74 results on '"Tomati V"'
Search Results
2. P004 Clinical consequences and functional impact of the rare S737F CFTR variant
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Terlizzi, V., primary, Pesce, E., additional, Lena, M.T., additional, Tomati, V., additional, Capurro, V., additional, Pastorino, C., additional, Bocciardi, R., additional, Centrone, C., additional, Taccetti, G., additional, Castellani, C., additional, and Pedemonte, N., additional more...
- Published
- 2023
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Catalog
3. WS05.02 CFTR rescue by lumacaftor (VX-809) induces an extensive reorganisation of mitochondria in the cystic fibrosis bronchial epithelium
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Armirotti, A., primary, Braccia, C., additional, Christopher, J., additional, Crook, O., additional, Breckels, L., additional, Queiroz, R., additional, Liessi, N., additional, Tomati, V., additional, Capurro, V., additional, Bandiera, T., additional, Baldassari, S., additional, Pedemonte, N., additional, and Lilley, K., additional more...
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- 2022
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4. Gain- and Loss-of-Function CFTR Alleles Are Associated with COVID-19 Clinical Outcomes
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Baldassarri, M, Zguro, K, Tomati, V, Pastorino, C, Fava, F, Croci, S, Bruttini, M, Picchiotti, N, Furini, S, Pedemonte, N, Gabbi, C, Renieri, A, Fallerini, C, Crotti, L, Baldassarri, Margherita, Zguro, Kristina, Tomati, Valeria, Pastorino, Cristina, Fava, Francesca, Croci, Susanna, Bruttini, Mirella, Picchiotti, Nicola, Furini, Simone, Pedemonte, Nicoletta, Gabbi, Chiara, Renieri, Alessandra, Fallerini, Chiara, Crotti,Lia, Baldassarri, M, Zguro, K, Tomati, V, Pastorino, C, Fava, F, Croci, S, Bruttini, M, Picchiotti, N, Furini, S, Pedemonte, N, Gabbi, C, Renieri, A, Fallerini, C, Crotti, L, Baldassarri, Margherita, Zguro, Kristina, Tomati, Valeria, Pastorino, Cristina, Fava, Francesca, Croci, Susanna, Bruttini, Mirella, Picchiotti, Nicola, Furini, Simone, Pedemonte, Nicoletta, Gabbi, Chiara, Renieri, Alessandra, Fallerini, Chiara, and Crotti,Lia more...
- Abstract
Carriers of single pathogenic variants of the CFTR (cystic fibrosis transmembrane conductance regulator) gene have a higher risk of severe COVID-19 and 14-day death. The machine learning post-Mendelian model pinpointed CFTR as a bidirectional modulator of COVID-19 outcomes. Here, we demonstrate that the rare complex allele [G576V;R668C] is associated with a milder disease via a gain-of-function mechanism. Conversely, CFTR ultra-rare alleles with reduced function are associated with disease severity either alone (dominant disorder) or with another hypomorphic allele in the second chromosome (recessive disorder) with a global residual CFTR activity between 50 to 91%. Furthermore, we characterized novel CFTR complex alleles, including [A238V;F508del], [R74W;D1270N;V201M], [I1027T;F508del], [I506V;D1168G], and simple alleles, including R347C, F1052V, Y625N, I328V, K68E, A309D, A252T, G542*, V562I, R1066H, I506V, I807M, which lead to a reduced CFTR function and thus, to more severe COVID-19. In conclusion, CFTR genetic analysis is an important tool in identifying patients at risk of severe COVID-19. more...
- Published
- 2022
5. IDENTIFICATION OF NEW TARGETS FOR ΔF508-CFTR RESCUE BY GENOME-WIDE SHORT INTERFERING RNA SCREENING: 232
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Pedemonte, N., Tomati, V., Sondo, E., Pesce, E., Caci, E., and Galietta, L. J.
- Published
- 2012
6. IDENTIFICATION OF NOVEL TARGETS FOR δF508-CFTR RESCUE BY RNA INTERFERENCE-MEDIATED SILENCING: 208★
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Pedemonte, N., Caci, E., Sondo, E., Tomati, V., and Galietta, L. J.
- Published
- 2011
7. EXPRESSION AND FUNCTION OF TMEM16A PROTEIN IN HETEROLOGOUS AND NATIVE EXPRESSION SYSTEMS: 118
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Sondo, E., Ferrera, L., Scudieri, P., Caci, E., Tomati, V., Gianotti, A., Bruno, S., Zegarra-Moran, O. L., Pedemonte, N., and Galietta, L. J.
- Published
- 2011
8. Erratum: Corrigendum Thymosin α-1 does not correct F508del-CFTR in cystic fibrosis airway epithelia (JCI Insight (2018) 3:3 (e98699) DOI: 10.1172/jci.insight.98699)
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Tomati V., Caci E., Ferrera L., Pesce E., Sondo E., Cholon D. M., Quinney N. L., Boyles S. E., Armirotti A., Ravazzolo R., Galietta L. J. V., Gentzsch M., Pedemonte N., Tomati, V., Caci, E., Ferrera, L., Pesce, E., Sondo, E., Cholon, D. M., Quinney, N. L., Boyles, S. E., Armirotti, A., Ravazzolo, R., Galietta, L. J. V., Gentzsch, M., and Pedemonte, N. more...
- Abstract
The unit for the concentration of peptide in the proliferation study was incorrectly reported in the Results and Methods sections and the Figure 4 legend. The corrected sentences, with sections indicated, are below. Results Evaluation of Tα-1 sequence and its effect on proliferation and apoptosis of MCF-7 breast cancer cells. Therefore, we plated MCF-7 at low density on 96-well plates suitable for confocal high-content imaging and evaluated cell proliferation for 72 hours following treatment with Tα-1 (100 μM) or scrambled peptide (100 μM). Methods Proliferation study. MCF-7 cells were plated at low density (10,000 cells/well) on 96-well plates suitable for high-content imaging. After 6 hours, cells were treated with the scrambled peptide (100 μM) or with Tα-1 (100 μM). Figure 4 legend (A) Dot plot showing proliferation of MCF-7 cells after 72-hour treatment with Tα-1 (100 μM) or scrambled peptide (100 μM, control). (B) Dot plot showing the number of apoptotic MCF-7 cells after 72-hour treatment with Tα-1 (100 μM) or scrambled peptide (100 μM, control). The article has been updated to reflect these changes. The authors regret the errors. more...
- Published
- 2019
9. The role of functional studies in the diagnosis and treatment of Cystic Fibrosis: comparing the case of the G970D and G970R mutation
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Amato F, Scudieri P, Musante I, Tomati V, Caci E, Comegna M, Maietta S, Manzoni F, Di Lullo AM, De Wachter E, Vanderhelst Eef, Terlizzi V, Braggion C, Castaldo G, Galietta LJV., SIFC, Amato, F, Scudieri, P, Musante, I, Tomati, V, Caci, E, Comegna, M, Maietta, S, Manzoni, F, DI LULLO, ANTONELLA MIRIAM, De Wachter, E, Vanderhelst, Eef, Terlizzi, V, Braggion, C, Castaldo, G, and Galietta, Ljv. more...
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Cystic Fibrosis, mutations - Abstract
More than 2000 CFTR mutations have been identified so far, but only few of them are clearly defined as CF-causing based on functional studies. We present a case of a rare mutation, the G970D, that has been shown using transfected cDNA in HEK293 cells to be sensitive to Ivacaftor. However, a similar missense mutant, G970R in the same codon, was found to be sensitive to potentiators in vitro, but not in vivo due to splicing alteration. Thus, we used several basic research methodologies to evaluate if this patient was eligible for treatment with Ivacaftor. Materials and methods 1) nasal epithelial cells (HNEC) were collected from patients to evaluate the effect of mutations on splicing by RT-PCR assay, 2) HNEC were expanded and polarized for evaluation of CFTR function by Ussing chamber system 3) the use of a minigene system was used to confirm in vitro the splicing pattern. Results Firstly, we used in silico tool to predict the physio-pathological effect of mutations, confirming that the G970R completely abolishes the canonical 5’ splice donor site of exon 17 resulting in a likely retention of intron 17. On the contrary, the G970D predicted not to affect splicing. This prediction was confirmed by RT-PCR analysis of mRNA extracted from HNEC cells and from in vitro minigene assay. Finally, the functional behavior of CFTR, from HNEC bearing the G970D, was evaluated by short-circuit recordings. The cells responded to cAMP agonist with an increase in trans-epithelial current and this current was nearly doubled by stimulation with Ivacaftor. Moreover, in cells that were also incubated with VX-809 (1 μM) for 24 hours, CFTR function was significantly enhanced, with a proportional increase of both cAMP- and potentiator-dependent responses. Conclusions Our results show that the G970R actually disrupts the RNA splicing, thus leading to a severely altered CFTR protein. This event explains the lack of success of treatment with potentiator. In contrast, the G970D does not alter RNA splicing. The resulting G970D mutation affects the gating and trafficking of the CFTR channel that can be targeted with VX-770 and VX-809. These results represent the evidence needed to justify the treatment of the patient with these drugs. Finally, our study is an interesting example of the precision medicine approach by emphasizing the role of appropriate in vitro studies, in this case focused on RNA analysis, to fully characterize the effects of rare CFTR mutations. We thank Telethon Foundation (TMLGCBX16TT) and Ministero della Salute (Rome, Italy) L.548/93 for the regional research funding quote 2008-2015. more...
