Molecular biology of CFTR and new therapies in cystic fibrosis da spostate

Author: alessandra mazzucco
Date: 09/01/2014


Alessandra Mazzucco
Mariela Valenti


As the most prevalent genetic disorder in the western population, cystic fibrosis (CF) affects approximately 80,000 people in the world. CF is a rare monogenic disorder that mainly affects individuals of Caucasian descent. In order to manifest CF an individual must inherit two defective CFTR genes (or alleles). Promotion of the CFTR gene normally results in expression of CFTR protein. The CFTR gene is located on the long arm of chromosome 7 at position 931.2. To date, over 1900 sequences variations in the CFTR gene have been reported, although a detailed understanding of how CFTR mutations impact channel dysfunction is limited to only a few of these.

Targeting a genetic defect:cystic fibrosi transmembrane conductance regulator modulator in cystic fibrosis

CFTR structure

Image 1: CFTR molecular structure

CFTR is a large transmembraneous protein that comprises 1480 aminoacids in five distinct domains: At the N-terminus a membrane-spanning domain (MSD1) with six transmembrane a-helices is located followed by the nucleotidebinding domain 1 (NBD1), a regulatory domain (R-domain), the second six-helix MSD (MSD2), and the NBD2 at the C-terminus. The two transmembrane domain form the channel pore.
The epithelial chloride ion channel belongs to the family of ATP-binding cassette (ABC) transporters and is predominantly expressed in polarized epithelia. Additionally, it can be found in cardiomyocytes, smooth muscles, and some blood cells. After genetranscription, translation of the mRNA into a protein, and glycosylation, wild-type CFTR is transported to the cellmembrane where it functions as an ion channel.

CFTR channel function is regulated by cyclic adenosine monophosphate (cAMP)-dependent phosphorylation at the regulatory domain by protein kinase A (PKA) or protein kinase C (PKC).
ATP binding to NBDs causes conformational changes and channel opening because it leads to dimerization of the NBDs to a head-to-tail dimer which is thought to induce unknown conformational changes that open the gate of the channel. Furthermore, the channel protein has ATPase activity and hydrolyzes ATP to ADP + Pi, which finishes the native gating cycle and induces closure of the channel pore. The R-domain contains at least eight motifs for PKA phophorylation, which predominantly increases channel gating activity. Hence the phosphorilation alters the a-helical structure of the R-domain and the binding of the latter to the NBD1. R-domain has to be phosphorylated and release from the NBD1 to enable NBD dimerization.
The structure and dynamics of the NBD1 and the R-domain are strongly connected and have to be further evaluated, which can eventually discover novel binding sites for CFTR modulators that improve channel gating.

Activity of CFTR is hence dependent on intracellular nucleotide concentrations. Besides chloride ions, gluconate and bicarbonate ions can also pass the membrane through CFTR. Several ion channels such as the ENaC have been shown to be dysregulated in CF although they are intact proteins. Furthermore, intracellular vesicular CFTR might be involved in pH regulation and in the control of exo/endocytosis.

Reparing mutated protein development of small molecules–targeting decets in the cystic fibrosi transmembrane conductance regulator

CFTR mutations

Image 2: CFTR mutations

Most of the defects in the CFTR gene only affect one to three nucleotides, leading to frameshifts, amino acid exchange as well as splice site or nonsense mutations. The defects can be divided into six classes although it has to be noticed that many mutations show characteristics of more than one class.

Class I comprehends mutations in the CFTR gene which cause complete loss of full-length protein synthesis. This can be due to translation stop at premature stop codons that are formed by nonsense mutations or impaired splicing because of splicing site mutations. Class I mutations are associated with more severe phenotypes since no functional CFTR is formed by the cells.

Mutations of class II such as F508del lead to the formation of immature CFTR protein that is misfolded or not glycosylated. This defect protein is either not released by the endoplasmic reticulum (ER) or fastly ubiquitinated and degraded in the proteasome. Furthermore, any F508del-CFTR protein reaching the cell surface may also have a defect in the opening and closing of the channel, known as a gating defect. Also class II mutations produce severe CF phenotypes. Around 90% of individuals with CF are heterozy­gous and 40%–50% are homozygous for the F508del.

