Luis Juan Vicente Galietta
@tigem.it
Telethon Institute of Genetics and Medicine
University of Napoli "Federico II"
RESEARCH INTERESTS
Ion channels, chloride channels, calcium signaling, drug discovery, cystic fibrosis
Scopus Publications
Scopus Publications
Claudia Mazio, Laura Sara Scognamiglio, Costantino Casale, Valeria Panzetta, Francesco Urciuolo, Luis J.V. Galietta, Giorgia Imparato, and Paolo A. Netti
Elsevier BV
Fabiana Ciciriello, Francesco Panariello, Paola Medino, Arianna Biffi, Federico Alghisi, Chiara Rosazza, Patrizia Annunziata, Valentina Bouchè, Antonio Grimaldi, Daniela Guidone,et al.
Elsevier BV
Mario Renda, Marilia Barreca, Anna Borrelli, Virginia Spanò, Alessandra Montalbano, Maria Valeria Raimondi, Roberta Bivacqua, Ilaria Musante, Paolo Scudieri, Daniela Guidone,et al.
Springer Science and Business Media LLC
AbstractF508del, the most frequent mutation in cystic fibrosis (CF), impairs the stability and folding of the CFTR chloride channel, thus resulting in intracellular retention and CFTR degradation. The F508del defect can be targeted with pharmacological correctors, such as VX-809 and VX-445, that stabilize CFTR and improve its trafficking to plasma membrane. Using a functional test to evaluate a panel of chemical compounds, we have identified tricyclic pyrrolo-quinolines as novel F508del correctors with high efficacy on primary airway epithelial cells from CF patients. The most effective compound, PP028, showed synergy when combined with VX-809 and VX-661 but not with VX-445. By testing the ability of correctors to stabilize CFTR fragments of different length, we found that VX-809 is effective on the amino-terminal portion of the protein that includes the first membrane-spanning domain (amino acids 1–387). Instead, PP028 and VX-445 only show a stabilizing effect when the second membrane-spanning domain is included (amino acids 1–1181). Our results indicate that tricyclic pyrrolo-quinolines are a novel class of CFTR correctors that, similarly to VX-445, interact with CFTR at a site different from that of VX-809. Tricyclic pirrolo-quinolines may represent novel CFTR correctors suitable for combinatorial pharmacological treatments to treat the basic defect in CF.
Simone Amistadi, Giulia Maule, Matteo Ciciani, Marjolein M. Ensinck, Liesbeth De Keersmaecker, Anabela S. Ramalho, Daniela Guidone, Martina Buccirossi, Luis J.V. Galietta, Marianne S. Carlon,et al.
Elsevier BV
Claudia Mazio, Laura S. Scognamiglio, Roberta Passariello, Valeria Panzetta, Costantino Casale, Francesco Urciuolo, Luis J. V. Galietta, Giorgia Imparato, and Paolo A. Netti
American Chemical Society (ACS)
Cystic fibrosis (CF) is one of the most frequent genetic diseases, caused by dysfunction of the CF transmembrane conductance regulator (CFTR) chloride channel. CF particularly affects the epithelium of the respiratory system. Therapies aim at rescuing CFTR defects in the epithelium, but CF genetic heterogeneity hinders the finding of a single and generally effective treatment. Therefore, in vitro models have been developed to study CF and guide patient therapy. Here, we show a CF model on-chip by coupling the feasibility of the human bronchial epithelium differentiated in vitro at the air–liquid interface and the innovation of microfluidics. We demonstrate that the dynamic flow enhanced cilia distribution and increased mucus quantity, thus promoting tissue differentiation in a short time. The microfluidic devices highlighted differences between CF and non-CF epithelia, as shown by electrophysiological measures, mucus quantity, viscosity, and the analysis of ciliary beat frequency. The described model on-chip may be a handy instrument for studying CF and setting up therapies. As a proof of principle, we administrated the corrector VX-809 on-chip and observed a decrease in mucus thickness and viscosity.
Valeria Tomati, Stefano Costa, Valeria Capurro, Emanuela Pesce, Cristina Pastorino, Mariateresa Lena, Elvira Sondo, Marco Di Duca, Federico Cresta, Simona Cristadoro,et al.
Elsevier BV
Michele Genovese, Martina Buccirossi, Daniela Guidone, Rossella De Cegli, Sergio Sarnataro, Diego di Bernardo, and Luis J. V. Galietta
Wiley
Pharmacological inhibitors of TMEM16A (ANO1), a Ca2+‐activated Cl− channel, are important tools of research and possible therapeutic agents acting on smooth muscle, airway epithelia and cancer cells. We tested a panel of TMEM16A inhibitors, including CaCCinh‐A01, niclosamide, MONNA, Ani9 and niflumic acid, to evaluate their possible effect on intracellular Ca2+.