- Published
- 2018
10. Genetic Inhibition of the Ubiquitin Ligase Rnf5 Attenuates Phenotypes Associated to F508del Cystic Fibrosis Mutation
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Tomati, V. (Valeria), Sondo, E. (Elvira), Armirotti, A. (Andrea), Caci, E. (Emanuela), Pesce, E. (Emanuela), Marini, M. (Monica), Gianotti, A. (Ambra), Ju Jeon, Y. (Young), Cilli, M. (Michele), Pistorio, A. (Angela), Mastracci, L. (Luca), Ravazzolo, R. (Roberto), Scholte, B.J. (Bob), Ronai, Z. (Ze'ev), Galietta, L.J.V. (Luis J. V.), Pedemonte, N. (Nicoletta), Tomati, V. (Valeria), Sondo, E. (Elvira), Armirotti, A. (Andrea), Caci, E. (Emanuela), Pesce, E. (Emanuela), Marini, M. (Monica), Gianotti, A. (Ambra), Ju Jeon, Y. (Young), Cilli, M. (Michele), Pistorio, A. (Angela), Mastracci, L. (Luca), Ravazzolo, R. (Roberto), Scholte, B.J. (Bob), Ronai, Z. (Ze'ev), Galietta, L.J.V. (Luis J. V.), and Pedemonte, N. (Nicoletta) more...
- Abstract
Cystic fibrosis (CF) is caused by mutations in the CFTR chloride channel. Deletion of phenylalanine 508 (F508del), the most frequent CF mutation, impairs CFTR trafficking and gating. F508del-CFTR mistrafficking may be corrected by acting directly on mutant CFTR itself or by modulating expression/activity of CFTR-interacting proteins, that may thus represent potential drug targets. To evaluate possible candidates for F508del-CFTR rescue, we screened a siRNA library targeting known CFTR interactors. Our analysis identified RNF5 as a protein whose inhibition promoted significant F508del-CFTR rescue and displayed an additive effect with the investigational drug VX-809. Significantly, RNF5 loss in F508del-CFTR transgenic animals ameliorated intestinal malabsorption and concomitantly led to an increase in CFTR activity in intestinal epithelial cells. In addition, we found that RNF5 is differentially expressed in human bronchial epithelia from CF vs. control patients. Our results identify RNF5 as a target for therapeutic modalities to antagonize mutant CFTR proteins. more...
- Published
- 2015
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11. Genetic Inhibition Of The Ubiquitin Ligase Rnf5 Attenuates Phenotypes Associated To F508del Cystic Fibrosis Mutation
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Tomati, V, Sondo, E, Armirotti, A, Caci, E, Pesce, E, Marini, M, Gianotti, A, Jeon, YJ, Cilli, M, Pistorio, A, Mastracci, L, Ravazzolo, R, Scholte, Bob, Ronai, Z, Galietta, LJV, Pedemonte, N, Tomati, V, Sondo, E, Armirotti, A, Caci, E, Pesce, E, Marini, M, Gianotti, A, Jeon, YJ, Cilli, M, Pistorio, A, Mastracci, L, Ravazzolo, R, Scholte, Bob, Ronai, Z, Galietta, LJV, and Pedemonte, N more...
- Abstract
Cystic fibrosis (CF) is caused by mutations in the CFTR chloride channel. Deletion of phenylalanine 508 (F508del), the most frequent CF mutation, impairs CFTR trafficking and gating. F508del-CFTR mistrafficking may be corrected by acting directly on mutant CFTR itself or by modulating expression/activity of CFTR-interacting proteins, that may thus represent potential drug targets. To evaluate possible candidates for F508del-CFTR rescue, we screened a siRNA library targeting known CFTR interactors. Our analysis identified RNF5 as a protein whose inhibition promoted significant F508del-CFTR rescue and displayed an additive effect with the investigational drug VX-809. Significantly, RNF5 loss in F508del-CFTR transgenic animals ameliorated intestinal malabsorption and concomitantly led to an increase in CFTR activity in intestinal epithelial cells. In addition, we found that RNF5 is differentially expressed in human bronchial epithelia from CF vs. control patients. Our results identify RNF5 as a target for therapeutic modalities to antagonize mutant CFTR proteins. more...
- Published
- 2015
12. A novel Bim-BH3-derived Bcl-XL inhibitor
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Ponassi, R, Biasotti, B, Tomati, V, Bruno, S, Poggi, A, Malacarne, D, Cimoli, G, Salis, A, Pozzi, S, Miglino, M, Damonte, G, Cozzini, P, Spyraki, F, Campanini, B, Bagnasco, L, Castagnino, N, Tortolina, L, Mumot, A, Frassoni, F, Daga, A, Cilli, M, Piccardi, F, Monfardini, I, Perugini, M, Zoppoli, G, Darrigo, C, Pesenti, Raffaele, and Parodi, S. more...
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Targeted therapy ,Bim-BH3 ,Apoptosis ,Leukaemia ,Multi-parameter flow cytometry - Published
- 2008
13. A novel Bim-BH3-derived Bcl-X(L) inhibitor: Biochemical characterization, in vitro, in vivo and ex-vivo anti-leukemic activity
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Ponassi R., Biasotti B., Tomati V., Bruno S., Poggi A., Malacarne D., Cimoli G., Salis A., Pozzi S., Miglino M., Damonte G., Cozzini P., Spyraki F., Campanini B., Bagnasco L., Castagnino N., Tortolina L., Mumot A., Frassoni F., Daga A., Cilli M., and Piccardi more...
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- 2008
14. C terminal region of protein kinase CK2a: how the structure can affect function and stability of the catalytic subunit
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Grasselli, Elena, Tomati, V., Berbasconi, M., Nicolini, C., and Vergani, Laura
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- 2004
15. Internalization via Antennapedia protein transduction domain of an scFv antibody toward c‐Myc protein
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Avignolo, C., primary, Bagnasco, L., additional, Biasotti, B., additional, Melchiori, A., additional, Tomati, V., additional, Bauer, I., additional, Salis, A., additional, Chiossone, L., additional, Mingari, M. C., additional, Orecchia, P., additional, Carnemolla, B., additional, Neri, D., additional, Zardi, L., additional, and Parodi, S., additional more...
- Published
- 2007
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16. Inhibition of a protein‐protein interaction between INI1 and c‐Myc by small peptidomimetic molecules inspired by Helix‐1 of c‐Myc: identification of a new target of potential antineoplastic interest
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Bagnasco, L., primary, Tortolina, L., additional, Biasotti, B., additional, Castagnino, N., additional, Ponassi, R., additional, Tomati, V., additional, Nieddu, E., additional, Stier, G., additional, Malacarne, D., additional, and Parodi, S., additional more...
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- 2007
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17. Internalization via Antennapedia protein transduction domain of an scFv antibody toward c-Myc protein.
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Avignolo, C., Bagnasco, L., Biasotti, B., Melchiori, A., Tomati, V., Bauer, I., Saris, A., Chiossone, L., Mingari, M. C., Orecchia, P., Carnemolla, B., Neri, D., Zardi, L., and Parodi, S.
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GENETIC transduction ,MYC proteins ,IMMUNOGLOBULINS ,CYTOPLASM ,CELL membranes - Abstract
We constructed a single-chain variable fragment miniantibody (G11-scFv) directed toward the transactivation domain of c-Myc, which is fused with the internalization domain Int of Antennapedia at its carboxyl terminus (a cargo-carrier construct). In ELISA experiments, an EC
50 for binding saturation was achieved at concentrations of G11-scFv-Int(-) of ~10-8 M. Internalization of a fluoresceinated FI-G11-scFv-Int(+) construct was observed in intact human cultured cells with confocal microscopy. After 5 h of incubation in medium containing 1 µM FI-G11-scFv-Int(+) or FI-G11-scFv-Int(-), fluorescence intensity was determined in individual cells, both for cytoplasmic and nuclear compartments: concentration levels of FI-G11-scFv-Int(+), relative to the extracellular culture medium concentration, were 4-5 times higher in the cytoplasm, 7-8 times higher in the nucleus, and 10 times higher in the nucleoli. In the same experimental conditions, the FI-G11-scFv-Int(-) construct was 3-4 times more concentrated outside of the cells than inside. Cell membranes kept their integrity after 5 h of incubation. The antiproliferative activity of our miniantibody was studied on HCT116 cells. Incubation with 4 µM G11-scFv-Int(+) for 4 days induced very significant statistical and biological growth inhibition, whereas Int alone was completely inactive. Miniantibodies capable of penetrating cell membranes dramatically broaden the potential for innovative therapeutic agents and attack of new targets. [ABSTRACT FROM AUTHOR] more...- Published
- 2008
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18. A novel Bim-BH3-derived Bcl-XL inhibitor: Biochemical characterization, in vitro, in vivo and ex-vivo anti-leukemic activity
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Ponassi R, Biasotti B, Tomati V, Bruno S, Poggi A, Malacarne D, Cimoli G, Salis A, Pozzi S, Miglino M, Damonte G, Cozzini P, Spyrakis F, Spyraki F, Campanini B, Bagnasco L, Castagnino N, Tortolina L, Mumot A, Frassoni F, Antonio Daga, Cilli M, Piccardi F, Monfardini I, Perugini M, Zoppoli G, D'Arrigo C, Pesenti R, and Parodi S more...
