jul 15.

Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, Via Archirafi 32, 90123, Palermo, Italy. Electronic address:

Deletion of phenylalanine at position 508 (F508del) in the CFTR protein, is the most common mutation causing cystic fibrosis (CF). F508del causes misfolding and rapid degradation of CFTR protein a defect that can be targeted with pharmacological agents termed "correctors". Correctors belong to various chemical classes but are generally small molecules based on nitrogen sulfur or oxygen heterocycles. The mechanism of action of correctors is generally unknown but there is experimental evidence that some of them can directly act on mutant CFTR improving folding and stability. Here we overview the characteristics of the various F508del correctors described so far to obtain indications on key chemical structures and modifications that are required for mutant protein rescue.
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http://dx.doi.org/10.1016/j.ejmech.2019.07.037DOI Listing
October 2019

Furocoumarins as multi-target agents in the treatment of cystic fibrosis.

Eur J Med Chem 2019 Oct 10;180:283-290. Epub 2019 Jul 10.

Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, Via Archirafi 32, 90123, Palermo, Italy. Electronic address:

Multi-target molecular entities, offer a path to progress both in understanding causes of disease and in defining effective small molecule treatments. Coumarin and its derivatives belong to an important group of natural compounds with diverse biological properties. They are found in vegetables and plants for which literature reports thousands of publications for the great variety of biological applications among which the photoprotective effects, thus being considered multi-targeting agents. Their furan condensed analogues constitute the family of furocoumarins, less represented in the literature, endowed with photosensitizing properties and often used for the treatment of skin diseases such as vitiligo and psoriasis. Despite the study of biological properties of linear and angular furocumarins dates back to ancient times, mainly as photosensitizers, these small molecules still represent an attractive scaffold for further development and applications in several therapeutic fields. The aim of the present review is to summarize the most promising chemical entities belonging to the class of furocumarins and coumarins, emerged in the last decades, and the methods used for their synthesis with a particular focus on main targets involved in the cystic fibrosis treatment.
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http://dx.doi.org/10.1016/j.ejmech.2019.07.025DOI Listing
October 2019

Bioactive Thymosin Alpha-1 Does Not Influence F508del-CFTR Maturation and Activity.

Sci Rep 2019 07 16;9(1):10310. Epub 2019 Jul 16.

UOC Genetica Medica, IRCCS Istituto Giannina Gaslini, Via Gerolamo Gaslini 5, 16147, Genova, Italy.

Deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is the most frequent mutation causing cystic fibrosis (CF). F508del-CFTR is misfolded and prematurely degraded. Recently thymosin a-1 (Tα-1) was proposed as a single molecule-based therapy for CF, improving both F508del-CFTR maturation and function by restoring defective autophagy. However, three independent laboratories failed to reproduce these results. Lack of reproducibility has been ascribed by the authors of the original paper to the use of DMSO and to improper handling. Here, we address these potential issues by demonstrating that Tα-1 changes induced by DMSO are fully reversible and that Tα-1 peptides prepared from different stock solutions have equivalent biological activity. Considering the negative results here reported, six independent laboratories failed to demonstrate F508del-CFTR correction by Tα-1. This study also calls into question the autophagy modulator cysteamine, since no rescue of mutant CFTR function was detected following treatment with cysteamine, while deleterious effects were observed when bronchial epithelia were exposed to cysteamine plus the antioxidant food supplement EGCG. Although these studies do not exclude the possibility of beneficial immunomodulatory effects of thymosin α-1, they do not support its utility as a corrector of F508del-CFTR.
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http://dx.doi.org/10.1038/s41598-019-46639-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635361PMC
July 2019

High-Throughput Screening for Modulators of CFTR Activity Based on Genetically Engineered Cystic Fibrosis Disease-Specific iPSCs.

Stem Cell Reports 2019 06 9;12(6):1389-1403. Epub 2019 May 9.

Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, 30625 Hannover, Germany; REBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany; Biomedical Research in Endstage and Obstructive Lung Disease (BREATH), Member of the German Center for Lung Research (DZL), 30625 Hannover, Germany. Electronic address:

Organotypic culture systems from disease-specific induced pluripotent stem cells (iPSCs) exhibit obvious advantages compared with immortalized cell lines and primary cell cultures, but implementation of iPSC-based high-throughput (HT) assays is still technically challenging. Here, we demonstrate the development and conduction of an organotypic HT Cl/I exchange assay using cystic fibrosis (CF) disease-specific iPSCs. The introduction of a halide-sensitive YFP variant enabled automated quantitative measurement of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) function in iPSC-derived intestinal epithelia. CFTR function was partially rescued by treatment with VX-770 and VX-809, and seamless gene correction of the p.Phe508del mutation resulted in full restoration of CFTR function. The identification of a series of validated primary hits that improve the function of p.Phe508del CFTR from a library of ∼42,500 chemical compounds demonstrates that the advantages of complex iPSC-derived culture systems for disease modeling can also be utilized for drug screening in a true HT format.
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http://dx.doi.org/10.1016/j.stemcr.2019.04.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6565754PMC
June 2019

Airway surface liquid acidification initiates host defense abnormalities in Cystic Fibrosis.