Miroslaw Zajac, Agathe Lepissier, Elise Dréano, Benoit Chevalier, Aurélie Hatton, Mairead Kelly-Aubert, Daniela Guidone, Gabrielle Planelles, Aleksander Edelman, Emmanuelle Girodon,et al.
Frontiers Media SA
Introduction: Cystic fibrosis (CF) is caused by defective Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) proteins. CFTR controls chloride (Cl−) and bicarbonate (HCO3−) transport into the Airway Surface Liquid (ASL). We investigated the impact of F508del-CFTR correction on HCO3− secretion by studying transepithelial HCO3− fluxes.Methods: HCO3− secretion was measured by pH-stat technique in primary human respiratory epithelial cells from healthy subjects (WT) and people with CF (pwCF) carrying at least one F508del variant. Its changes after CFTR modulation by the triple combination VX445/661/770 and in the context of TNF-α+IL-17 induced inflammation were correlated to ASL pH and transcriptional levels of CFTR and other HCO3− transporters of airway epithelia such as SLC26A4 (Pendrin), SLC26A9 and NBCe1.Results: CFTR-mediated HCO3− secretion was not detected in F508del primary human respiratory epithelial cells. It was rescued up to ∼ 80% of the WT level by VX-445/661/770. In contrast, TNF-α+IL-17 normalized transepithelial HCO3− transport and increased ASL pH. This was related to an increase in SLC26A4 and CFTR transcript levels. VX-445/661/770 induced an increase in pH only in the context of inflammation. Effects on HCO3− transport were not different between F508del homozygous and F508del compound heterozygous CF airway epithelia.Conclusion: Our studies show that correction of F508del-CFTR HCO3− is not sufficient to buffer acidic ASL and inflammation is a key regulator of HCO3− secretion in CF airways. Prediction of the response to CFTR modulators by theratyping should take into account airway inflammation.
Michele Genovese, Daniela Guidone, Martina Buccirossi, Anna Borrelli, Alejandra Rodriguez-Gimeno, Fabio Bertozzi, Tiziano Bandiera, and Luis J V Galietta
Oxford University Press (OUP)
Abstract Pharmacological modulators of the Ca2+ signaling cascade are important research tools and may translate into novel therapeutic strategies for a series of human diseases. We carried out a screening of a maximally diverse chemical library using the Ca2+-sensitive Cl− channel TMEM16A as a functional readout. We found compounds that were able to potentiate UTP-dependent TMEM16A activation. Mechanism of action of these compounds was investigated by a panel of assays that looked at intracellular Ca2+ mobilization triggered by extracellular agonists or by caged-IP3 photolysis, PIP2 breakdown by phospholipase C, and ion channel activity on nuclear membrane. One compound appears as a selective potentiator of inositol triphosphate receptor type 1 (ITPR1) with a possible application for some forms of spinocerebellar ataxia. A second compound is instead a potentiator of the P2RY2 purinergic receptor, an activity that could promote fluid secretion in dry eye and chronic obstructive respiratory diseases.
Daniela Guidone, Martina Buccirossi, Paolo Scudieri, Michele Genovese, Sergio Sarnataro, Rossella De Cegli, Federico Cresta, Vito Terlizzi, Gabrielle Planelles, Gilles Crambert,et al.
American Society for Clinical Investigation
The fluid covering the surface of airway epithelia represents a first barrier against pathogens. The chemical and physical properties of the airway surface fluid are controlled by the activity of ion channels and transporters. In cystic fibrosis (CF), loss of CFTR chloride channel function causes airway surface dehydration, bacterial infection, and inflammation. We investigated the effects of IL-17A plus TNF-α, 2 cytokines with relevant roles in CF and other chronic lung diseases. Transcriptome analysis revealed a profound change with upregulation of several genes involved in ion transport, antibacterial defense, and neutrophil recruitment. At the functional level, bronchial epithelia treated in vitro with the cytokine combination showed upregulation of ENaC channel, ATP12A proton pump, ADRB2 β-adrenergic receptor, and SLC26A4 anion exchanger. The overall result of IL-17A/TNF-α treatment was hyperviscosity of the airway surface, as demonstrated by fluorescence recovery after photobleaching (FRAP) experiments. Importantly, stimulation with a β-adrenergic agonist switched airway surface to a low-viscosity state in non-CF but not in CF epithelia. Our study suggests that CF lung disease is sustained by a vicious cycle in which epithelia cannot exit from the hyperviscous state, thus perpetuating the proinflammatory airway surface condition.