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Molecular Biology ,Developmental Biology ,Cell Biology ,media_common.quotation_subject ,Molecular Sequence Data ,Transplantation, Heterologous ,bcl-X Protein ,Bcl-xL ,Mice, SCID ,Mitochondrion ,Flow cytometry ,Mice ,chemistry.chemical_compound ,Mice, Inbred NOD ,Proto-Oncogene Proteins ,Tumor Cells, Cultured ,medicine ,Animals ,Humans ,Cytotoxic T cell ,Amino Acid Sequence ,Lymphocytes ,multi-parameter flow cytometry ,Internalization ,Cells, Cultured ,bcl-2-Associated X Protein ,media_common ,Bcl-2-Like Protein 11 ,biology ,medicine.diagnostic_test ,apoptosis ,Membrane Proteins ,targeted therapy ,Protein Structure, Tertiary ,Cell biology ,Leukemia, Myeloid, Acute ,Bim-BH3 ,chemistry ,Apoptosis ,leukaemia ,biology.protein ,Female ,biological phenomena, cell phenomena, and immunity ,Growth inhibition ,Apoptosis Regulatory Proteins ,Peptides ,Neoplasm Transplantation ,Ex vivo - Abstract
BH3-only members of the Bcl-2 family exert a fundamental role in apoptosis induction. This work focuses on the development of a novel peptidic molecule based on the BH3 domain of Bim. The antiapoptotic molecule Bcl-X(L), involved in cancer development/progression and tumour resistance to cytotoxic drugs, is a target for Bim. According to a rational study of the structural interactions between wt Bim-BH3 and Bcl-X(L), we replaced specific residues of Bim-BH3 with natural and non-natural aminoacids and added an internalizing sequence, thus increasing dramatically the inhibitory activity of our modified Bim-BH3 peptide, called 072RB. Confocal microscopy and flow cytometry demonstrated cellular uptake and internalization of 072RB, followed by co-localization with mitochondria. Multiparameter flow cytometry demonstrated that the 072RB dose-dependent growth inhibition of leukaemia cell lines was due to apoptotic cell death. No effect was observed when cells were treated with the internalizing vector alone or a mutated control peptide (single aminoacid substitution L94A). Ex-vivo derived leukemic cells from acute myeloid leukaemia (AML) patients underwent cell death when cultured in vitro in the presence of 072RB. Conversely, no significant cytotoxic effect was observed when 072RB was administered to cultures of peripheral blood mononuclear cells, either resting or PHA-stimulated, and bone marrow cells of normal donors. Xenografts of human AML cells in NOD/SCID mice displayed a significant delay of leukemic cell growth upon treatment with 072RB administered intravenously (15 mg/Kg three times, 48 hours after tumour cell injection). Altogether, these observations support the therapeutic potentials of this novel BH3 mimetic. more...
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19. 351 Beyond elexacaftor/tezacaftor/ivacaftor: mechanistic insights to maximize N1303K-CFTR rescue.
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Pranke, I., Capurro, V., Chevalier, B., Pesce, E., Tomati, V., Pastorino, C., Hatton, A., Dreano, E., Kelly-Aubert, M., Guerrera, C., Galietta, L., Girodon, E., Burgel, P., Sermet-Gaudelus, I., Hinzpeter, A., and Pedemonte, N. more...
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- 2024
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20. Partial Rescue of F508del-CFTR Stability and Trafficking Defects by Double Corrector Treatment
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Arianna Venturini, Daniela Guidone, Ilaria Musante, Sine Mandrup Bertozzi, Cristina Pastorino, Valeria Tomati, Tiziano Bandiera, Nicoletta Pedemonte, Valeria Capurro, Federico Zara, Luis J. V. Galietta, Fabio Bertozzi, Anna Borrelli, Elvira Sondo, Mario Renda, Alessandro Giraudo, Capurro, V., Tomati, V., Sondo, E., Renda, M., Borrelli, A., Pastorino, C., Guidone, D., Venturini, A., Giraudo, A., Bertozzi, S. M., Musante, I., Bertozzi, F., Bandiera, T., Zara, F., Galietta, L. J. V., and Pedemonte, N. more...
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Protein Folding ,elexacaftor ,Indoles ,Pyrrolidines ,Cystic Fibrosis ,Pyridines ,Chloride Channel ,Pyridine ,Mutant ,Aminopyridines ,Aminophenol ,Cystic Fibrosis Transmembrane Conductance Regulator ,Aminophenols ,medicine.disease_cause ,Cystic fibrosis ,Pyrrolidine ,Ubiquitin ,Drug Combination ,Biology (General) ,Spectroscopy ,Mutation ,conformational stability ,biology ,Chemistry ,General Medicine ,Small molecule ,Computer Science Applications ,Cell biology ,chloride secretion ,Drug Combinations ,Chloride channel ,Quinolines ,Human ,Primary bronchial cell ,QH301-705.5 ,Bronchi ,Article ,Catalysis ,Inorganic Chemistry ,Chloride Channels ,allosteric folding correction ,medicine ,Humans ,Benzodioxoles ,Physical and Theoretical Chemistry ,QD1-999 ,Molecular Biology ,Cystic Fibrosi ,Epithelial Cell ,Organic Chemistry ,Allosteric folding correction ,Chloride secretion ,Conformational stability ,Elexacaftor ,Primary bronchial cells ,VX-445 ,Epithelial Cells ,Pyrazoles ,Subcellular localization ,medicine.disease ,primary bronchial cells ,Aminopyridine ,Indole ,Pyrazole ,biology.protein ,Benzodioxole ,Function (biology) - Abstract
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. more...
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- 2021
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21. Discovery of a picomolar potency pharmacological corrector of the mutant CFTR chloride channel
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Elvira Sondo, Alejandra Rodríguez-Gimeno, Federico Sorana, Ambra Gianotti, Luis J. V. Galietta, Loretta Ferrera, Fabio Bertozzi, Emanuela Caci, Nicoletta Pedemonte, Paolo Scudieri, Francesco Berti, Emanuela Pesce, Tiziano Bandiera, Paolo Di Fruscia, Valeria Tomati, Pedemonte, N., Bertozzi, F., Caci, E., Sorana, F., Fruscia, P. D., Tomati, V., Ferrera, L., Rodriguez-Gimeno, A., Berti, F., Pesce, E., Sondo, E., Gianotti, A., Scudieri, P., Bandiera, T., and Galietta, L. J. V. more...
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Drug ,congenital, hereditary, and neonatal diseases and abnormalities ,endocrine system ,media_common.quotation_subject ,Cystic Fibrosis Transmembrane Conductance Regulator ,Bronchi ,Pharmacology ,Cystic fibrosis ,Biochemistry ,Chemical library ,Cell Line ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,medicine ,Potency ,Humans ,Research Articles ,030304 developmental biology ,media_common ,EC50 ,0303 health sciences ,Multidisciplinary ,Chemistry ,SciAdv r-articles ,Epithelial Cells ,Potentiator ,respiratory system ,medicine.disease ,Small molecule ,High-Throughput Screening Assays ,Pharmaceutical Preparations ,030220 oncology & carcinogenesis ,Chloride channel ,Mutant Proteins ,Research Article - Abstract
We identified a small molecule that, at picomolar concentrations, rescues mutant CFTR chloride channel from protein degradation., 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. more...
- Published
- 2019
22. Bioactive Thymosin Alpha-1 Does Not Influence F508del-CFTR Maturation and Activity
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Andrea Armirotti, Daniela Guidone, Elizabeth Matthes, Alan S. Verkman, John W. Hanrahan, Deborah M. Cholon, Clarissa Braccia, Puay-Wah Phuan, Gergely L. Lukacs, Nicoletta Pedemonte, Valeria Tomati, Luis J. V. Galietta, Guido Veit, Martina Gentzsch, Armirotti, A., Tomati, V., Matthes, E., Veit, G., Cholon, D. M., Phuan, P. -W., Braccia, Giuseppe, Guidone, D., Gentzsch, M., Lukacs, G. L., Verkman, A. S., Galietta, L. J. V., Hanrahan, J. W., and LUZ PEDEMONTE, Benjiamin more...
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0301 basic medicine ,Cystic Fibrosis ,Thymalfasin ,Cells ,Mutant ,Primary Cell Culture ,lcsh:Medicine ,Cystic Fibrosis Transmembrane Conductance Regulator ,Phenylalanine ,Cystic fibrosis ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Congenital ,0302 clinical medicine ,Rare Diseases ,medicine ,Autophagy ,Animals ,Humans ,lcsh:Science ,Lung ,Cells, Cultured ,Chloride channels ,Sequence Deletion ,Multidisciplinary ,Cultured ,biology ,Chemistry ,lcsh:R ,Thymosin ,Biological activity ,medicine.disease ,Cystic fibrosis transmembrane conductance regulator ,3. Good health ,Cell biology ,Protein Transport ,030104 developmental biology ,Mutation ,biology.protein ,MCF-7 Cells ,Cysteamine ,lcsh:Q ,030217 neurology & neurosurgery - Abstract
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. more...