Class III CFTR mutations affect chloride channel regulation/gating. CFTR protein production and trafficking are normal, but when the protein arrives at the cell surface it does not respond to cAMP stimulation. This prevents binding of ATP and/or phosphorylation. The CFTR protein fails to open in response to intracellular signals at the cell surface, and remains closed for the majority of the time. The missense mutation Gly551Asp ( G551D ) is the third most common CFTR mutation and causes a glycine-to-aspartate substitution at residue 551.7,16 Approximately 4%–5% of all patients with CF have the Gly551Asp mutation on at least one allele,6,7 although this mutation is more frequent in those of Celtic (Scottish, Irish, English) origin.
This mutation is located in the intracellular NBD1 and leads to an exchange of glycin through aspartic acid. The exchange of glycin through aspartic acid and therby introduction of a large negatively charged side chain within the binding site for ATP is thought to dirups the nucleotide-binding pocket of the NBD1, that fails dimerization. However, in vitro experiments showed a remaining low activity of phosphorylated G551D-CFTR without activation by ATP. Additionally, it was observed that small molecules can improve activation and gating of phosphorylated G551D-CFTR in presence of forskolin, which stimulates the adenylylcyclase and induces the production of cAMP, in vitro and that even in absence of an activating stimulus by forskolin channel function can be improved with CFTR potentiators .

In contrast to class I -- III mutations that generally show a complete loss of CFTR function and severe CF, classes IV and V produce milder phenotypes since some CFTR channel activity is maintained. In class IV, the conductance of mutated CFTR for chloride ions is reduced, while class V is characterized by reduced biosynthesis of CFTR.

Class VI mutations are rare and not yet fully characterized.
Their protein product is unstable at the membrane or the transport capacity for other ions than chloride is reduced, which generally leads to severe phenotypes.

Reparing mutated protein development of small molecules–targeting decets in the cystic fibrosi transmembrane conductance regulator

Development, clinical utility, and place of Ivacaftor in the treatment of cystic fibrosis

Clinical manifestation

Defects in the CFTR gene lead to reduced surface expression or disturbed functionality of the ion channel, which in turn impairs the transport of chloride and other ions out of epithelial cells.
In the lung, the reduced ion transport causes surface dehydration and production of viscous and dehydrated mucus which affects the ciliary clearance of the mucus; the accumulating mucus leads to airway obstruction, that could cause permanent inflammation that damage lung tissue on one hand and is a breeding ground for microorganisms on the other. Patients suffer from recurring infections with predominantly Haemophilus influenzae, Staphylococcus aureus, Pseudomonas aeruginosa, and often Aspergillus fumigatus. Finally lead to respiratory failure, end-stage lung disease, and death.

Besides the interrupted transport of anions, several other ion transport systems that might be regulated by CFTR are disturbed in CF, especially the amiloride-sensitive epithelial sodium channel (ENaC) that shows increased activity in CF. CFTR is also responsible for chloride absorption in sweat glands, which opens the possibility of measuring chloride concentrations in sweat as biomarker of CFTR function. Sweat chloride levels are usually elevated (60 mmol/L) in patients with CF, whereas people without CF have sweat chloride levels ,40 mmol/L.

While the airways are the main site of CF symptoms, other tissues are also affected such as exocrine glands, pancreas, and kidneys. In more than 85% of patients, pancreatic failure and malabsorption in the intestinal tract are observed.
However, although the understanding and pharmacotherapy of CF have strongly improved over the years, thereis still no satisfactory therapy and no cure.

Development, clinical utility, and place of Ivacaftor in the treatment of cystic fibrosis


Since the identification of the mutant CFTR gene as the cause of CF, there has been a significant effort to harness gene therapy to correct the mutation at a cellular level. Traditional gene therapy would aim to place a copy of normal CFTR into the patients cells. The power of this approach is that it could potentially be curative for all CF patients regardless of the genotype. Despite the theoretical allure of gene therapy, the reality of bringing this technology to the clinical setting has proven a significant challenge.

The clinical developmental pipeline predominantly contains anti-inflammatory and anti-infective agents. Besides these, there are approaches to improve mucus hydration by different mechanisms than improving CFTR function and the attempt to dissolve the accumulating mucus .
However, these symptomatic therapies do not address the primary and basic defect of the disease. Therefore, the development of CFTR modulators is the most promising strategy for CF therapy at present. .

