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Novel therapeutic approaches

Novel therapeutic approaches addressing the basic defect in cystic fibrosis

This article summarises recent advances in the discovery and mechanism of action of novel compounds aimed at tackling CFTR dysfunction, the basic defect in cystic fibrosis

Professor Margarida D Amaral PhD Assistant Professor Carlos M Farinha University of Lisbon, Faculty of Sciences, BioFIG – Centre for Biodiversity, Functional and Integrative Genomics, Campo Grande, C8 bdg, 1749-016 Lisbon, Portugal email:

Cystic fibrosis (CF) is the most common lethal autosomic recessive disorder among Caucasians, affecting one in every 2500–6000 newborn. Clinically, CF is characterised by a major involvement of the airways, with recurrent respiratory infections (the main cause of mortality and morbidity), pancreatic insufficiency, elevated salt concentration in the sweat and male infertility.

CF is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), one of the largest genes of the human genome, spanning a region of about 190 kb (kilobases) in the long arm of chromosome 7. This gene encodes the CFTR protein, which functions as a cAMP-regulated chloride (Cl-

) channel at the apical membrane

of epithelial cells. CFTR protein has a complex structure (Figure 1), which includes two transmembrane domains (TMD1 and TMD2) that together form the pore of the channel, two nucleotide-binding domains (NBD1 and NBD2) – which bind ATP to form a 'sandwich' that pushes the TMDs to go apart and thus open the channel pore to conduct Cl-

ions, and a unique regulatory domain (RD) – containing multiple phosphorylation sites, whose modification is also needed for protein function.

CFTR mutations

More than 1900 mutations have been reported in the CFTR gene, but a single

Figure 1: Regulation of the CFTR Cl- channel opening. This schematic model shows the regulation of CFTR through cAMP-dependent phosphorylation at the R domain (RD) and ATP binding and hydrolysis at the NBDs. Before the opening of the channel (A), phosphorylation by the cAMP-dependent protein kinase (PKA) has to occur at the RD in its multiple consensus phosphorylation sites. Following RD phosphorylation, ATP binds at the interface of the two NBD and these assemble to form a dimer forcing the channel pore to open (B) and Cl- ions are transported. Channel closure is determined by protein phosphatases (PPases) that dephosphorylate the R domain and return CFTR to its inactive state (A). In and Out denote the intra- and extracellular sides of the cell membrane. Abbreviations: MSD, membrane-spanning domain; NBD, nucleotide-binding domain; P phosphorylation of the R domain; PKA, protein kinase A; PPase, protein phosphatase; RD, regulatory domain

one – the deletion of phenylalanine residue at position 508 (F508del) – is present in 90% of CF patients in at least one allele. CFTR mutations are associated with distinct cellular phenotypes that ultimately lead to the absence or reduction of CFTR-dependent Cl-

at the

plasma membrane. According to its cellular effect, mutations have been grouped into six distinct classes (Figure 2):1,2 – Class I – Mutations in this class completely impair the synthesis of CFTR protein, leading to complete absence of protein in the cell. These are mainly nonsense (or 'stop') mutations, that is, mutations leading to production of an incomplete CFTR protein which is non-functional.

– Class II – The protein is retained intracellularly at the endoplasmic reticulum, never achieving its correct location at the cell surface, thus being unable to fulfil its role as a Cl-


The most common mutation – F508del – is included in this class. – Class III – Mutations in this class lead to defective channel regulation. The protein is located at the plasma membrane but the channel does not open, being thus unable to conduct Cl-

. Most of

these mutations are located in the NBDs of CFTR. The second most common mutation disease-causing mutation, G551D, is in this class. – Class IV – In this group, mutant CFTR protein exhibits defective conductance,


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