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- 2019
23. Pharmacological rescue of the G85E CFTR variant by preclinical and approved modulators.
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Tomati V, Capurro V, Pesce E, Pastorino C, Sondo E, Lena M, Borrelli A, Cresta F, Pantano S, Collini F, Ripani P, Terlizzi V, Fevola C, Costa S, Lucanto MC, Zara F, Bandiera T, Bocciardi R, Castellani C, Galietta LJV, and Pedemonte N more...
- Abstract
Introduction: Cystic Fibrosis (CF) is a genetic disease due to loss-of-function mutations of the CFTR channel. F508del is the most frequent mutation (70% of alleles in Italy), while other mutations have much lower frequency. Among them, G85E (0.4% frequency globally, 1.13% in Italy) emerges as a mutation characterized by a severe CFTR folding and trafficking defect., Methods: To investigate the pharmacological responsiveness of the G85E-CFTR variant, we performed a functional and biochemical characterization in heterologous expression systems and ex vivo models based on patient-derived human nasal epithelial cells (HNEC)., Results: Our study demonstrated that treatment of primary airway cells with elexacaftor and tezacaftor causes a significant (although modest) rescue of CFTR function, that reaches 15%-25% of the activity measured in non-CF epithelia. A detrimental effect of chronic treatment with ivacaftor, further limiting G85E rescue, was also observed. A higher rescue of CFTR function, up to 25%-35% of the normal CFTR activity, with no evidence of negative effects upon chronic potentiator treatment, can be achieved by combining elexacaftor with ARN23765, a novel type 1 corrector endowed with very high potency. Importantly, dose-response relationships suggest that G85E might alter the binding of type 1 correctors, possibly affecting their affinity for the target., Discussion: In conclusion, our studies suggest that novel combinations of modulators, endowed with higher efficacy leading to increased rescue of G85E-CFTR, are needed to improve the clinical benefit in patients for this variant., Competing Interests: TB, NP, and LJVG are inventors on the patent application WO 2018/167690 A1, filed by Fondazione Istituto Italiano di Tecnologia, IRCCS Istituto Giannina Gaslini, and Fondazione per la Ricerca sulla Fibrosi Cistica, which claims ARN23765. This application has matured into two issued patents: US 10,745,407 B2 and US 10,968,225 B2. C.C. is the Scientific Director of the Fondazione per la Ricerca sulla Fibrosi Cistica, an Assignee of the US patents reported above. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Tomati, Capurro, Pesce, Pastorino, Sondo, Lena, Borrelli, Cresta, Pantano, Collini, Ripani, Terlizzi, Fevola, Costa, Lucanto, Zara, Bandiera, Bocciardi, Castellani, Galietta and Pedemonte.) more...
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- 2024
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24. Tezacaftor is a direct inhibitor of sphingolipid delta-4 desaturase enzyme (DEGS).
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Ciobanu DZ, Liessi N, Tomati V, Capurro V, Bertozzi SM, Summa M, Bertorelli R, Loberto N, Dobi D, Aureli M, Nobbio L, Bandiera T, Pedemonte N, Bassi R, and Armirotti A
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- Humans, Mice, Animals, Pyrazoles pharmacology, Quinolones pharmacology, Cells, Cultured, Cystic Fibrosis drug therapy, Cystic Fibrosis genetics, Epithelial Cells drug effects, Epithelial Cells metabolism, Bronchi drug effects, Bronchi metabolism, Oxidoreductases metabolism, Oxidoreductases genetics, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Ceramides, Pyridines, Pyrrolidines, Aminophenols, Benzodioxoles pharmacology, Indoles pharmacology
- Abstract
Background: We recently demonstrated that 48 h exposure of primary human bronchial epithelial (hBE) cells, obtained from both CF (F508del homozygous) and non-CF subjects, to the triple drug combination Elexacaftor/Tezacaftor/Ivacaftor (ETI) results in a CFTR genotype-independent modulation of the de novo synthethic pathway of sphingolipids, with an accumulation of dihydroceramides (dHCer). Since dHCer are converted into ceramides (Cer) by the action of a delta-4 sphingolipid desaturase (DEGS) enzyme, we aimed to better understand this off-target effect of ETI (i.e., not related to CFTR rescue) METHODS: hBE cells, both F508del and wild-type, were cultured to create fully differentiated bronchial epithelia. We analyzed Cer and dHCer using an LC-MS based method previously developed by our lab. DEGS expression levels in differentiated hBE cells lysates were quantified by western blot analysis., Results: We demonstrated that 1) dHCer accumulate in hBE with time following prolonged ETI exposure, that 2) similar inhibition occurs in wild-type primary human hepatocytes and that 3) this does not result in an alteration of DEGS expression. We then proved that 4) ETI is a direct inhibitor of DEGS, that 5) Tezacaftor is the molecule responsible for this effect, that 6) the inhibition is concentration dependent. Finally, after repeated oral administration of ETI to naïve, non-CF, mice, we observed a slight accumulation of dHCer in the brain., Conclusions: We believe that further investigations on Tezacaftor should be envisaged, particularly for the use of ETI during pregnancy, breastfeeding and in the early stages of development. DEGS dysfunction and dHCer accumulation causes impairment in the development of the nervous system, due to a derangement in myelin formation and maintenance., Competing Interests: Declaration of competing interest The Authors declare that no conflict of interest exists., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.) more...
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- 2024
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25. Sources of biases in the in vitro testing of nanomaterials: the role of the biomolecular corona.
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Castagnola V, Tomati V, Boselli L, Braccia C, Decherchi S, Pompa PP, Pedemonte N, Benfenati F, and Armirotti A
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- Humans, Animals, Proteomics methods, Lipidomics methods, Cattle, Nanostructures chemistry, Protein Corona chemistry
- Abstract
The biological fate of nanomaterials (NMs) is driven by specific interactions through which biomolecules, naturally adhering onto their surface, engage with cell membrane receptors and intracellular organelles. The molecular composition of this layer, called the biomolecular corona (BMC), depends on both the physical-chemical features of the NMs and the biological media in which the NMs are dispersed and cells grow. In this work, we demonstrate that the widespread use of 10% fetal bovine serum in an in vitro assay cannot recapitulate the complexity of in vivo systemic administration, with NMs being transported by the blood. For this purpose, we undertook a comparative journey involving proteomics, lipidomics, high throughput multiparametric in vitro screening, and single molecular feature analysis to investigate the molecular details behind this in vivo / in vitro bias. Our work indirectly highlights the need to introduce novel, more physiological-like media closer in composition to human plasma to produce realistic in vitro screening data for NMs. We also aim to set the basis to reduce this in vitro - in vivo mismatch, which currently limits the formulation of NMs for clinical settings. more...
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- 2024
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26. De novo variants in DENND5B cause a neurodevelopmental disorder.
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Scala M, Tomati V, Ferla M, Lena M, Cohen JS, Fatemi A, Brokamp E, Bican A, Phillips JA 3rd, Koziura ME, Nicouleau M, Rio M, Siquier K, Boddaert N, Musante I, Tamburro S, Baldassari S, Iacomino M, Scudieri P, Rosenfeld JA, Bellus G, Reed S, Al Saif H, Russo RS, Walsh MB, Cantagrel V, Crunk A, Gustincich S, Ruggiero SM, Fitzgerald MP, Helbig I, Striano P, Severino M, Salpietro V, Pedemonte N, and Zara F more...
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- Humans, Brain metabolism, Guanine Nucleotide Exchange Factors genetics, Guanine Nucleotide Exchange Factors metabolism, Lipids, rab GTP-Binding Proteins metabolism, Neurodevelopmental Disorders genetics, Neurodevelopmental Disorders metabolism, Epilepsy genetics, Epilepsy metabolism, Intellectual Disability genetics, Intellectual Disability metabolism
- Abstract
The Rab family of guanosine triphosphatases (GTPases) includes key regulators of intracellular transport and membrane trafficking targeting specific steps in exocytic, endocytic, and recycling pathways. DENND5B (Rab6-interacting Protein 1B-like protein, R6IP1B) is the longest isoform of DENND5, an evolutionarily conserved DENN domain-containing guanine nucleotide exchange factor (GEF) that is highly expressed in the brain. Through exome sequencing and international matchmaking platforms, we identified five de novo variants in DENND5B in a cohort of five unrelated individuals with neurodevelopmental phenotypes featuring cognitive impairment, dysmorphism, abnormal behavior, variable epilepsy, white matter abnormalities, and cortical gyration defects. We used biochemical assays and confocal microscopy to assess the impact of DENND5B variants on protein accumulation and distribution. Then, exploiting fluorescent lipid cargoes coupled to high-content imaging and analysis in living cells, we investigated whether DENND5B variants affected the dynamics of vesicle-mediated intracellular transport of specific cargoes. We further generated an in silico model to investigate the consequences of DENND5B variants on the DENND5B-RAB39A interaction. Biochemical analysis showed decreased protein levels of DENND5B mutants in various cell types. Functional investigation of DENND5B variants revealed defective intracellular vesicle trafficking, with significant impairment of lipid uptake and distribution. Although none of the variants affected the DENND5B-RAB39A interface, all were predicted to disrupt protein folding. Overall, our findings indicate that DENND5B variants perturb intracellular membrane trafficking pathways and cause a complex neurodevelopmental syndrome with variable epilepsy and white matter involvement., Competing Interests: Declaration of interests A.C. is an employee of GeneDx, LLC. The Department of Molecular and Human Genetics at Baylor College of Medicine receives revenue from clinical genetic testing completed at Baylor Genetics Laboratories., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.) more...