Gene therapy in cystic fibrosis

Repairing mutated proteins - development of small molecules targeting defects in the cystic fibrosis transmembrane conductance regulator

CFTR modulators

Image 3: CFTR modulators and mutations

As stated, the most promising experimental drugs for CF are agents targeting the underlying defect in CF, namely improving the channel function of defective CFTR. At present, there are two strategies to enhance the activity of mutated CFTR depending on the existing type of CFTR defect and mutation:

CFTR potentiators interact with mutated CFTR channels that already reached the surface and enhance their activity and gating.
CFTR correctors help mutated CFTR proteins to reach the cell surface that are normally subject to degradation, thereby improving the protein trafficking and channel density at the membrane. The most prominent mutation that might be treatable with such correctors is F508del.

Reparing mutated protein development of small molecules–targeting decets in the cystic fibrosi transmembrane conductance regulator

CFTR Potentiator

Potentiators are CFTR modulators that increase the time CFTR spends in the open channel configuration, with increased chloride transport. The most important is Ivacaftor (VX-770), the first potentiator to gain Food and Drug Administration approval is used for CF patients with the G551D mutation. Though it is the second most common mutation identified as a cause of CF, G551D is present in only about 4% of CF patients.

The G551D mutation, also known as the Celtic mutation, and it is caused by substitution of the amino acid glycine by aspartate at position 551 in the nucleotide binding domain-1 of the CFTR gene. It abolishes ATP-dependent gating (Fig.1), resulting in an open probability that is approximately 100-fold lower than that of wild-type channels. It is associated with a severe phenotype, characterised by pulmonary dysfunction and pancreatic insufficiency.

Image 4 - Postulated mechanism of action of ivacaftor (VX-770): (a) w.t. and G551D-CFTR are phosphorylated by protein kinase A (PKA) or other kinases; (b) binding of ATP then induces gating in w.t. CFTR but not in G551D-CFTR; © VX-770 directly binds to phosphorylated w.t. and G551D-CFTR and can open (potentiate) the channel pore without binding of ATP by unknown conformational changes; (d) VX-770 can also potentiate phosphorylated and ATP-activated w.t. CFTR and enhance gating. According to Eckford et al. (2012)

Clinical Trial of Ivacaftor

In a phase II clinical trial the safety and adverse event profile of ivacaftor was assessed as a primary end-point. Secondary end-points included improvements in CFTR-mediated ion transport (measured by nasal potential difference and sweat chloride concentrations), pulmonary status (measured by FEV1) and health-related quality of life (measured by the Cystic Fibrosis Questionnaire revised (CFQ-R)).

The study showed that ivacaftor was well tolerated and the authors recommended follow-up clinical trials to further investigate the drug’s efficacy in CF patients with the G551DCFTR mutation.

The most commonly occurring events were fever, cough, nausea, pain and rhinorrhoea. Most importantly, ivacaftor treatment showed improvements in channel function in both the nasal and the sweat gland epithelium. Moreover, a trend towards normalization of sweat chloride and nasal potential difference levels was associated with improvements in lung function. In some treated subjects the sweat chloride levels decreased to below the diagnostic cut-off for CF (60 mEq*L-1). This was the first study of a CF therapy to demonstrate normalization of sweat chloride.
Very interestingly, measures of lung function showed significant improvements: the median change from baseline in FEV1 was 8.7% predicted (range 2.3–31.3, p50.008). A clinically important improvement in health-related quality of life (CFQ-R scores) was also reported in ivacaftor treated patients, although this was not significant compared to placebo.
This proof-of-concept phase II study therefore confirmed the ability of the drug to restore G551D-dependant CFTR chloride transport. Taken together, these findings supported further clinical investigation of ivacaftor to determine long-term efficacy and safety as a targeted therapy for CF patients with the G551D mutation.

The phase III clinical trials investigate the efficacy of Ivacaftor in adults and children with the G551-CFTR mutation, with 2 studies: STRIVE (Evaluating the Efficacy and Safety of Treatment with VX-770 in CF patients with G551D Mutation) and ENVISION (Evaluation of Efficacy and Safety of VX-770 in children six to eleven years old with CF).
At the end of the 48-week study period patients were offered the opportunity to rollover into an optional open-label study: PERSIST (An Open-Label Rollover Study to Evaluate the Safety and Efficacy of VX-770 in Cystic Fibrosis patients), designed to monitor the long-term impact of ivacaftor treatment over 96 weeks.