Amber R. Philp, Fernando Miranda, Ambra Gianotti, Agustín Mansilla, Paolo Scudieri, Ilaria Musante, Génesis Vega, Carlos D. Figueroa, Luis J. V. Galietta, José M. Sarmiento,et al.
American Physiological Society
Ion channels are potentially exploitable as pharmacological targets to treat asthma. This study evaluated the role of KCa3.1 channels, encoded by Kcnn4, in regulating the gene expression of mouse airway epithelium and the development of asthma traits. We used the ovalbumin (OVA) challenge as an asthma model in wild-type and Kcnn4−/− mice, performed histological analysis, and measured serum IgE to evaluate asthma traits. We analyzed gene expression of isolated epithelial cells of trachea or bronchi using mRNA sequencing and gene ontology and performed Ussing chamber experiments in mouse trachea to evaluate anion secretion. Gene expression of epithelial cells from mouse airways differed between trachea and bronchi, indicating regional differences in the inflammatory and transepithelial transport properties of proximal and distal airways. We found that Kcnn4 silencing reduced mast cell numbers, mucus, and collagen in the airways, and reduced the amount of epithelial anion secretion in the OVA-challenged animals. In addition, gene expression was differentially modified in the trachea and bronchi, with Kcnn4 genetic silencing significantly altering the expression of genes involved in the TNF pathway, supporting the potential of KCa3.1 as a therapeutic target for asthma.
Anita Golec, Iwona Pranke, Paolo Scudieri, Kate Hayes, Elise Dreano, Fiona Dunlevy, Aurelie Hatton, Damian G. Downey, Luis Galietta, and Isabelle Sermet
Elsevier BV
Luis J.V. Galietta
Elsevier BV
Elvira Sondo, Federico Cresta, Cristina Pastorino, Valeria Tomati, Valeria Capurro, Emanuela Pesce, Mariateresa Lena, Michele Iacomino, Ave Maria Baffico, Domenico Coviello,et al.
MDPI AG
Loss-of-function mutations of the CFTR gene cause cystic fibrosis (CF) through a variety of molecular mechanisms involving altered expression, trafficking, and/or activity of the CFTR chloride channel. The most frequent mutation among CF patients, F508del, causes multiple defects that can be, however, overcome by a combination of three pharmacological agents that improve CFTR channel trafficking and gating, namely, elexacaftor, tezacaftor, and ivacaftor. This study was prompted by the evidence of two CF patients, compound heterozygous for F508del and a minimal function variant, who failed to obtain any beneficial effects following treatment with the triple drug combination. Functional studies on nasal epithelia generated in vitro from these patients confirmed the lack of response to pharmacological treatment. Molecular characterization highlighted the presence of an additional amino acid substitution, L467F, in cis with the F508del variant, demonstrating that both patients were carriers of a complex allele. Functional and biochemical assays in heterologous expression systems demonstrated that the double mutant L467F-F508del has a severely reduced activity, with negligible rescue by CFTR modulators. While further studies are needed to investigate the actual prevalence of the L467F-F508del allele, our results suggest that this complex allele should be taken into consideration as plausible cause in CF patients not responding to CFTR modulators.
Loretta Ferrera, Floriana Cappiello, Maria Rosa Loffredo, Elena Puglisi, Bruno Casciaro, Bruno Botta, Luis J. V. Galietta, Mattia Mori, and Maria Luisa Mangoni
Springer Science and Business Media LLC
AbstractMutations in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein lead to persistent lung bacterial infections, mainly due to Pseudomonas aeruginosa, causing loss of respiratory function and finally death of people affected by CF. Unfortunately, even in the era of CFTR modulation therapies, management of pulmonary infections in CF remains highly challenging especially for patients with advanced stages of lung disease. Recently, we identified antimicrobial peptides (AMPs), namely Esc peptides, with potent antipseudomonal activity. In this study, by means of electrophysiological techniques and computational studies we discovered their ability to increase the CFTR-controlled ion currents, by direct interaction with the F508del-CFTR mutant. Remarkably, this property was not explored previously with any AMPs or peptides in general. More interestingly, in contrast with clinically used CFTR modulators, Esc peptides would give particular benefit to CF patients by combining their capability to eradicate lung infections and to act as promoters of airway wound repair with their ability to ameliorate the activity of the channel with conductance defects. Overall, our findings not only highlighted Esc peptides as the first characterized AMPs with a novel property, that is the potentiator activity of CFTR, but also paved the avenue to investigate the functions of AMPs and/or other peptide molecules, for a new up-and-coming pharmacological approach to address CF lung disease.