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- 2024
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27. SUMOylation Inhibition Enhances Protein Transcription under CMV Promoter: A Lesson from a Study with the F508del-CFTR Mutant.
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Borgo C, D'Amore C, Capurro V, Tomati V, Pedemonte N, Bosello Travain V, and Salvi M
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- Humans, Cytomegalovirus, Mutation, Promoter Regions, Genetic drug effects, Cystic Fibrosis drug therapy, Cystic Fibrosis genetics, Cystic Fibrosis metabolism, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Sumoylation drug effects
- Abstract
Cystic fibrosis (CF) is a genetic disorder caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator ( CFTR ), a selective anion channel expressed in the epithelium of various organs. The most frequent mutation is F508del. This mutation leads to a misfolded CFTR protein quickly degraded via ubiquitination in the endoplasmic reticulum. Although preventing ubiquitination stabilizes the protein, functionality is not restored due to impaired plasma membrane transport. However, inhibiting the ubiquitination process can improve the effectiveness of correctors which act as chemical chaperones, facilitating F508del CFTR trafficking to the plasma membrane. Previous studies indicate a crosstalk between SUMOylation and ubiquitination in the regulation of CFTR. In this study, we investigated the potential of inhibiting SUMOylation to increase the effects of correctors and enhance the rescue of the F508del mutant across various cell models. In the widely used CFBE41o-cell line expressing F508del-CFTR, inhibiting SUMOylation substantially boosted F508del expression, thereby increasing the efficacy of correctors. Interestingly, this outcome did not result from enhanced stability of the mutant channel, but rather from augmented cytomegalovirus (CMV) promoter-mediated gene expression of F508del-CFTR. Notably, CFTR regulated by endogenous promoters in multiple cell lines or patient cells was not influenced by SUMOylation inhibitors. more...
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- 2024
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28. Innovative Strategy toward Mutant CFTR Rescue in Cystic Fibrosis: Design and Synthesis of Thiadiazole Inhibitors of the E3 Ligase RNF5.
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Brusa I, Sondo E, Pesce E, Tomati V, Gioia D, Falchi F, Balboni B, Ortega Martínez JA, Veronesi M, Romeo E, Margaroli N, Recanatini M, Girotto S, Pedemonte N, Roberti M, and Cavalli A
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- Humans, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Ubiquitin-Protein Ligases metabolism, Structure-Activity Relationship, Aminophenols, Benzodioxoles pharmacology, Mutation, DNA-Binding Proteins metabolism, Cystic Fibrosis drug therapy, Cystic Fibrosis genetics, Thiadiazoles pharmacology, Thiadiazoles therapeutic use
- Abstract
In cystic fibrosis (CF), deletion of phenylalanine 508 (F508del) in the CF transmembrane conductance regulator (CFTR) is associated to misfolding and defective gating of the mutant channel. One of the most promising CF drug targets is the ubiquitin ligase RNF5, which promotes F508del-CFTR degradation. Recently, the first ever reported inhibitor of RNF5 was discovered, i.e., the 1,2,4-thiadiazol-5-ylidene inh-2 . Here, we designed and synthesized a series of new analogues to explore the structure-activity relationships (SAR) of this class of compounds. SAR efforts ultimately led to compound 16 , which showed a greater F508del-CFTR corrector activity than inh-2 , good tolerability, and no toxic side effects. Analogue 16 increased the basal level of autophagy similar to what has been described with RNF5 silencing. Furthermore, co-treatment with 16 significantly improved the F508del-CFTR rescue induced by the triple combination elexacaftor/tezacaftor/ivacaftor in CFBE41o
- cells. These findings validate the 1,2,4-thiadiazolylidene scaffold for the discovery of novel RNF5 inhibitors and provide evidence to pursue this unprecedented strategy for the treatment of CF. more...- Published
- 2023
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29. The combination elexacaftor/tezacaftor/ivacaftor (ETI) modulates the de novo synthethic pathway of ceramides in a genotype-independent manner.
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Liessi N, Tomati V, Capurro V, Loberto N, Garcia-Aloy M, Franceschi P, Aureli M, Pedemonte N, and Armirotti A
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- Humans, Ceramides, Genotype, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Benzodioxoles, Aminophenols, Mutation, Cystic Fibrosis drug therapy, Cystic Fibrosis genetics
- Abstract
We report here how the triple combination of drugs elexacaftor/tezacaftor/ivacaftor (ETI) alters the balance of the de-novo synthethic pathway of sphingolipids in primary cells of human bronchial epithelium. The treatment with ETI roughly doubles the levels of dihydrosphingolipids, possibly by modulating the delta(4)-desaturase enzymes that convert dihydroceramides into ceramides. This appears to be an off-target effect of ETI, since it occurs in a genotype-independent manner, for both cystic fibrosis (CF) and non-CF subjects., Competing Interests: Declaration of Competing Interest The Authors declare that no conflict of interest exists. Andrea Armirotti, Ph.D. (on behalf of all authors)., (Copyright © 2023 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.) more...
- Published
- 2023
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30. Patient's dermal fibroblasts as disease markers for visceral myopathy.
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Viti F, Pramotton FM, Martufi M, Magrassi R, Pedemonte N, Nizzari M, Zanacchi FC, De Michele B, Alampi M, Zambito M, Santamaria G, Bajetto A, Sardar S, Tomati V, Gandullia P, Giampietro C, Florio T, Beltrame F, Vassalli M, and Ceccherini I more...
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- Humans, Actins genetics, Actins metabolism, Muscle Contraction, Phenotype, Muscle, Smooth metabolism, Intestinal Pseudo-Obstruction diagnosis, Intestinal Pseudo-Obstruction genetics, Intestinal Pseudo-Obstruction metabolism
- Abstract
Visceral myopathy (VSCM) is a rare genetic disease, orphan of pharmacological therapy. VSCM diagnosis is not always straightforward due to symptomatology similarities with mitochondrial or neuronal forms of intestinal pseudo-obstruction. The most prevalent form of VSCM is associates with variants in the gene ACTG2, encoding the protein gamma-2 actin. Overall, VSCM is a mechano-biological disorder, in which different genetic variants lead to similar alterations to the contractile phenotype of enteric smooth muscles, resulting in the emergence of life-threatening symptoms. In this work we analyzed the morpho-mechanical phenotype of human dermal fibroblasts from patients affected with VSCM, demonstrating that they retain a clear signature of the disease when compared with different controls. We evaluated several biophysical traits of fibroblasts, and we show that a measure of cellular traction forces can be used as a non-specific biomarker of the disease. We propose that a simple assay based on traction forces could be designed to provide a valuable support for clinical decision or pre-clinical research., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Federica Viti reports financial support was provided by Fondazione Alessandra Bono. Isabella Ceccherini reports financial support was provided by Fondazione Alessandra Bono. Costanza Giampietro reports financial support was provided by Swiss National Science Foundation., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.) more...
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- 2023
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31. Rescue by elexacaftor-tezacaftor-ivacaftor of the G1244E cystic fibrosis mutation's stability and gating defects are dependent on cell background.
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Tomati V, Costa S, Capurro V, Pesce E, Pastorino C, Lena M, Sondo E, Di Duca M, Cresta F, Cristadoro S, Zara F, Galietta LJV, Bocciardi R, Castellani C, Lucanto MC, and Pedemonte N
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- Humans, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Aminophenols pharmacology, Benzodioxoles pharmacology, Mutation, Cystic Fibrosis drug therapy, Cystic Fibrosis genetics, Cystic Fibrosis metabolism
- Abstract
Background: Cystic fibrosis is caused by mutations impairing expression, trafficking, stability and/or activity of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. The G1244E mutation causes a severe gating defect that it is not completely rescued by ivacaftor but requires the use of a second compound (a co-potentiator). Recently, it has been proposed that the corrector elexacaftor may act also as a co-potentiator., Methods: By using molecular, biochemical and functional analyses we performed an in-depth characterization of the G1244E-CFTR mutant in heterologous and native cell models., Results: Our studies demonstrate that processing and function of the mutant protein, as well as its pharmacological sensitivity, are markedly dependent on cell background. In heterologous expression systems, elexacaftor mainly acted on G1244E-CFTR as a co-potentiator, thus ameliorating the gating defect. On the contrary, in the native nasal epithelial cell model, elexacaftor did not act as a co-potentiator, but it increased mature CFTR expression possibly by improving mutant's defective stability at the plasma membrane., Conclusions: Our study highlights the importance of the cell background in the evaluation of CFTR modulator effects. Further, our results draw attention to the need for the development of novel potentiators having different mechanisms with respect to ivacaftor to improve channel activity for mutants with severe gating defect., Competing Interests: Conflicts of Interest The authors declare no conflict of interest., (Copyright © 2022. Published by Elsevier B.V.) more...
