cystic fibrosis and protein synthesis case study

Biology lesson plans, worksheets, tutorials and resources for teachers and students.

Case study: cf mutations.

cystic fibrosis and protein synthesis case study

This case study is a follow-up to the Cystic Fibrosis Case Study where students explore how changes in transport proteins affects the movement of ions, resulting in a build-up of chloride ions and the symptoms of the disease.

Students were introduced to the idea that different mutations can cause differences in the transport proteins, but in the first version, the origin of these mutations was not discussed.

Eventually, students get to the chapter on DNA, RNA, and protein synthesis, so it’s a good time to circle back to the CF case and explore how mutations in DNA can affect the protein made by the ribosomes. Students should already have some background in the central dogma, but a review may be in order to remind students how to transcribe DNA to RNA and then use a codon chart to determine the sequence of amino acids. This practice worksheet on using codon charts is something they may have done in freshman biology.

This case explore frameshift mutations, missense mutations, and nonsense mutations. Students are given a section of DNA to transcribe and compare it to mutant DNA. Students should see that changes in DNA can result in changes in the synthesized protein, though some changes are more profound than others. The link below is a Google Doc designed for remote learning but will work for in-class lessons. An original in-class version is also available, where it doesn’t have the colored text boxes.

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Week 6 Lecture Quizzes: Protein Synthesis (case: Cystic Fibrosis)

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Class 1 CF Mutations

Michael wilschanski.

1 Pediatric Gastroenterology, Hadassah Hospitals Hebrew University, Jerusalem, Israel

Since the discovery of the gene that causes Cystic Fibrosis, our knowledge of how mutations in this gene cause the varied pathophysiological manifestations of this disease has increased substantially. This knowledge has led to the possibility of new therapeutic approaches aimed at the basic defect. Class I mutations of CFTR include premature termination codons (PTCs) or stop codons. In the last 10 years there has been a concerted international effort to utilize the concept of read-through of the stop codon producing full length functioning CFTR protein. This author considers that this approach will result in clinical trials in CF patients carrying these mutations.

Class I mutations include PTCs or nonsense codons. A nonsense mutation is a single point alteration in DNA that results in the inappropriate presence of a UAA, UAG, or UGA stop codon in the protein-coding region of the corresponding messenger RNA (mRNA) transcript. Such a stop codon causes premature cessation of translation, with protein truncation leading to loss of function and consequent disease. Nonsense mutations are responsible for about 10% of cystic fibrosis cases worldwide. However, in Israel, nonsense mutations are the cause of cystic fibrosis in most patients (Kerem et al., 1997 ). As such mutations produce little functional CFTR, these patients usually have a phenotype of CF with exocrine pancreatic insufficiency.

The increased understanding of ribosomal function, the process of translation, and small molecules that change the interaction between the ribosome and mRNA have led to the identification of several agents that are capable of suppressing PTCs. This has resulted in a novel strategy to treat CF and other genetic disorders caused by PTCs by restoring full length protein.

Aminoglycoside antibiotics were the first drugs demonstrated to suppress PTCs in disease-causing mutations, allowing the translation of full length proteins (Hermann, 2007 ). Aminoglycosides are antibacterial agents, their mode of action is interfering with normal translation via binding to the bacteria 16S rRNA. There is reduced discrimination between cognate and near-cognate tRNA hence reducing translational fidelity. Eventually, there is accumulation of truncated and non-functioning proteins resulting in bacterial cell death.

Gorini and Kataja ( 1964 ) demonstrated that aminoglycosides may suppress PTCs and lead to full length translation in E. coli . Aminoglycosides may also bind to human 18S rRNA subunit reducing discrimination of near-cognate tRNAs. This interaction is less stable than in bacteria but may be sufficient to lead to an insertion of a near-cognate aminoacyl-tRNA into the ribosomal A site that is subsequently incorporated into the polypeptide chain.