Sci Rep 2019 04 24;9(1):6516. Epub 2019 Apr 24.

Inserm U1151 - CNRS UMR 8253 - Institut Necker Enfants Malades, Université Paris Sorbonne, Paris, France.

Cystic fibrosis (CF) is caused by defective Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein. Morbidity is mainly due to early airway infection. We hypothesized that S. aureus clearance during the first hours of infection was impaired in CF human Airway Surface Liquid (ASL) because of a lowered pH. The ASL pH of human bronchial epithelial cell lines and primary respiratory cells from healthy controls (WT) and patients with CF was measured with a pH microelectrode. The antimicrobial capacity of airway cells was studied after S. aureus apical infection by counting surviving bacteria. ASL was significantly more acidic in CF than in WT respiratory cells. This was consistent with a defect in bicarbonate secretion involving CFTR and SLC26A4 (pendrin) and a persistent proton secretion by ATP12A. ASL demonstrated a defect in S. aureus clearance which was improved by pH normalization. Pendrin inhibition in WT airways recapitulated the CF airway defect and increased S. aureus proliferation. ATP12A inhibition by ouabain decreased bacterial proliferation. Antimicrobial peptides LL-37 and hBD1 demonstrated a pH-dependent activity. Normalizing ASL pH might improve innate airway defense in newborns with CF during onset of S. aureus infection. Pendrin activation and ATP12A inhibition could represent novel therapeutic strategies to normalize pH in CF airways.
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http://dx.doi.org/10.1038/s41598-019-42751-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6482305PMC
April 2019

Peripheral localization of the epithelial sodium channel in the apical membrane of bronchial epithelial cells.

Exp Physiol 2019 06 8;104(6):866-875. Epub 2019 Apr 8.

Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy.

New Findings: What is the central question of this study? What is the precise subcellular localization of the epithelial sodium channel (ENaC) in human airway epithelium? What is the main finding and its importance? ENaC protein has an unexpected localization in the peripheral region of the apical membrane of bronchial epithelial cells, very close to tight junctions. This may be important for the mechanism of Na absorption ABSTRACT: The epithelial sodium channel (ENaC) has a key role in absorbing fluid across the human airway epithelium. Altered activity of ENaC may perturb the process of mucociliary clearance, thus impairing the innate defence mechanisms against microbial agents. The proteins forming ENaC are present on the apical membrane of the epithelium. However, their precise localization is unknown. In the present study, we used two antibodies recognizing the α and β ENaC subunits. Both antibodies revealed a restricted localization of ENaC in the peripheral region of the apical membrane of cultured bronchial epithelial cells, close to but not overlapping with tight junctions. In contrast, the cystic fibrosis transmembrane conductance regulator chloride channel was more diffusely expressed on the whole apical membrane. Modulation of ENaC activity by aprotinin or elastase resulted in a decrease or increase in the peripheral localization, respectively. Our results suggest that sodium absorption is mainly occurring close to tight junctions where this cation may be rapidly expelled by the Na /K pump present in lateral membranes. This arrangement of channels and pumps may limit Na build-up in other regions of the cells.
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http://dx.doi.org/10.1113/EP087590DOI Listing
June 2019

Two CFTR mutations within codon 970 differently impact on the chloride channel functionality.

Hum Mutat 2019 06 28;40(6):742-748. Epub 2019 Mar 28.

Cell Biology and Disease Mechanisms Program, Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy.