- Published
- 2023
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32. Clinical Consequences and Functional Impact of the Rare S737F CFTR Variant and Its Responsiveness to CFTR Modulators.
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Terlizzi V, Pesce E, Capurro V, Tomati V, Lena M, Pastorino C, Bocciardi R, Zara F, Centrone C, Taccetti G, Castellani C, and Pedemonte N
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- Adult, Humans, Adolescent, Retrospective Studies, Benzodioxoles pharmacology, Benzodioxoles therapeutic use, Nasal Mucosa, Cell Line, Mutation, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Cystic Fibrosis drug therapy, Cystic Fibrosis genetics, Cystic Fibrosis diagnosis
- Abstract
S737F is a Cystic Fibrosis (CF) transmembrane conductance regulator (CFTR) missense variant. The aim of our study was to describe the clinical features of a cohort of individuals carrying this variant. In parallel, by exploiting ex vivo functional and molecular analyses on nasal epithelia derived from a subset of S737F carriers, we evaluated its functional impact on CFTR protein as well as its responsiveness to CFTR modulators. We retrospectively collected clinical data of all individuals bearing at least one S737F CFTR variant and followed at the CF Centre of Tuscany region (Italy). Nasal brushing was performed in cooperating individuals. At study end clinical data were available for 10 subjects (mean age: 14 years; range 1-44 years; 3 adult individuals). Five asymptomatic subjects had CF, 2 were CRMS/CFSPID and 3 had an inconclusive diagnosis. Ex vivo analysis on nasal epithelia demonstrated different levels of CF activity. In particular, epithelia derived from asymptomatic CF subjects and from one of the subjects with inconclusive diagnosis showed reduced CFTR activity that could be rescued by treatment with CFTR modulators. On the contrary, in the epithelia derived from the other two individuals with an inconclusive diagnosis, the CFTR-mediated current was similar to that observed in epithelia derived from healthy donors. In vitro functional and biochemical analysis on S737F-CFTR expressed in immortalized bronchial cells highlighted a modest impairment of the channel activity, that was improved by treatment with ivacaftor alone or in combination with tezacaftor/elexacaftor. Our study provide evidence towards the evaluation of CFTR function on ex vivo nasal epithelial cell models as a new assay to help clinicians to classify individuals, in presence of discordance between clinical picture, sweat test and genetic profile. more...
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- 2023
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33. Gain- and Loss-of-Function CFTR Alleles Are Associated with COVID-19 Clinical Outcomes.
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Baldassarri M, Zguro K, Tomati V, Pastorino C, Fava F, Croci S, Bruttini M, Picchiotti N, Furini S, Pedemonte N, Gabbi C, Renieri A, and Fallerini C
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- Humans, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Alleles, Heterozygote, Cystic Fibrosis pathology, COVID-19 genetics
- Abstract
Carriers of single pathogenic variants of the CFTR (cystic fibrosis transmembrane conductance regulator) gene have a higher risk of severe COVID-19 and 14-day death. The machine learning post-Mendelian model pinpointed CFTR as a bidirectional modulator of COVID-19 outcomes. Here, we demonstrate that the rare complex allele [G576V;R668C] is associated with a milder disease via a gain-of-function mechanism. Conversely, CFTR ultra-rare alleles with reduced function are associated with disease severity either alone (dominant disorder) or with another hypomorphic allele in the second chromosome (recessive disorder) with a global residual CFTR activity between 50 to 91%. Furthermore, we characterized novel CFTR complex alleles, including [A238V;F508del], [R74W;D1270N;V201M], [I1027T;F508del], [I506V;D1168G], and simple alleles, including R347C, F1052V, Y625N, I328V, K68E, A309D, A252T, G542*, V562I, R1066H, I506V, I807M, which lead to a reduced CFTR function and thus, to more severe COVID-19. In conclusion, CFTR genetic analysis is an important tool in identifying patients at risk of severe COVID-19. more...
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- 2022
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34. CFTR Rescue by Lumacaftor (VX-809) Induces an Extensive Reorganization of Mitochondria in the Cystic Fibrosis Bronchial Epithelium.
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Braccia C, Christopher JA, Crook OM, Breckels LM, Queiroz RML, Liessi N, Tomati V, Capurro V, Bandiera T, Baldassari S, Pedemonte N, Lilley KS, and Armirotti A
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- Aminopyridines, Benzodioxoles, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Epithelium metabolism, Humans, Infant, Newborn, Mitochondria metabolism, Proteome metabolism, Cystic Fibrosis metabolism
- Abstract
Background: Cystic Fibrosis (CF) is a genetic disorder affecting around 1 in every 3000 newborns. In the most common mutation, F508del, the defective anion channel, CFTR, is prevented from reaching the plasma membrane (PM) by the quality check control of the cell. Little is known about how CFTR pharmacological rescue impacts the cell proteome., Methods: We used high-resolution mass spectrometry, differential ultracentrifugation, machine learning and bioinformatics to investigate both changes in the expression and localization of the human bronchial epithelium CF model (F508del-CFTR CFBE41o-) proteome following treatment with VX-809 (Lumacaftor), a drug able to improve the trafficking of CFTR., Results: The data suggested no stark changes in protein expression, yet subtle localization changes of proteins of the mitochondria and peroxisomes were detected. We then used high-content confocal microscopy to further investigate the morphological and compositional changes of peroxisomes and mitochondria under these conditions, as well as in patient-derived primary cells. We profiled several thousand proteins and we determined the subcellular localization data for around 5000 of them using the LOPIT-DC spatial proteomics protocol., Conclusions: We observed that treatment with VX-809 induces extensive structural and functional remodelling of mitochondria and peroxisomes that resemble the phenotype of healthy cells. Our data suggest additional rescue mechanisms of VX-809 beyond the correction of aberrant folding of F508del-CFTR and subsequent trafficking to the PM. more...
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- 2022
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35. Targeting of Ubiquitin E3 Ligase RNF5 as a Novel Therapeutic Strategy in Neuroectodermal Tumors.
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Principi E, Sondo E, Bianchi G, Ravera S, Morini M, Tomati V, Pastorino C, Zara F, Bruno C, Eva A, Pedemonte N, and Raffaghello L
- Abstract
RNF5, an endoplasmic reticulum (ER) E3 ubiquitin ligase, participates to the ER-associated protein degradation guaranteeing the protein homeostasis. Depending on tumor model tested, RNF5 exerts pro- or anti-tumor activity. The aim of this study was to elucidate the controversial role of RNF5 in neuroblastoma and melanoma, two neuroectodermal tumors of infancy and adulthood, respectively. RNF5 gene levels are evaluated in publicly available datasets reporting the gene expression profile of melanoma and neuroblastoma primary tumors at diagnosis. The therapeutic effect of Analog-1, an RNF5 pharmacological activator, was investigated on in vitro and in vivo neuroblastoma and melanoma models. In both neuroblastoma and melanoma patients the high expression of RNF5 correlated with a better prognostic outcome. Treatment of neuroblastoma and melanoma cell lines with Analog-1 reduced cell viability by impairing the glutamine availability and energy metabolism through inhibition of F
1 Fo ATP-synthase activity. This latter event led to a marked increase in oxidative stress, which, in turn, caused cell death. Similarly, neuroblastoma- and melanoma-bearing mice treated with Analog-1 showed a significant delay of tumor growth in comparison to those treated with vehicle only. These findings validate RNF5 as an innovative drug target and support the development of Analog-1 in early phase clinical trials for neuroblastoma and melanoma patients. more...- Published
- 2022
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36. Targeting the E1 ubiquitin-activating enzyme (UBA1) improves elexacaftor/tezacaftor/ivacaftor efficacy towards F508del and rare misfolded CFTR mutants.
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Borgo C, D'Amore C, Capurro V, Tomati V, Sondo E, Cresta F, Castellani C, Pedemonte N, and Salvi M
- Subjects
- Cells, Cultured, Cystic Fibrosis metabolism, Cystic Fibrosis Transmembrane Conductance Regulator chemistry, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Drug Synergism, Drug Therapy, Combination, Enzyme Inhibitors administration & dosage, Humans, Mutation, Protein Folding drug effects, Sequence Deletion, Aminophenols administration & dosage, Benzodioxoles administration & dosage, Cystic Fibrosis drug therapy, Cystic Fibrosis genetics, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Indoles administration & dosage, Pyrazoles administration & dosage, Pyridines administration & dosage, Pyrimidines administration & dosage, Pyrrolidines administration & dosage, Quinolones administration & dosage, Sulfides administration & dosage, Sulfonamides administration & dosage, Ubiquitin-Activating Enzymes antagonists & inhibitors
- Abstract
The advent of Trikafta (Kaftrio in Europe) (a triple-combination therapy based on two correctors-elexacaftor/tezacaftor-and the potentiator ivacaftor) has represented a revolution for the treatment of patients with cystic fibrosis (CF) carrying the most common misfolding mutation, F508del-CFTR. This therapy has proved to be of great efficacy in people homozygous for F508del-CFTR and is also useful in individuals with a single F508del allele. Nevertheless, the efficacy of this therapy needs to be improved, especially in light of the extent of its use in patients with rare class II CFTR mutations. Using CFBE41o- cells expressing F508del-CFTR, we provide mechanistic evidence that targeting the E1 ubiquitin-activating enzyme (UBA1) by TAK-243, a small molecule in clinical trials for other diseases, boosts the rescue of F508del-CFTR induced by CFTR correctors. Moreover, TAK-243 significantly increases the F508del-CFTR short-circuit current induced by elexacaftor/tezacaftor/ivacaftor in differentiated human primary airway epithelial cells, a gold standard for the pre-clinical evaluation of patients' responsiveness to pharmacological treatments. This new combinatory approach also leads to an improvement in CFTR conductance on cells expressing other rare CF-causing mutations, including N1303K, for which Trikafta is not approved. These findings show that Trikafta therapy can be improved by the addition of a drug targeting the misfolding detection machinery at the beginning of the ubiquitination cascade and may pave the way for an extension of Trikafta to low/non-responding rare misfolded CFTR mutants., (© 2022. The Author(s).) more...