Alan S. Verkman and Luis J. V. Galietta
American Physiological Society
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.
Arianna Venturini, Anna Borrelli, Ilaria Musante, Paolo Scudieri, Valeria Capurro, Mario Renda, Nicoletta Pedemonte, and Luis J. V. Galietta
MDPI AG
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 CFTR 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.
Valeria Capurro, Valeria Tomati, Elvira Sondo, Mario Renda, Anna Borrelli, Cristina Pastorino, Daniela Guidone, Arianna Venturini, Alessandro Giraudo, Sine Mandrup Bertozzi,et al.
MDPI AG
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.
Tiziano Bandiera and Luis J.V. Galietta
Elsevier BV
Vito Terlizzi, Felice Amato, Chiara Castellani, Beatrice Ferrari, Luis J. V. Galietta, Giuseppe Castaldo, and Giovanni Taccetti
Wiley
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.
Virginia Spanò, Marilia Barreca, Vincenzo Cilibrasi, Michele Genovese, Mario Renda, Alessandra Montalbano, Luis Juan Vicente Galietta, and Paola Barraja
MDPI AG
Cystic fibrosis (CF) is a genetic disease caused by mutations that impair the function of the CFTR chloride channel. The most frequent mutation, F508del, causes misfolding and premature degradation of CFTR protein. This defect can be overcome with pharmacological agents named “correctors”. So far, at least three different classes of correctors have been identified based on the additive/synergistic effects that are obtained when compounds of different classes are combined together. The development of class 2 correctors has lagged behind that of compounds belonging to the other classes. It was shown that the efficacy of the prototypical class 2 corrector, the bithiazole corr-4a, could be improved by generating conformationally-locked bithiazoles. In the present study, we investigated the effect of tricyclic pyrrolothiazoles as analogues of constrained bithiazoles. Thirty-five compounds were tested using the functional assay based on the halide-sensitive yellow fluorescent protein (HS-YFP) that measured CFTR activity. One compound, having a six atom carbocyle central ring in the tricyclic pyrrolothiazole system and bearing a pivalamide group at the thiazole moiety and a 5-chloro-2-methoxyphenyl carboxamide at the pyrrole ring, significantly increased F508del-CFTR activity. This compound could lead to the synthesis of a novel class of CFTR correctors.
Mafalda Concilli, Raffaella Petruzzelli, Silvia Parisi, Federico Catalano, Francesco Sirci, Francesco Napolitano, Mario Renda, Luis J. V. Galietta, Diego Di Bernardo, and Roman S. Polishchuk
Proceedings of the National Academy of Sciences
Significance Wilson disease is a disorder of copper homeostasis caused by mutations in ATP7B . The most frequent mutation of ATP7B results in an H1069Q substitution that affects the localization and stability of the protein product. By interrogating the interactome of ATP7B-H1069Q, we found that this mutant shows stronger interaction with HSP70, which drives mutant degradation. Using an HSP70 inhibitor for structural similarity searches, we identified a Food and Drug Administration-approved drug that increases ATP7B-H1069Q stability in cells and thus improves ATP7B function. This pharmacoproteomic strategy provides an effective shortcut from understanding cellular mechanisms operating in Wilson disease to rapid identification of safe pharmacological tools and therefore might be expanded for drug repurposing to counteract other genetic disorders.
Virginia Spanò, Arianna Venturini, Michele Genovese, Marilia Barreca, Maria Valeria Raimondi, Alessandra Montalbano, Luis J.V. Galietta, and Paola Barraja
Elsevier BV
Nicoletta Brindani, Ambra Gianotti, Simone Giovani, Francesca Giacomina, Paolo Di Fruscia, Federico Sorana, Sine Mandrup Bertozzi, Giuliana Ottonello, Luca Goldoni, Ilaria Penna,et al.
American Chemical Society (ACS)
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-1H-pyrido[4,3-b]indole core as a novel chemotype of potentiators. In-depth structure–activity relationship studies led to the discovery of enantiomerically pure 39 endowed with a good efficacy in rescuing the gating defect of F508del- and G551D-CFTR and a promising in vitro druglike profile. The in vivo characterization of γ-carboline 39 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.
Paolo Scudieri, Ilaria Musante, Arianna Venturini, Daniela Guidone, Michele Genovese, Federico Cresta, Emanuela Caci, Alessandro Palleschi, Marco Poeta, Francesca Santamaria,et al.
MDPI AG
The airway epithelium contains ionocytes, a rare cell type with high expression of Forkhead Box I1 (FOXI1) transcription factor and Cystic Fibrosis Transmembrane conductance Regulator (CFTR), 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.