Howard et al. ( 1996 ) described PTC suppression by the synthetic aminoglycoside geneticin (G418) to restore function in HeLa cells expressing nonsense codons in 1996. This pivotal work was extended to four nonsense mutations of cftr who were expressed by the human airway cell line IB3-1.In this study, the commonly used aminoglycoside, gentamicin, was incubated with these cells and full length protein was produced (Bedwell et al., 1997 ).

Animal Models

Two mouse models have been developed that contain PTCs including the mdx mouse model of Duchenne Muscular Dystrophy and the G542X-hCFTR mouse which is a transgenic model of CF. Barton-Davis et al. ( 1999 ) reported suppression of PTC in the dystrophin gene of the mdx mouse by gentamicin. Intra-peritoneal injection of gentamicin restored the full length dystrophin protein in both skeletal and cardiac muscle. Similar studies in the G542X-hCFTR mouse model with gentamicin injections caused full length functional CFTR protein in intestinal tissues. There was also a tendency to increased survival in these mice (Du et al., 2002 ).

Clinical Trials

The preclinical studies mentioned above have led to a number of clinical trials designed to test both proof of principle and efficacy in patients with genetic diseases caused by PTCs. As stated earlier, about 60% of CF patient in Israel carry PTCs or Class I mutations. An initial open label pilot study showed a significant improvement of Nasal Potential Difference measurements (NPD) after the instillation of gentamicin nose drops (Wilschanski et al., 2001 ). This was followed by a double-blind, placebo-controlled study on 24 patients which included NPD measurement and membrane localization by immuno-fluorescent staining utilizing an anti-body directed against the C-terminus of CFTR (Wilschanski et al., 2003 ). These studies utilized nasal gentamicin administered for 2 weeks which resulted in significant improvements of basal PD and chloride secretion representing CFTR function in the treatment arms compared with placebo. Together with this immuno-fluorescent staining was positive in the treatment group. These results were specific for patients with Class I mutations with no effect in the control group of patients homozygous for the Delta F508 mutation. In both studies, the vast majority of patients with PTCs expressed at least one copy of the W1282X CFTR mutation which is highly prevalent in CF patients of Ashkenazi Jewish descent. In a study performed in the USA, intravenous administration of gentamicin administered for 1 week also resulted in NPD improvement representing CFTR function in four out of five patients with Class I CF mutations (Clancy et al., 2001 ). Sermet-Gaudilus et al. ( 2007 ) reported similar results following 15 days of systemic gentamicin treatment in six out of nine CF patients carrying the Y122X mutation. In all these studies there was a variability of response with some patients not responding to gentamicin. Linde et al. showed that this NMD variability may be related to nonsense-mediated mRNA decay (NMD) – the major machinery evolved to protect against harmful products of nonsense mutations. This is a post-transcriptional translation-dependent surveillance mechanism that prevents the synthesis of proteins carrying PTCs. NMD has been shown to degrade transcripts carrying disease-causing nonsense or frameshift mutations. It is the efficiency of NMD which affect the level of transcripts carrying PTCs, which govern the response to read-through treatment. Response to gentamicin was found only in patients with a higher level of transcripts (Linde et al., 2007 ). Down regulation of NMD in cells carrying the W1282X mutation increased the level of CFTR nonsense transcripts and enhanced the CFTR chloride channel activity in response to gentamicin. This may have a critical clinical correlation in the read-through of PTCs in various diseases. However, the inconvenience of parenteral administration and the potential for serious toxic effects preclude long-term systemic use of gentamicin for suppression of nonsense mutations.