- Published
- 2022
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37. The L467F-F508del Complex Allele Hampers Pharmacological Rescue of Mutant CFTR by Elexacaftor/Tezacaftor/Ivacaftor in Cystic Fibrosis Patients: The Value of the Ex Vivo Nasal Epithelial Model to Address Non-Responders to CFTR-Modulating Drugs.
- Author
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Sondo E, Cresta F, Pastorino C, Tomati V, Capurro V, Pesce E, Lena M, Iacomino M, Baffico AM, Coviello D, Bandiera T, Zara F, Galietta LJV, Bocciardi R, Castellani C, and Pedemonte N
- Subjects
- Alleles, Aminophenols, Benzodioxoles pharmacology, Benzodioxoles therapeutic use, Drug Combinations, Humans, Indoles, Mutation, Pyrazoles, Pyridines, Pyrrolidines, Quinolones, Cystic Fibrosis drug therapy, Cystic Fibrosis genetics, Cystic Fibrosis Transmembrane Conductance Regulator metabolism
- Abstract
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. more...
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- 2022
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38. Journey on VX-809-Based Hybrid Derivatives towards Drug-like F508del-CFTR Correctors: From Molecular Modeling to Chemical Synthesis and Biological Assays.
- Author
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Parodi A, Righetti G, Pesce E, Salis A, Tomati V, Pastorino C, Tasso B, Benvenuti M, Damonte G, Pedemonte N, Cichero E, and Millo E
- Abstract
Cystic fibrosis (CF) is a genetic disease affecting the lungs and pancreas and causing progressive damage. CF is caused by mutations abolishing the function of CFTR, a protein whose role is chloride's mobilization in the epithelial cells of various organs. Recently a therapy focused on small molecules has been chosen as a main approach to contrast CF, designing and synthesizing compounds acting as misfolding (correctors) or defective channel gating (potentiators). Multi-drug therapies have been tested with different combinations of the two series of compounds. Previously, we designed and characterized two series of correctors, namely, hybrids, which were conceived including the aminoarylthiazole (AAT) core, merged with the benzodioxole carboxamide moiety featured by VX-809. In this paper, we herein proceeded with molecular modeling studies guiding the design of a new third series of hybrids, featuring structural variations at the thiazole moiety and modifications on position 4. These derivatives were tested in different assays including a YFP functional assay on models F508del-CFTR CFBE41o-cells, alone and in combination with VX-445, and by using electrophysiological techniques on human primary bronchial epithelia to demonstrate their F508del-CFTR corrector ability. This study is aimed (i) at identifying three molecules ( 9b, 9g, and 9j ), useful as novel CFTR correctors with a good efficacy in rescuing the defect of F508del-CFTR; and (ii) at providing useful information to complete the structure-activity study within all the three series of hybrids as possible CFTR correctors, supporting the development of pharmacophore modelling studies, taking into account all the three series of hybrids. Finally, in silico evaluation of the hybrids pharmacokinetic (PK) properties contributed to highlight hybrid developability as drug-like correctors. more...
- Published
- 2022
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39. Partial Rescue of F508del-CFTR Stability and Trafficking Defects by Double Corrector Treatment.
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Capurro V, Tomati V, Sondo E, Renda M, Borrelli A, Pastorino C, Guidone D, Venturini A, Giraudo A, Mandrup Bertozzi S, Musante I, Bertozzi F, Bandiera T, Zara F, Galietta LJV, and Pedemonte N
- Subjects
- Aminophenols pharmacology, Aminopyridines pharmacology, Benzodioxoles pharmacology, Bronchi drug effects, Bronchi metabolism, Chloride Channels genetics, Chloride Channels metabolism, Cystic Fibrosis drug therapy, Cystic Fibrosis genetics, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Drug Combinations, Epithelial Cells metabolism, Humans, Indoles pharmacology, Protein Folding drug effects, Pyrazoles metabolism, Pyridines metabolism, Pyrrolidines metabolism, Quinolines pharmacology, Cystic Fibrosis Transmembrane Conductance Regulator drug effects, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Pyrazoles pharmacology, Pyridines pharmacology, Pyrrolidines pharmacology
- Abstract
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. more...- Published
- 2021
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40. Discovery of novel VX-809 hybrid derivatives as F508del-CFTR correctors by molecular modeling, chemical synthesis and biological assays.
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Parodi A, Righetti G, Pesce E, Salis A, Tasso B, Urbinati C, Tomati V, Damonte G, Rusnati M, Pedemonte N, Cichero E, and Millo E
- Subjects
- Aminopyridines chemical synthesis, Benzodioxoles chemical synthesis, Cell Line, Cystic Fibrosis Transmembrane Conductance Regulator chemistry, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Drug Design, Humans, Molecular Docking Simulation, Mutation, Protein Binding, Protein Domains, Aminopyridines metabolism, Aminopyridines pharmacology, Benzodioxoles metabolism, Benzodioxoles pharmacology, Cystic Fibrosis Transmembrane Conductance Regulator metabolism
- Abstract
Cystic fibrosis (CF) is the autosomal recessive disorder most recurrent in Caucasian populations. It is caused by different mutations in the cystic fibrosis transmembrane regulator protein (CFTR) gene, with F508del being the most common. During the last years, small-molecule therapy chosen to contrast CF relied on compounds that correct CFTR misfolding and ER retention (correctors such as VX-809), or defective channel gating (potentiators such as VX-770). Combination therapy with the two series of drugs has been applied, leading to the approval of several multi-drugs such as Orkambi. Despite this, this treatment proved to be only partially effective making the search for novel modulators an urgent need to contrast CF. Recently, we reported compound 2a as reference compound of a series of aminoarylthiazole-VX-809 hybrid derivatives exhibiting promising F508del-CFTR corrector ability. Herein, we report exploring the docking mode of the prototype VX-809 and of 2a in order to derive useful guidelines for the rational design of novel optimized analogues. To demonstrate experimentally their effective F508del-CFTR-binding and rescuing potential, the most promising derivatives had been synthesized and evaluated in biological assays including YFP functional assay on F508del-CFTR CFBE41o-cells, trans epithelial electrical resistance (TEER) and surface plasmon resonance (SPR). This multidisciplinary strategy led to the discovery of a second series of hybrids including 7j and 7m endowed with higher potency than the prototype., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.) more...
- Published
- 2020
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41. CFTR processing, trafficking and interactions.
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Amaral MD, Hutt DM, Tomati V, Botelho HM, and Pedemonte N
- Subjects
- Humans, Mutation, Proteostasis drug effects, Cystic Fibrosis drug therapy, Cystic Fibrosis genetics, Cystic Fibrosis metabolism, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Genetic Therapy methods, Ion Transport drug effects, Ion Transport genetics, Membrane Transport Modulators pharmacology
- Abstract
Mutations associated with cystic fibrosis (CF) have complex effects on the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The most common CF mutation, F508del, disrupts the processing to and stability at the plasma membrane and function as a Cl
- channel. CFTR is surrounded by a dynamic network of interacting components, referred to as the CFTR Functional Landscape, that impact its synthesis, folding, stability, trafficking and function. CFTR interacting proteins can be manipulated by functional genomic approaches to rescue the trafficking and functional defects characteristic of CF. Here we review recent efforts to elucidate the impact of genetic variation on the ability of the nascent CFTR polypeptide to interact with the proteostatic environment. We also provide an overview of how specific components of this protein network can be modulated to rescue the trafficking and functional defects associated with the F508del variant of CFTR. The identification of novel proteins playing key roles in the processing of CFTR could pave the way for their use as novel therapeutic targets to provide synergistic correction of mutant CFTR for the greater benefit of individuals with CF., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2019. Published by Elsevier B.V.) more...- Published
- 2020
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42. Discovery of a picomolar potency pharmacological corrector of the mutant CFTR chloride channel.
