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LABORATORY OF: Molecular Physiopathology of Ion Channels
CONTACT PERSON: Prof.Luis Galietta (Gaslini Institute)
Phone +39 010 5737534 E-mail: galietta@unige.it

Description of Laboratory and Expertise:

The major research field of our group is the functional, pharmacological, and molecular characterization of ion channels in epithelial cells. In particular, we are interested in identifying pharmacological modulators (activators, inhibitors, chemical chaperones) of the CFTR chloride channel that could be helpful for the treatment of cystic fibrosis and other CFTR-related diseases.

Abstract of Activities:

Cystic fibrosis (CF) is one of the most frequent genetic diseases, characterized by a progressive and irreversible damage to the lungs and the pancreas. CF is caused by mutations that abolish the function of the CFTR protein, which is needed to transport chloride in many epithelial cells. On the other hand, CFTR hyperactivity is also responsible for deadly secretory diarrhea in third world countries and is involved in the enlargement of cysts in polycystic kidney disease. Therefore, pharmacological CFTR modulators could be very important as therapeutic agents for serious genetic and non-genetic diseases. In our laboratory, we search for small molecules able to modulate CFTR activity and biogenesis by screening large collections of chemical compounds with an automated assay based on the halide-sensitive yellow fluorescent protein. After screening, active compounds are further evaluated using electrophysiological techniques.

Detailed Research Activities:

Cystic fibrosis (CF) is the most frequent autosomic recessive genetic disease in Caucasian populations (1/3,500 in Italy). There are no effective treatments able to reverse or stop the progression of CF. The gene affected in CF codes for a plasma membrane chloride channel, CFTR, which is localized in the apical membrane of various epithelial cells. CFTR belongs to the superfamily of ATP-binding cassette (ABC) proteins and is made of two homologous halves, each one consisting of six transmembrane helices and one nucleotide binding domain (NBD), connected by a regulatory domain, which contains various phosphorylation sites for cAMP-dependent protein kinase A. Indeed, protein kinase-mediated phosphorylation is required to activate CFTR. Upon phosphorylation, binding of two molecules of ATP to the NBDs evokes the opening of CFTR pore and consequently chloride transport. In agreement with other ABC proteins, it is possible that CFTR activation involves dimerization of the NBDs.
There are more than 1,300 CF mutations reported so far. Mutations causing CF affect different domains of CFTR protein and have been grouped in five classes according to the molecular mechanism through which they cause total or partial loss of function. Class II mutations, like F508del, cause a severe deficit in protein trafficking due to misfolding and/or defective interactions with molecular chaperones. Accordingly the mutant CFTR protein is trapped in the endoplasmic reticulum and subsequently degraded. Class III mutations (e.g. G551D and G1349D) allow normal trafficking to the apical membrane but impair almost totally the process of CFTR pore opening (gating defect) with a dramatically low open channel probability even under conditions of maximal cAMP elevation. Interestingly, the F508del mutation shows also a gating defect.
Given the absence of other effective treatments, there has been an increasing interest in the possibility to restore the defective CFTR function by pharmacological means. For class III mutants, a single drug (called potentiator), able to increase CFTR open channel probability, could be enough to provide therapeutic restoration of epithelial chloride transport. For class F508del (the most frequent CF mutation), combination of two drugs (a corrector and a potentiator), could be needed to address the trafficking and gating defects, respectively.

CFTR is also involved in other genetic and non-genetic diseases. Massive activation of CFTR in the intestine by bacterial toxins (e.g. cholera toxin) causes deadly secretory diarrhea, a dramatic problem in third world countries. CFTR is probably also involved in the enlargements of cysts in polycystic kidney disease. In contrast to CF, such diseases could benefit from selective CFTR inhibitors.

To identify small organic molecules able to restore or inhibit CFTR function, we have chosen a strategy based on the high-throughput screening of chemical libraries. The screening is performed using the assay based on the halide-sensitive yellow fluorescent protein for rapid evaluation of compound activity in living cells. Results obtained with the fluorescence assay are confirmed by measuring CFTR chloride currents in Ussing chambers or patch-clamp experiments. Various classes of active compounds have been identified so far, many of them in collaboration with Prof. A.S. Verkman (University of California San Francisco). Such compounds include: phenylglycines, sulfonamides, benzothiophenes, benzofurans, 1,4-dihydropyridines (DHPs) as potentiators, and bithiazoles as correctors. In particular, DHPs were specifically identified in our laboratory through an approached based on the screening of FDA/EMEA-approved drugs.
Indeed, we have found that some anti-hypertensive DHPs are potent stimulators (potentiators) of mutant CFTR channel activity. The mechanism of CFTR activation does not involve interaction with voltage-dependent calcium channels, the primary target of DHPs as anti-hypertensive agents, but probably direct binding of DHPs to CFTR protein itself. In a recent study, by performing a structure-activity relationship on 333 DHPs, we have identified specific modifications of DHP structure that highly increase the selectivity for CFTR versus calcium channels. The major effort at the moment is towards the optimization of DHPs and the identification of novel F508del correctors.
Our laboratory is also interested in molecular and functional characterization of other epithelial chloride channels.