The primary efficacy end-point in both STRIVE and ENVISION was the absolute change from baseline to week 24 in FEV1%pred (Image 5)

Image 5 - Results from the the STRIVE study of forced expiratory volume in 1 s (FEV1) absolute change from baseline. Treatment effect from baseline to week 24: +10.6%, p,0.0001. Treatment effect from baseline to week 48: +10.5%, p50.0001. Data are presented as mean (95% CI). pred: prediced.

Secondary end-points included the change in FEV1 % pred from baseline to week 48, time-to-first pulmonary exacerbation and patient-reported respiratory symptoms through to weeks 24 and 48 (Image 6).

Image 6 - Time to first pulmonary exacerbation in adults treated with ivacaftor or placebo according to the modified Fuch’s criteria. The dotted lines represent weeks 24 (168 days) and 48 (336 days), respectively. Week 24: hazard ratio50.40, p50.0016. Week 48: hazard ratio5 +10.5%, p50.0001.

Other secondary end-points included change in weight and sweat chloride concentration (a measure of CFTR channel function) from baseline through to weeks 24 and 48 (Image 7).

Image 7 - Sweat chloride concentrations from baseline to week 48 of the study. Treatment effect from baseline to week 24: -47.9 mmol*L-1, p,0.0001. Treatment effect from baseline to week 48: -48.1 mmol*L-1, p,0.0001. Data are presented as mean (95% C) diagnostic threshold.

Upon completion of the 48-week treatment period, patients were eligible to rollover into the PERSIST study to receive open-label ivacaftor for an additional 96 weeks.

The preliminary analysis observed that improvements in FEV1 reported during STRIVE were sustained for an additional 12 weeks beyond the initial 48-week study period (treatment difference 9.4% points). An improvement in FEV1 was also noted for those who switched from placebo to ivacaftor at the beginning of PERSIST. The magnitude of this improvement at 15 days after the start of active treatment was similar to that reported in STRIVE, thus, further supporting the beneficial effect of ivacaftor.
Preliminary results from PERSIST also strengthened evidence that ivacaftor has a beneficial effect on CF patients with severe disease. Eight patients in the group who switched from placebo to ivacaftor at the start of PERSIST had an FEV1,40% pred (indicating severe disease). In this subgroup, the mean¡ FEV1 pred on day 1 of STRIVE was 34.5 ± 3.7. This improved to a mean¡SD absolute change of 10.8 ± 8.7% at day 15 and a mean ± SD absolute change of 13.0 ± 10.5% at week 12. Similarly, the reductions in the number and duration of pulmonary exacerbations of patients initially in the placebo group after entering the open label study were similar to those reductions observed in subjects in the placebo-controlled ivacaftor study.

Gene therapy in cystic fibrosis

Ivacaftor treatment in patients with cystic fibrosis and the G551D-CFTR mutation

CFTR corrector

Since F508del is the most common (at least one allele in 90% of patients) mutation causing CF and among the most serious defects, research has strongly focused on developing pharmacological agents to rescue this mutant.
The F508del mutation disrupts folding of nascent CFTR, causing retention in the endoplasmic reticulum and subsequent proteasomal degradation. The result is minimal protein escaping intracellular degradation.

Ivacaftor monotherapy has been trialed in patients with F508del CFTR with much less success than in those with G551D. Efficacy results from an early clinical study in which patients homozygous for the F508del mutation were randomized to receive Ivacaftor or placebo. Treatment with Ivacaftor showed no improvement in the primary outcome measure: there were no increase in predicted FEV1 after 16 weeks, nor in any secondary outcome measure such as weight gain or improvement in quality of life. There was no clinically meaningful change in sweat chloride levels, although there was a slight decrease from baseline in the Ivacaftor group.

CFTR correctors increase F508del CFTR protein at the plasma membrane. According to a phase II multicenter, randomize, double blind, placebo controlled study Ivacaftor in combination with other small molecule compounds , VX-809 CFTR corrector - Lumacaftor, showed efficacy in vitro studies by enhancing in adult homozygous F508del processing and chloride transport in cultured human bronchial epithelial cells. However, the effect was much less than that seen with iva­caftor, and only 14% of epithelial cells showed improvement in chloride processing and secretion. Although VX-809 was found to be safe for patients, there was no impact on any clinically significant outcome.