Recently a novel agent PTC124 or Ataluren was developed through an extensive high throughput screening program using a luciferase based system (Welch et al., 2007 ). The molecule is a 1,2,4-oxadiazole benzoic acid and is reported to interact with mammalian ribosomes in a manner distinct from aminoglycosides. Ataluren does not have antibiotic activity and is orally bioavailable. Studies in myocytes isolated from the mdx mouse defined target doses and exposures to rescue dystrophin function. After treatment with, full length dystrophin was localized in skeletal and cardiac tissue. In the G542X-hCFTR mouse oral and intra-peritoneal administration led to detectable full length CFTR localization at the apical cell membrane of intestinal glandular cells by immuno-fluorescent staining together with improved chloride conductance as assayed by trans-epithelial ion transport (Du et al., 2008 ). Correction of CFTR chloride transport was incomplete. Less than 30% of the short-circuit current that was observed in wild-type mice occurred in the CF mice. This suggests that potential clinical benefit would only need partial restoration of protein function.

Phase I studies in healthy volunteers established the initial safety profile for Ataluren, and defined dosing regimens to achieve target trough plasma concentrations (of 2–10 μg/mL) that are known to be active in preclinical models.

Our group reported a phase II clinical trial of PTC124 in 23 patients with cystic fibrosis (Kerem et al., 2008 ). This open label study included two consecutive 28-day cycles, each of 14 days of treatment followed by 14 days of washout. In the first cycle, patients received daily postprandial doses of 4, 4, and 8 mg/kg. The doses were increased in the second cycle to 10, 10, and 20 mg/kg. Convincing changes in NPD were observed in more than half the patients in the first cycle. Interestingly, this effect was seen in only about a third of the patients in the second cycle. Coupled to this finding, modest but statistically significant improvements in lung function and bodyweight were observed after the first cycle which, in general, persisted to the end of the second cycle. Following this study, 19 of these patients were enrolled in a 12 week open label extension study. NPD improvements were reported over time in both the higher and lower dose treatment groups including four patients who did not respond to PTC124 in the 2 week study. This was accompanied by modest improvements in pulmonary function and a significant reduction in quantitative cough assessment (Wilschanski et al., 2011 ; Figure ​ Figure1). 1 ). A similar phase 2 study was performed on adults in the United States which did not reach statistical significance in nasal potential difference measurements. This may be due to the multitude of sites performing the trial each having relatively few patients and the different mutations carried by the patients.

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Change in mean pulmonary function tests . Data are presented as mean ± SEM. (A) FEV1, forced expiratory volume in 1 s. (B) FVC, forced vital capacity.

A similar phase 2a study was carried out in children in France and Belgium. Twenty-two children aged 6–18 years of age completed a dose-ranging crossover study. There was significant improvement in NPD and nasal epithelial CFTR protein by immunofluorescence (Sermet-Gaudelus et al., 2010 ).

The development of agents that suppress premature stop codons, such as Ataluren, is not without theoretical risk, because there are at least two potential concerns about its mode of action. First, Ataluren might lead to erroneous suppression of native stop codons, and second, Ataluren might disrupt NMD. Encouragingly, Ataluren seems to be remarkably selective for premature, rather than native, stop codons, and it seems to restrict its action to those ribosomes that are involved in productive translation of proteins rather than those that are involved in NMD. These preclinical findings were supported by the observation that CFTR mRNA levels are largely unaffected by Ataluren treatment (unlike after gentamicin administration).

Suppression of PTCs with small molecules is emerging as a rational approach to treat a variety of genetic disorders including CF. Following these positive findings, a multinational Phase 3 placebo-controlled efficacy trials is currently underway. These studies provide hope that a treatment strategy could be applied to the basic defect rather than downstream manifestations of the disease.

Every person has two copies of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. A person must inherit two copies of the CFTR gene that contain mutations — one copy from each parent — to have cystic fibrosis.

cystic fibrosis and protein synthesis case study

In people with CF, mutations in the CFTR gene can disrupt the normal production or functioning of the CFTR protein found in the cells of the lungs and other parts of the body.

People who inherit one copy of the CFTR gene that contains a mutation and one normal copy are considered  CF carriers . CF carriers do not have the disease but can pass their copy of the defective gene on to their children.

Our Genetic Encyclopedia

Each of our cells contains genetic information that provides the body with coded instructions to make proteins, which determine how the body looks, develops, and works.