Pharmacological rescue of mutant cystic fibrosis transmembrane conductance regulator (CFTR) in cystic fibrosis (CF) depends on the specific defect caused by different mutation classes. We asked whether a patient with the rare p.Gly970Asp (c.2909G>A) mutation could benefit from CFTR pharmacotherapy since a similar missense mutant p.Gly970Arg (c.2908G>C) was previously found to be sensitive to potentiators in vitro but not in vivo. By complementary DNA transfection, we found that both mutations are associated with defective CFTR function amenable to pharmacological treatment. However, analysis of messenger RNA (mRNA) from patient's cells revealed that c.2908G>C impairs RNA splicing whereas c.2909G>A does not perturb splicing and leads to the expected p.Gly970Asp mutation. In agreement with these results, nasal epithelial cells from the p.Gly970Asp patient showed significant improvement of CFTR function upon pharmacological treatment. Our results underline the importance of controlling the effect of CF mutation at the mRNA level to determine if the pharmacotherapy of CFTR basic defect is appropriate.
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http://dx.doi.org/10.1002/humu.23741DOI Listing
June 2019

The Autophagy Inhibitor Spautin-1 Antagonizes Rescue of Mutant CFTR Through an Autophagy-Independent and USP13-Mediated Mechanism.

Front Pharmacol 2018 13;9:1464. Epub 2018 Dec 13.

Telethon Institute of Genetics and Medicine, Pozzuoli, Italy.

The mutation F508del, responsible for a majority of cystic fibrosis cases, provokes the instability and misfolding of the CFTR chloride channel. Pharmacological recovery of F508del-CFTR may be obtained with small molecules called correctors. However, treatment with a single corrector and only leads to a partial rescue, a consequence of cell quality control systems that still detect F508del-CFTR as a defective protein causing its degradation. We tested the effect of spautin-1 on F508del-CFTR since it is an inhibitor of USP10 deubiquitinase and of autophagy, a target and a biological process that have been associated with cystic fibrosis and mutant CFTR. We found that short-term treatment of cells with spautin-1 downregulates the function and expression of F508del-CFTR despite the presence of corrector VX-809, a finding obtained in multiple cell models and assays. In contrast, spautin-1 was ineffective on wild type CFTR. Silencing and upregulation of USP13 (another target of spautin-1) but not of USP10, had opposite effects on F508del-CFTR expression/function. In contrast, modulation of autophagy with known activators or inhibitors did not affect F508del-CFTR. Our results identify spautin-1 as a novel chemical probe to investigate the molecular mechanisms that prevent full rescue of mutant CFTR.
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http://dx.doi.org/10.3389/fphar.2018.01464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6300570PMC
December 2018

A European regulatory perspective on cystic fibrosis: current treatments, trends in drug development and translational challenges for CFTR modulators.

Eur Respir Rev 2018 Jun 13;27(148). Epub 2018 Apr 13.

European Medicines Agency, London, UK.

In this article we analyse the current authorised treatments and trends in early drug development for cystic fibrosis (CF) in the European Union for the time period 2000-2016. The analysis indicates a significant improvement in the innovation and development of new potential medicines for CF, shifting from products that act on the symptoms of the disease towards new therapies targeting the cause of CF. However, within these new innovative medicines, results for CF transmembrane conductance regulator (CFTR) modulators indicate that one major challenge for turning a CF concept product into an actual medicine for the benefit of patients resides in the fact that, although pre-clinical models have shown good predictability for certain mutations, a good correlation to clinical end-points or biomarkers ( forced expiratory volume in 1 s and sweat chloride) for all mutations has not yet been achieved. In this respect, the use of alternative end-points and innovative nonclinical models could be helpful for the understanding of those translational discrepancies. Collaborative endeavours to promote further research and development in these areas as well as early dialogue with the regulatory bodies available at the European competent authorities are recommended.
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http://dx.doi.org/10.1183/16000617.0124-2017DOI Listing
June 2018

Speeding Up the Identification of Cystic Fibrosis Transmembrane Conductance Regulator-Targeted Drugs: An Approach Based on Bioinformatics Strategies and Surface Plasmon Resonance.

Molecules 2018 Jan 8;23(1). Epub 2018 Jan 8.

Institute for Biomedical Technologies, National Research Council (ITB-CNR), 20090 Segrate, Italy.

Cystic fibrosis (CF) is mainly caused by the deletion of Phe 508 (ΔF508) in the cystic fibrosis transmembrane conductance regulator (CFTR) protein that is thus withheld in the endoplasmic reticulum and rapidly degraded by the ubiquitin/proteasome system. New drugs able to rescue ΔF508-CFTR trafficking are eagerly awaited. An integrated bioinformatics and surface plasmon resonance (SPR) approach was here applied to investigate the rescue mechanism(s) of a series of CFTR-ligands including VX809, VX770 and some aminoarylthiazole derivatives (AAT). Computational studies tentatively identified a large binding pocket in the ΔF508-CFTR nucleotide binding domain-1 (NBD1) and predicted all the tested compounds to bind to three sub-regions of this main pocket. Noticeably, the known CFTR chaperone keratin-8 (K8) seems to interact with some residues located in one of these sub-pockets, potentially interfering with the binding of some ligands. SPR results corroborated all these computational findings. Moreover, for all the considered ligands, a statistically significant correlation was determined between their binding capability to ΔF508-NBD1 measured by SPR and the pockets availability measured by computational studies. Taken together, these results demonstrate a strong agreement between the in silico prediction and the SPR-generated binding data, suggesting a path to speed up the identification of new drugs for the treatment of cystic fibrosis.
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http://dx.doi.org/10.3390/molecules23010120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6017603PMC
January 2018