Applications and Developments:

Pharmacological modulators of CFTR chloride channel may be beneficial to develop drugs for the treatment of cystic fibrosis and other genetic and non-genetic CFTR-related diseases. Ligands of CFTR (potentiators and inhibitors) are also very important as research tools to understand CFTR protein structure-activity relationship.

Ongoing collaborations:

Prof. A.S. Verkman, University of California San Francisco
Identification and characterization of CFTR inhibitors and correctors.

Prof. Mauro Mazzei, Department of Pharmaceutical Sciences, University of Genova
Optimization of 1,4-dyhydropyridines.

Most recent and significant publications:

Pedemonte N, Diena T, Caci E, Nieddu E, Mazzei M, Ravazzolo R, Zegarra-Moran O, Galietta LJV Antihypertensive 1,4-dihydropyridines as correctors of the cystic fibrosis transmembrane conductance regulator channel gating defect caused by cystic fibrosis mutations. Mol Pharmacol. 2005 Dec;68(6):1736-46

Pedemonte N, Sonawane ND, Taddei A, Hu J, Zegarra-Moran O, Suen YF, Robins LI, Dicus CW, Willenbring D, Nantz MH, Kurth MJ, Galietta LJ, Verkman AS. Phenylglycine and sulfonamide correctors of defective delta F508 and G551D cystic fibrosis transmembrane conductance regulator chloride-channel gating. Mol Pharmacol. 2005 May;67(5):1797-807

Pedemonte N, Lukacs GL, Du K, Caci E, Zegarra-Moran O, Galietta LJ, Verkman AS Small-molecule correctors of defective DeltaF508-CFTR cellular processing identified by high-throughput screening. J Clin Invest. 2005 Sep;115(9):2564-71.

Verkman AS, Lukacs GL, Galietta LJ CFTR chloride channel drug discovery--inhibitors as antidiarrheals and activators for therapy of cystic fibrosis. Curr Pharm Des. 2006;12(18):2235-47.

Rhoden KJ, Cianchetta S, Stivani V, Portulano C, Galietta LJ, Romeo G. Cell-based imaging of sodium iodide symporter activity with the yellow fluorescent protein variant YFP-H148Q/I152L. Am J Physiol 2007 292: C814-C823

Diena T, Melani R, Caci E, Pedemonte N, Sondo E, Zegarra-Moran O, Galietta LJ Block of CFTR-dependent chloride currents by inhibitors of multidrug resistance-associated proteins.Eur J Pharmacol. 2007 Apr 10;560(2-3):127-31

Pedemonte N, Caci E, Sondo E, Caputo A, Rhoden K, Pfeffer U, Di Candia M, Bandettini R, Ravazzolo R, Zegarra-Moran O, Galietta LJV Thiocyanate transport in resting and IL-4-stimulated human bronchial epithelial cells: role of pendrin and anion channels. J Immunol. 2007 Apr 15;178(8):5144-53.

Zegarra-Moran O, Monteverde M, Galietta LJ, Moran O Functional analysis of mutations in the putative binding site for cystic fibrosis transmembrane conductance regulator potentiators. Interaction between activation and inhibition. J Biol Chem. 2007 Mar 23;282(12):9098-104

Sonawane ND, Zhao D, Zegarra-Moran O, Galietta LJ, Verkman AS Lectin conjugates as potent, nonabsorbable CFTR inhibitors for reducing intestinal fluid secretion in cholera.Gastroenterology. 2007 Apr;132(4):1234-44.

Pedemonte N, Boido D, Moran O, Giampieri M, Mazzei M, Ravazzolo R, Galietta LJ Structure-activity relationship of 1,4-dihydropyridines as potentiators of the cystic fibrosis transmembrane conductance regulator chloride channel. Mol Pharmacol. 2007 Jul;72(1):197-207