Genetic information is stored in chromosomes, which can be thought of as different volumes of our genetic encyclopedia. Humans have 23 pairs of chromosomes. Each pair is made up of one copy of a chromosome from the mother and one from the father.

Most cells in the body have a full copy of the genetic encyclopedia, which includes 23 pairs of chromosomes. However, eggs and sperm have only a single copy of each chromosome, rather than the pairs found in other cells in the body.

Each chromosome is made up of many genes, which are the entries, or topics, in the encyclopedia. The genes supply the body with instructions for making proteins.

All of this genetic information that makes up our genes is in code and stored as a molecule called deoxyribonucleic acid (DNA). The DNA code is made up of letters that spell out the entries of our genetic encyclopedia.

If we all shared 100 percent of our genetic material then we would all be as alike as identical twins are. But not every single letter in the genetic encyclopedia is the same in all of us, which helps to explain why we are not all exactly alike.

How Changes in the CFTR Gene Affect the Body

Different types of changes, or mutations, in our genes affect the body in different ways. Within our genetic encyclopedia, there are small differences in our genes. Sometimes the differences are minor or do not affect a person's health — like two encyclopedia entries that are worded in slightly different ways but still say the same thing. In these cases, people's genes may differ or the protein made by the gene is slightly different — resulting in a different eye color — but the genes and the proteins work correctly. 

At other times, the change in a gene may cause the protein to not work or not be made at all. Cystic fibrosis is caused by mutations in the gene that produces the cystic fibrosis transmembrane conductance regulator (CFTR) protein. This protein is responsible for regulating the flow of salt and fluids in and out of the cells in different parts of the body. 

Mutations in the CFTR gene cause the CFTR protein to malfunction or not be made at all, leading to a buildup of thick mucus, which in turn leads to persistent lung infections, destruction of the pancreas, and complications in other organs.

Cystic fibrosis is an example of a recessive disease. That means a person must have a mutation in both copies of the CFTR gene to have CF. If someone has a mutation in only one copy of the CFTR gene and the other copy is normal, he or she does not have CF and is a CF carrier. About 10 million people in the United States are CF carriers.

CF carriers can pass their copy of the CFTR gene mutation to their children. Each time two CF carriers have a child together, the chances are: 

People with CF can also pass copies of their CFTR gene mutations to their children. If someone with CF has a child with a CF carrier, the chances are:

Children of two carriers may be CF carriers like their parents. In a family with four children, it is possible that none of the children, some of the children, or all of the children will have CF. Each baby has the same chance to inherit CFTR mutations from both parents, no matter whether any of the other siblings are carriers or have CF. When someone with CF has children with a CF carrier, the children will either be CF carriers or have CF.

This infographic shows how a person gets CF from their parents. It shows that when two people who are carriers have a child, there is a 25% chance of having a child with CF. When one parent has CF and one parent is a carrier, there is a 50% chance of having a child with CF.

Carrier Testing for Cystic Fibrosis Article | 3 min read

Types of CFTR Mutations Article | 9 min read

cystic fibrosis and protein synthesis case study

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cystic fibrosis and protein synthesis case study

Molecular basis of cystic fibrosis: from bench to bedside

Maria Cristina Dechecchi 1# , Anna Tamanini 1# , Giulio Cabrini 2

1 Laboratory of Analysis, Section of Molecular Pathology, University Hospital of Verona , Verona , Italy ; 2 Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona , Verona , Italy

Contributions : (I) Conception and design: G Cabrini; (II) Administrative support: None; (III) Provision of study materials or patients: None; (IV) Collection and assembly of data: MC Dechecchi, A Tamanini; (V) Data analysis and interpretation: MC Dechecchi, A Tamanini; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors.

#These authors contributed equally to this work.