Synthesis and biological evaluation of novel thiazole- VX-809 hybrid derivatives as F508del correctors by QSAR-based filtering tools.

Eur J Med Chem 2018 Jan 8;144:179-200. Epub 2017 Dec 8.

Department of Experimental Medicine, Section of Biochemistry, University of Genoa, Viale Benedetto XV 1, 16132 Genoa, Italy; Center of Excellence for Biomedical Research (CEBR), University of Genoa, Viale Benedetto XV 9, 16132 Genoa, Italy. Electronic address:

The most common CF mutation, F508del, impairs the processing and gating of CFTR protein. This deletion results in the improper folding of the protein and its degradation before it reaches the plasma membrane of epithelial cells. Present correctors, like VX809 only induce a partial rescue of the mutant protein. Our previous studies reported a class of compounds, called aminoarylthiazoles (AATs), featuring an interesting activity as correctors. Some of them show additive effect with VX809 indicating a different mechanism of action. In an attempt to construct more interesting molecules, it was thought to generate chemically hybrid compounds, blending a portion of VX809 merged to the thiazole scaffold. This approach was guided by the development of QSAR analyses, which were performed based on the F508del correctors so far disclosed in the literature. This strategy was aimed at exploring the key requirements turning in the corrector ability of the collected derivatives and allowed us to derive a predictive model guiding for the synthesis of novel hybrids as promising correctors. The new molecules were tested in functional and biochemical assays on bronchial CFBE41o-cells expressing F508del-CFTR showing a promising corrector activity.
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http://dx.doi.org/10.1016/j.ejmech.2017.12.030DOI Listing
January 2018

High-throughput screening identifies FAU protein as a regulator of mutant cystic fibrosis transmembrane conductance regulator channel.

J Biol Chem 2018 01 20;293(4):1203-1217. Epub 2017 Nov 20.

From the Unità Operativa Complessa (U.O.C.) Genetica Medica, Istituto Giannina Gaslini, 16147 Genova, Italy,

In cystic fibrosis, deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel causes misfolding and premature degradation. One possible approach to reducing the detrimental health effects of cystic fibrosis could be the identification of proteins whose suppression rescues F508del-CFTR function in bronchial epithelial cells. However, searches for these potential targets have not yet been conducted, particularly in a relevant airway background using a functional readout. To identify proteins associated with F508del-CFTR processing, we used a high-throughput functional assay to screen an siRNA library targeting 6,650 different cellular proteins. We identified 37 proteins whose silencing significantly rescued F508del-CFTR activity, as indicated by enhanced anion transport through the plasma membrane. These proteins included FAU, UBE2I, UBA52, MLLT6, UBA2, CHD4, PLXNA1, and TRIM24, among others. We focused our attention on FAU, a poorly characterized protein with unknown function. FAU knockdown increased the plasma membrane targeting and function of F508del-CFTR, but not of wild-type CFTR. Investigation into the mechanism of action revealed a preferential physical interaction of FAU with mutant CFTR, leading to its degradation. FAU and other proteins identified in our screening may offer a therapeutically relevant panel of drug targets to correct basic defects in F508del-CFTR processing.
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http://dx.doi.org/10.1074/jbc.M117.816595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5787799PMC
January 2018

Substituted 2-Acylaminocycloalkylthiophene-3-carboxylic Acid Arylamides as Inhibitors of the Calcium-Activated Chloride Channel Transmembrane Protein 16A (TMEM16A).

J Med Chem 2017 06 24;60(11):4626-4635. Epub 2017 May 24.

Department of Chemistry and Biochemistry, San Francisco State University , San Francisco, California 94132-4136, United States.