Another ongoing multicenter, double-blinded, placebo controlled Phase II trial is examining the therapeutic efficacy and safety of VX-661, another CFTR corrector, both alone and in combination with Ivacaftor in adults homozygous for the F508del mutation. Initial reports have observed dose-dependent mean relative improvements of up to 9% in FEV1 in patients receiving the combination treatment at high dose versus placebo at day 28.
One unanswered question in regards to mutation specific therapy in CF is whether both mutations need to be
targeted in patients who are heterozygous for the CFTR genotype.

Ivacaftor treatment in patients with cystic fibrosis and the G551D-CFTR mutation

Reparing mutated protein development of small molecules–targeting decets in the cystic fibrosis transmembrane conductance regulator

Gene therapy in cystic fibrosis

PTC mutation suppressor

Besides CFTR correctors and potentiators, there are premature termination codon therapeutics. PTCs result when single base-pair substitutions create an erroneous stop codon within the open reading frame of a gene. Suppressors of PTCs, such as aminoglycoside antibiotics, are able to bind eukaryotic ribosomes and cause the insertion of a near cognate amino-acyl transfer RNA into the ribosomal A site.
This process can allow the ribosome to ‘readthrough’ the PTC and produce some full length protein and has been extensively tested in proof of concept studies utilizing aminoglycosides to suppress PTCs (gentamicin, amikacin, geneticin).
There has been demonstrated efficacy in vitro, in animal models of CF and muscular dystrophy, as well as in small numbers of CF patients. Currently, there is one oral compound, Ataluren (PTC Therapeutics), in clinical trials to treat CF caused by PTCs. Ataluren was studied in three phase II, randomized, dose ascending open label trials in CF.
Each study demonstrated short-term tolerability of ataluren (the primary drug related toxicity was to the kidney), and 2 demonstrated improvements in CFTR function (across a number of PTC mutations) as measured by the nasal potential difference (NPD-a CFTR bioassay). One study also demonstrated improvements in CFTR localization to the nasal cell membrane, and another demonstrated improvement in cough over 3 months. The third study failed to demonstrate improvements in NPD, and all 3 studies were limited by small numbers and absence of placebo groups. It was then studied in a large phase III randomized, 48-week, double blind, placebo controlled trial designed to test safety, efficacy, and tolerability.
The results have only been reported in an abstract and showed that Ataluren was associated with a trend toward slower loss of FEV1 and (22.5% Ataluren vs 25.5% placebo) and fewer pulmonary exacerbations.

Ataluren seems to be more effective on patients who received detobramycine inhalation. In this subgroup the results were more significative (FEV1 increase of 6,7% at week 48). More studies need to be conducted to evaluate the efficacy of this molecule.

Gene therapy in cystic fibrosis

Therapeutic update in cystic fibrosis


Ivacaftor is a promising therapeutic option for CF patients with the G551D-CFTR mutation. The studies demonstrated rapid, dramatic and sustained improvements in FEV1, self-reported outcomes of respiratory symptoms, nutritional status and reduced pulmonary exacerbations by 50%.
Efficacy outcomes in children were consistent with those in the older population despite milder disease at baseline, suggesting that early disease might be reversible or at least preventable.
A significant change in sweat chloride levels mirrored improvements in lung function, as a proof-of-concept of the drug mechanism of action.
There were no important safety concerns for 48 weeks administration. Initial analysis of an ongoing longer term open-label study suggests that the efficacy of Ivacaftor will be maintained in the long-term.

At present, efforts to treat the Phe508del CFTR mutation with ivacaftor or other small-molecule compounds have not shown adequate therapeutic efficacy. Further trials are currently underway and their results are awaited. Importantly, as future therapies aim to prevent rather than improve existing organ damage, FEV1 is unlikely to be a sensitive enough endpoint for future trials. The reliability of other endpoints, even established measure­ments such as sweat chloride, need to be examined further as clinical endpoints.

Such studies validate the knowledge that mutation-specific therapy can ultimately make a difference in the clinical setting.
Such innovative multisystem treatment of an underlying defect demonstrates how future therapeutic choices could be realistically driven by personalised genetic information. This represents an important milestone in CF and will pave the way for the development of other disease-modifying drugs in respiratory medicine and beyond.

Ivacaftor treatment in patients with cystic fibrosis and the G551D-CFTR mutation

Development, clinical utility, and place of Ivacaftor in the treatment of cystic fibrosis

2014-01-09T12:37:34 - alessandra mazzucco

Alessandra Mazzucco - Mariela Valenti

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