Abstract: Cystic fibrosis (CF), is an autosomal recessive disease affecting different organs. The lung disease, characterized by recurrent and chronic bacterial infection and inflammation since infancy, is the main cause of morbidity and precocious mortality of these individuals. The innovative therapies directed to repair the defective CF gene should account for the presence of more than 200 disease-causing mutations of the CF transmembrane conductance regulator ( CFTR ) gene. The review will recall the different experimental approaches in discovering CFTR protein targeted molecules, such as the high throughput screening on chemical libraries to discover correctors and potentiators of CFTR protein, dual-acting compounds, read-through molecules, splicing defects repairing tools, CFTR “amplifiers”.

Keywords: Cystic fibrosis (CF); personalized medicine; cystic fibrosis transmembrane conductance regulator correctors (CFTR correctors); CFTR potentiators

Submitted Jun 09, 2018. Accepted for publication Jun 26, 2018.

doi: 10.21037/atm.2018.06.48


Cystic fibrosis (CF), the most common life-threatening rare disease among Caucasians, is an autosomal recessive genetic disease occurring in approximately one in 3,000–4,000 live birth as based on neonatal screening ( 1 ). Although several organs are involved, manifestation of CF disease in the airway tract is the main cause of mortality and morbidity in these patients ( 2 ). From the first description of a disease of the exocrine pancreas associated with lung symptoms in 1938 ( 3 ), survival of CF patients increased to a median age of 40 years, thanks to antibiotic therapies and correcting the intestinal malabsorption ( 4 , 5 ). After the identification of defective CF transmembrane conductance regulator ( CFTR ) gene, in 1989 ( 6 - 8 ), therapeutic approaches on the management of symptoms had a turning point which opened many hopes towards CFTR gene-targeted strategies ( 9 ), a field of investigation full of promises in steady progress.

Role of CFTR protein

CF disease is due to the defect of the CFTR gene located on chromosome 7 ( 6 ). CFTR gene encodes a protein encompassing the cellular membrane with two membrane-spanning domains (MSD), each constituted by six alpha-helices, two cytoplasmic domains, each binding one ATP molecule, termed nucleotide binding domains (NBD), a regulatory (R) domain with several consensus sequences for phosphorylation by protein kinase A (PKA) and protein kinase C (PKC). CFTR protein belongs to the family of ATP-binding cassette (ABC) transmembrane proteins ( 10 ). It is a chloride ion transporter localized at the apical membrane of several polarized epithelia ( 11 , 12 ), although other small molecules seem to be transported by CFTR ( 13 ), including ATP ( 14 - 17 ). As a chloride transporter, CFTR plays a critical role in the hydration of the mucus at the surface of the airway tract ( 18 ). Moreover, it favors mucus tethering and detachment through alkalinization with bicarbonate ( 19 , 20 ). De-hydration of airway surface fluid is a critical feature in the onset of the neutrophil dominated inflammatory and infective milieu of CF airways, which begins in the early months of life ( 21 ). CFTR-mediated ion transport requires binding of ATP on NBFs and phosphorylation of the R domain by protein kinase A ( 22 - 25 ) and protein kinase C ( 26 - 28 ).

Lung disease in CF

Defective ion transport mediated by CFTR reduces airway surface liquid hydration, which impairs mucociliary clearance, one of the basic innate immune defense mechanism of the respiratory tract ( 18 , 21 ). CF lung disease is characterized by an exaggerated inflammatory response accompanied by a huge number of neutrophils in the lumen of bronchi ( 21 ). However, these neutrophils are unable to completely clear bacteria; thus, repeated infections, mainly by Haemophilus influenzae and Staphylococcus aureus , pave the way to a chronic settlement of Pseudomonas aeruginosa . In addition, neutrophils release proteases, mainly elastases, reactive oxygen species and neutrophil extracellular traps thus worsening respiratory function and progressive tissue destruction and ultimately leading to respiratory insufficiency, reduced quality and expectancy of life ( 21 , 29 - 34 ). Experiments performed in different model systems in vitro , ex vivo and in vivo animal models have not yet clarified whether recruitment of neutrophils in the bronchial lumen, precedes or follows bacterial infection ( 35 - 37 ). To combat respiratory insufficiency, CF patients are treated with antibiotics and anti-inflammatories and soon or later, they undergo lung transplantation, which provides a dramatic improvement in the quality of life and some extension of survival ( 38 - 40 ).