Transmembrane protein 16A (TMEM16A), also called anoctamin 1 (ANO1), is a calcium-activated chloride channel expressed widely mammalian cells, including epithelia, vascular smooth muscle tissue, electrically excitable cells, and some tumors. TMEM16A inhibitors have been proposed for treatment of disorders of epithelial fluid and mucus secretion, hypertension, asthma, and possibly cancer. Herein we report, by screening, the discovery of 2-acylaminocycloalkylthiophene-3-carboxylic acid arylamides (AACTs) as inhibitors of TMEM16A and analysis of 48 synthesized analogs (10ab-10bw) of the original AACT compound (10aa). Structure-activity studies indicated the importance of benzene substituted as 2- or 4-methyl, or 4-fluoro, and defined the significance of thiophene substituents and size of the cycloalkylthiophene core. The most potent compound (10bm), which contains an unusual bromodifluoroacetamide at the thiophene 2-position, had IC of ∼30 nM, ∼3.6-fold more potent than the most potent previously reported TMEM16A inhibitor 4 (Ani9), and >10-fold improved metabolic stability. Direct and reversible inhibition of TMEM16A by 10bm was demonstrated by patch-clamp analysis. AACTs may be useful as pharmacological tools to study TMEM16A function and as potential drug development candidates.
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http://dx.doi.org/10.1021/acs.jmedchem.7b00020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5516794PMC
June 2017

Intermolecular Interactions in the TMEM16A Dimer Controlling Channel Activity.

Sci Rep 2016 12 8;6:38788. Epub 2016 Dec 8.

U.O.C. Genetica Medica, Istituto Giannina Gaslini, Genova, Italy.

TMEM16A and TMEM16B are plasma membrane proteins with Ca-dependent Cl channel function. By replacing the carboxy-terminus of TMEM16A with the equivalent region of TMEM16B, we obtained channels with potentiation of channel activity. Progressive shortening of the chimeric region restricted the "activating domain" to a short sequence close to the last transmembrane domain and led to TMEM16A channels with high activity at very low intracellular Ca concentrations. To elucidate the molecular mechanism underlying this effect, we carried out experiments based on double chimeras, Forster resonance energy transfer, and intermolecular cross-linking. We also modeled TMEM16A structure using the Nectria haematococca TMEM16 protein as template. Our results indicate that the enhanced activity in chimeric channels is due to altered interaction between the carboxy-terminus and the first intracellular loop in the TMEM16A homo-dimer. Mimicking this perturbation with a small molecule could be the basis for a pharmacological stimulation of TMEM16A-dependent Cl transport.
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http://dx.doi.org/10.1038/srep38788DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5144075PMC
December 2016

Phenylquinoxalinone CFTR activator as potential prosecretory therapy for constipation.

Transl Res 2017 04 15;182:14-26.e4. Epub 2016 Oct 15.

Departments of Medicine and Physiology, University of California San Francisco, San Francisco, Calif. Electronic address:

Constipation is a common condition for which current treatments can have limited efficacy. By high-throughput screening, we recently identified a phenylquinoxalinone activator of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel that stimulated intestinal fluid secretion and normalized stool output in a mouse model of opioid-induced constipation. Here, we report phenylquinoxalinone structure-activity analysis, mechanism of action, animal efficacy data in acute and chronic models of constipation, and functional data in ex vivo primary cultured human enterocytes. Structure-activity analysis was done on 175 phenylquinoxalinone analogs, including 15 synthesized compounds. The most potent compound, CFTR-J027, activated CFTR with EC ∼ 200 nM, with patch-clamp analysis showing a linear CFTR current-voltage relationship with direct CFTR activation. CFTR-J027 corrected reduced stool output and hydration in a mouse model of acute constipation produced by scopolamine and in a chronically constipated mouse strain (C3H/HeJ). Direct comparison with the approved prosecretory drugs lubiprostone and linaclotide showed substantially greater intestinal fluid secretion with CFTR-J027, as well as greater efficacy in a constipation model. As evidence to support efficacy in human constipation, CFTR-J027 increased transepithelial fluid transport in enteroids generated from normal human small intestine. Also, CFTR-J027 was rapidly metabolized in vitro in human hepatic microsomes, suggesting minimal systemic exposure upon oral administration. These data establish structure-activity and mechanistic data for phenylquinoxalinone CFTR activators, and support their potential efficacy in human constipation.
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http://dx.doi.org/10.1016/j.trsl.2016.10.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5453637PMC
April 2017

TMEM16 Proteins: Membrane Channels with Unusual Pores.

Biophys J 2016 11;111(9):1821-1822

Unità Operativa Complessa Genetica Medica, Istituto Giannina Gaslini, Genova, Italy. Electronic address:

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http://dx.doi.org/10.1016/j.bpj.2016.09.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5103025PMC
November 2016
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