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Pedemonte N, Bertozzi F, Caci E, Sorana F, Di Fruscia P, Tomati V, Ferrera L, Rodríguez-Gimeno A, Berti F, Pesce E, Sondo E, Gianotti A, Scudieri P, Bandiera T, and Galietta LJV
- Subjects
- Bronchi pathology, Cell Line, Cystic Fibrosis Transmembrane Conductance Regulator chemistry, Epithelial Cells metabolism, High-Throughput Screening Assays, Humans, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Mutant Proteins metabolism, Pharmaceutical Preparations metabolism
- Abstract
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
50 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., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).) more...- Published
- 2020
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43. Bioactive Thymosin Alpha-1 Does Not Influence F508del-CFTR Maturation and Activity.
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Armirotti A, Tomati V, Matthes E, Veit G, Cholon DM, Phuan PW, Braccia C, Guidone D, Gentzsch M, Lukacs GL, Verkman AS, Galietta LJV, Hanrahan JW, and Pedemonte N
- Subjects
- Animals, Autophagy, Cells, Cultured, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Humans, MCF-7 Cells, Primary Cell Culture, Protein Transport drug effects, Cystic Fibrosis genetics, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Sequence Deletion, Thymalfasin pharmacology
- Abstract
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. more...
- Published
- 2019
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44. SWATH label-free proteomics for cystic fibrosis research.
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Braccia C, Tomati V, Caci E, Pedemonte N, and Armirotti A
- Subjects
- Biomedical Research, Bronchi, Cells, Cultured, Epithelial Cells, Humans, Respiratory Mucosa cytology, Cystic Fibrosis diagnosis, Proteomics methods
- Abstract
Background: Label-free proteomics is a powerful tool for biological investigation. The SWATH protocol, relying on the Pan Human ion library, currently represents the state-of-the-art methodology for this kind of analysis. We recently discovered that this tool is not perfectly suitable for proteomics research in the CF field, as it lacks assays for several proteins crucial for the CF biology, including CFTR., Methods: We extensively investigated the proteome of a very popular model for in vitro research on CF, CFBE41o-, and we used the corresponding data to improve the power of SWATH proteomics for CF investigation. We then used this improved tool to explore in depth the proteome of primary bronchial epithelial (BE) cells deriving from four CF individuals compared with that of four corresponding non-CF controls. By means of advanced bioinformatics tools, we outlined the presence of a number of protein networks being significantly altered by CF., Results: Our analysis on patients' BE cells identified 154 proteins dysregulated by the CF pathology (94 upregulated and 60 downregulated). Some known CFTR interactors are present among them, but our analysis also revealed the alteration of other proteins not previously known to be related with CF., Conclusions: The present work outlines the power of SWATH label free proteomics applied to CF research., (Copyright © 2018. Published by Elsevier B.V.) more...
- Published
- 2019
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45. Two CFTR mutations within codon 970 differently impact on the chloride channel functionality.
- Author
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Amato F, Scudieri P, Musante I, Tomati V, Caci E, Comegna M, Maietta S, Manzoni F, Di Lullo AM, De Wachter E, Vanderhelst E, Terlizzi V, Braggion C, Castaldo G, and Galietta LJV
- Subjects
- Codon, Cystic Fibrosis metabolism, HEK293 Cells, Humans, Phenotype, RNA Splicing, Transfection, Cystic Fibrosis genetics, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Point Mutation
- Abstract
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., (© 2019 Wiley Periodicals, Inc.) more...
- Published
- 2019
- Full Text
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46. Thymosin α-1 does not correct F508del-CFTR in cystic fibrosis airway epithelia.
- Author
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Tomati V, Caci E, Ferrera L, Pesce E, Sondo E, Cholon DM, Quinney NL, Boyles SE, Armirotti A, Ravazzolo R, Galietta LJ, Gentzsch M, and Pedemonte N
- Published
- 2019
- Full Text
- View/download PDF
47. The Autophagy Inhibitor Spautin-1 Antagonizes Rescue of Mutant CFTR Through an Autophagy-Independent and USP13-Mediated Mechanism.
- Author
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Pesce E, Sondo E, Ferrera L, Tomati V, Caci E, Scudieri P, Musante I, Renda M, Baatallah N, Servel N, Hinzpeter A, di Bernardo D, Pedemonte N, and Galietta LJV
- Abstract
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 in vivo and in vitro 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. more...
- Published
- 2018
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48. Pharmacological Inhibition of the Ubiquitin Ligase RNF5 Rescues F508del-CFTR in Cystic Fibrosis Airway Epithelia.
- Author
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Sondo E, Falchi F, Caci E, Ferrera L, Giacomini E, Pesce E, Tomati V, Mandrup Bertozzi S, Goldoni L, Armirotti A, Ravazzolo R, Cavalli A, and Pedemonte N
- Subjects
- Animals, Apoptosis drug effects, Cell Proliferation drug effects, Cell Survival drug effects, Cystic Fibrosis metabolism, Cystic Fibrosis pathology, DNA-Binding Proteins metabolism, Disease Models, Animal, Dose-Response Relationship, Drug, Enzyme Inhibitors chemistry, Epithelial Cells metabolism, Humans, Mice, Models, Molecular, Molecular Structure, Phenylalanine genetics, Structure-Activity Relationship, Ubiquitin-Protein Ligases metabolism, Benzamidines pharmacology, Cystic Fibrosis drug therapy, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, DNA-Binding Proteins antagonists & inhibitors, Enzyme Inhibitors pharmacology, Epithelial Cells drug effects, Phenylalanine metabolism, Thiadiazoles pharmacology, Ubiquitin-Protein Ligases antagonists & inhibitors
- Abstract
In cystic fibrosis (CF), deletion of phenylalanine 508 (F508del) in the CFTR channel is associated with misfolding and premature degradation of the mutant protein. Among the known proteins associated with F508del-CFTR processing, the ubiquitin ligase RNF5/RMA1 is particularly interesting. We previously demonstrated that genetic suppression of RNF5 in vivo leads to an attenuation of intestinal pathological phenotypes in CF mice, validating the relevance of RNF5 as a drug target for CF. Here, we used a computational approach, based on ligand docking and virtual screening, to discover inh-02, a drug-like small molecule that inhibits RNF5. In in vitro experiments, treatment with inh-02 modulated ATG4B and paxillin, both known RNF5 targets. In immortalized and primary bronchial epithelial cells derived from CF patients homozygous for the F508del mutation, long-term incubation with inh-02 caused significant F508del-CFTR rescue. This work validates RNF5 as a drug target for CF, providing evidence to support its druggability., (Copyright © 2018 Elsevier Ltd. All rights reserved.) more...
- Published
- 2018
- Full Text
- View/download PDF
49. Thymosin α-1 does not correct F508del-CFTR in cystic fibrosis airway epithelia.
- Author
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Tomati V, Caci E, Ferrera L, Pesce E, Sondo E, Cholon DM, Quinney NL, Boyles SE, Armirotti A, Ravazzolo R, Galietta LJ, Gentzsch M, and Pedemonte N
- Subjects
- Anions metabolism, Bronchi cytology, Bronchi pathology, Cell Line, Tumor, Cystic Fibrosis genetics, Cystic Fibrosis pathology, Epithelial Cells metabolism, Humans, Primary Cell Culture, Respiratory Mucosa cytology, Respiratory Mucosa pathology, Thymalfasin therapeutic use, Cystic Fibrosis drug therapy, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Epithelial Cells drug effects, Protein Folding drug effects, Thymalfasin pharmacology
- Abstract
In cystic fibrosis (CF), deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel causes misfolding and premature degradation. Considering the numerous effects of the F508del mutation on the assembly and processing of CFTR protein, combination therapy with several pharmacological correctors is likely to be required to treat CF patients. Recently, it has been reported that thymosin α-1 (Tα-1) has multiple beneficial effects that could lead to a single-molecule-based therapy for CF patients with F508del. Such effects include suppression of inflammation, improvement in F508del-CFTR maturation and gating, and stimulation of chloride secretion through the calcium-activated chloride channel (CaCC). Given the importance of such a drug, we aimed to characterize the underlying molecular mechanisms of action of Tα-1. In-depth analysis of Tα-1 effects was performed using well-established microfluorimetric, biochemical, and electrophysiological techniques on epithelial cell lines and primary bronchial epithelial cells from CF patients. The studies, which were conducted in 2 independent laboratories with identical outcome, demonstrated that Tα-1 is devoid of activity on mutant CFTR as well as on CaCC. Although Tα-1 may still be useful as an antiinflammatory agent, its ability to target defective anion transport in CF remains to be further investigated. more...
- Published
- 2018
- Full Text
- View/download PDF
50. High-throughput screening identifies FAU protein as a regulator of mutant cystic fibrosis transmembrane conductance regulator channel.
- Author
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Tomati V, Pesce E, Caci E, Sondo E, Scudieri P, Marini M, Amato F, Castaldo G, Ravazzolo R, Galietta LJV, and Pedemonte N
- Subjects
- Bronchi pathology, Cell Membrane pathology, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Epithelial Cells pathology, Humans, Proteolysis, Ribosomal Proteins genetics, Bronchi metabolism, Cell Membrane metabolism, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Epithelial Cells metabolism, Mutation, Ribosomal Proteins metabolism
- Abstract
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., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.) more...
- Published
- 2018
- Full Text
- View/download PDF
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