The CFTR gene mutations

The 250-kb gene, located in chromosome 7, is structured in 27 exons. An International Worldwide Consortium of laboratories of molecular genetics extensively analyzed sequence variants and to date, over 2,000 sequence variations have been reported, at least 200 of them being associated with the disease (see the Cystic Fibrosis Mutation Database of the Cystic Fibrosis Gene Analysis Consortium, ) ( 41 , 42 ). Deletion of the phenylalanine in position 508 of the polypeptide chain, known as Phe508del or F508del, is the most common CFTR mutation, affecting from 50% to 90% of the chromosomes of CF patients along different geographical areas ( 43 ). Besides F508del CFTR , most CF causing mutations are missense variants (42%), nonsense (10%), frameshift (15%), splicing (13%), in frame deletion/insertion (2%) and promoter (0.5%) mutations ( 42 , 43 ).

The molecular defects of CFTR protein

F508del CFTR mutation ( 8 ) leads to the synthesis of an immature, non-glycosylated protein unable to localize on the plasma membrane ( 44 ). In-depth studies on the consequences of the different mutations on CFTR protein have allowed to simplify functional defect mechanisms ( 45 ), now schematized into the six classes ( 2 ), as shown in Figure 1 and described as follows:

cystic fibrosis and protein synthesis case study

Class I—“No protein”

These mutations affect protein synthesis, due to stop-codon (nonsense) mutations in which the CFTR mRNA is degraded through a process termed nonsense-mediated decay. This class includes G542X mutation (common in Mediterranean coastal area), R1162X (common in North-eastern Italy and Catalonia), W1282X (affecting about 40% chromosomes in Ashkenazi Jews).

Class II—“No traffic”

These mutations affect CFTR protein processing, due to protein misfolding, which is recognized by the endoplasmic reticulum (ER) quality control machinery leading to protein degradation. This class includes the most common F508del mutation, N1303K, R560T, A561E and R1066C.

Class III—“No function”

These mutations, also termed as “gating defect”, affect the activation of ion transport function, although CFTR is correctly glycosylated and located at the plasma membrane. This class includes G551D mutation.

Class IV—“Less function”

These mutations reduce chloride ions transported through pore channel, due to mutations the arginine located in the MSDs, which are involved in the flow of chloride through the plasma membrane. This class includes R117H and R334W.

Class V—“Less protein”

These mutations significantly reduce the amount of wild-type CFTR protein at the plasma membrane, mainly due aberrant splicing of RNA, leading to a non-functional protein. This class includes 3849-10 kb C>T and 3262-26A>G.

Class VI—“Less stable”

These mutations affect the stability and/or anchoring of CFTR protein at the plasma membrane. This class includes F508del CFTR rescued (rF508del) by correctors.

Notably, many CFTR causing mutations are not classified in one of these six classes and in some cases, mutations present more than one class defect, e.g., F508del mutation has a processing defect (class II), a gating defect (class III) and a reduced stability at the plasma membrane after being rescued (class VI). Despite simplistic, this classification has focused research of novel drugs towards different protein defects thus allowing development of personalized medicine, i.e., specific treatments tailored on CF genotype ( 46 ).

High throughput screening (HTS) in search of new CFTR protein targeted molecules

In search of CFTR modulators, large scale chemical libraries comprising thousands of compounds were tested. Initial challenge was to set up simple and rapid technological tools to study the effect of each molecule on chloride channel activity. In this respect, three different HTS assays have been developed, as reviewed and depicted in Figure 2 ( 50 ). Starting from the SPQ molecule, whose emission intensity is modulated by intracellular collisional quenching, other halide-sensitive fluorescent probes have been developed, such as MQAE, a membrane permeable dye retained inside the cells by cleavage of acetyl ester residues ( 47 , 51 - 53 ). A second assay, based on membrane depolarization dependent on chloride channels activation under proper experimental conditions has been set up. In this assay, membrane depolarization can be detected by measuring variations of fluorescence of membrane-potential sensitive dyes, due to the quantum yield change upon different polarity of the cellular environment ( 54 , 55 ). We developed the membrane-potential sensitive probe bis-oxonol to detect CFTR correction after transferring with viral vectors the wild type CF gene in CF bronchial epithelial cells ( 48 ). This assay was then accomplished by an HTS of more than 100,000 compounds that lead to the discovery of the first two small molecules became drugs for CF patients: VX-770 and VX-809 ( 56 - 58 ). A third tool, based on dynamic quenching of a yellow-fluorescent protein (YFP) made sensitive to intracellular chloride-ion concentration was set up ( 59 ) and further improved by mutations that render YFP very sensitive to chloride ion ( 49 , 60 ). F508del CFTR correctors and G551D CFTR potentiators were discovered by this assay ( 61 - 64 ). Interestingly, also a potent CFTR-specific inhibitor ( 65 ), currently used to inhibit CFTR function in vitro assays, was discovered and proposed to target the hyper-secretory diarrhea mediated by hyper-functional CFTR protein, induced by cholera toxin ( 65 ).

cystic fibrosis and protein synthesis case study

The first molecules reaching the chemist’s bench

CFTR correctors are the molecules able to rescue the class II defective CFTR, e.g., F508del CFTR and CFTR potentiators those activating the chloride transport in Class III gating-defective CFTR, e.g., G551D ( 2 ). This terminology allows to define the effect of each molecule and recalls the experimental conditions utilized in the screening. As a matter of fact, HTS for discovery correctors is performed in F508del CFTR expressing cells, incubated for 24–48 hours with the testing molecules whereas HTS for potentiators is carried out in G551D CFTR expressing cells, acutely treated with such compounds ( 50 ). Firstly, CFTR modulators were identified by academic groups ( 61 - 65 ), however these molecules did not undergo a pharmaceutical development from preclinical to clinical trials. Importantly, the biotech company Vertex Pharmaceuticals published its first “CFTR corrector” VX-809 (Lumacaftor) ( 56 , 58 ) and its first “CFTR potentiator” VX-770 (Ivacaftor, trade name Kalydeco) ( 57 ), few years later.

These molecules underwent a quick drug development passing from in vitro assays ( 56 - 58 ) directly to clinical trials in CF patients. Food and Drug Administration approved VX-770 in 2012 and VX-809 in 2015, for the treatment of CF patients carrying specific CFTR mutations.

VX-770 has proven excellent efficacy in children over six years of age and adults with G551D mutation in at least one allele ( 66 - 68 ), as demonstrated by an average 10% increase of forced expiratory volume in 1 second (FEV 1 ), decrease of pulmonary exacerbations, weight increase and normalization of sweat electrolytes ( 67 ), also patients with very low residual lung function (e.g., FEV 1 <40%) ( 69 , 70 ) or carrying class III mutations other than G551D ( 71 ). Unfortunately, the advantages of this drug are limited to very few CF patients as G551D mutation is very rare ( 43 , 72 ).

On the contrary, treatment with VX-809 in F508del CFTR homozygous patients did not produce any improvement of FEV 1 ( 73 ). These disappointed results led to development of VX-770 and VX-809 combined formulation, named Orkambi that was tested in CF patients homozygous for the F508del CFTR mutation, providing some benefits in lung function ( 74 ). These data were reproduced in a large international multicentric clinical trial showing a 2–3% increase of FEV 1 in respect to placebo after 24 weeks of treatment with Orkambi ( 75 ), although benefits were less evident in compound heterozygous CF patients carrying F508del CFTR in one allele ( 76 ). Different investigations in vitro have been pursued in order to understand these clinical limitations. For instance, it has been found that VX-770 negatively interacts with the rescued F508del CFTR protein by VX-809, thus reducing plasma membrane stability ( 77 , 78 ). How this interaction translates in CF patients is presently debated ( 79 , 80 ).

Molecules for class I “No protein” defects

As mentioned above, class I mutations cause CFTR mRNA degradation through nonsense-mediated decay. Discovery of molecules able to read-through the premature stop codons for treating CF patients carrying class I mutations started after the observation that aminoglycoside antibiotics can correct this defect ( 81 , 82 ). In this respect, the aminoglycoside gentamycin, previously used for the treatment of bacterial infections ( 83 - 86 ) was investigated. In order to avoid toxicity of aminoglycosides, gentamycin was then replaced by the analogue ataluren, produced by PTC Therapeutics ( 87 - 89 ). Unfortunately, a long-term placebo-controlled double-blind phase 3 study showed no improvement in the primary endpoint FEV 1 in CF patients , despite initial promising findings in several clinical trials ( 90 ). This led PTC Therapeutics discontinuing the development of ataluren in CF, leaving wide open the need of compounds targeting class I mutations ( 91 ).

Molecules for class II “No traffic” defects

Several correctors to rescue the class II defective CFTR, e.g., F508del CFTR, have been discovered by different academic groups in the United States and Europe ( 64 , 92 - 115 ). However very few of them underwent pharmaceutical development. Thus, different pharmaceutical companies are investing their own resources in pre-clinical discovery of new correctors.

The encouraging advancements obtained with VX-809 prompted Vertex Pharmaceuticals to explore new correctors, such as VX-661 (Tezacaftor) in association with VX-770 (116-118). F508del CFTR homozygous patients, treated with this combination ameliorated lung function ( 116 - 118 ). In addition to VX-809 and VX-661, several other correctors discovered by Vertex Pharmaceuticals (VX-152, VX-440, VX-445, VX-659) and by other companies, such as Genzyme/Sanofi, Pfizer and Reata (FDL169, GLPG2222, PTI-428, PTI-801), entered in phase 1/2 clinical trials ( ).

Molecules for class III “No function” defects

Treatment of F508del CFTR homozygous patients requires both correctors and potentiators to rescue the gating defect also present in F508del CFTR protein ( 119 ).

Approval of VX-770 for CF patients with G551D mutation ( 56 ), is one of the major breakthroughs for CF cure ( 66 - 68 ). Nevertheless, negative interactions between VX-770 and VX-809 ( 77 , 78 ) prompted academic groups to search novel potentiators that do not present these limitations ( 120 ). In parallel, other companies launched phase 2 and phase 1 clinical trials on new potentiators ( ). Very interestingly, dual-acting compounds, i.e., corrector and potentiator activity, may be a very appealing therapeutic perspective for CF treatment (see below).

Molecules for class IV “Less function” defects

This mutated CFTR protein displays low ion conductance that could be repaired by increasing protein expression at the plasma membrane or potentiating its open state period. In this regard, clinical trials with VX-770 (Ivacaftor, Kalydeco) in CF patients carrying the R117H mutation showed some benefit in lung function of adults with stable disease ( 121 ). This evidence supports further testing of potentiators in patients with CFTR class IV mutations.

Molecules for class V “Less protein” defects

As detailed above, class V mutations reduce the expression of functional CFTR. As a consequence of abnormal splicing both aberrant and normal transcripts are produced. To repair this defect, increase CF gene transcription as well as CFTR correctors and potentiators could represent useful remedies ( 122 ).

Molecules for class VI “Less stable” defects

Less stable CFTR protein needs to strengthen its anchoring at the plasma membrane. Importantly, rescued F508del CFTR protein by correctors displays increased turnover due to its removal by the peripheral quality control machinery and Disabled-2 (Dab2)-dependent ubiquitination ( 123 - 126 ), further worsened by P . aeruginosa chronic infection that decreases, the expression of critical proteins, such as Na + /H + exchanger regulatory factor 1 (NHERF1) ( 127 - 129 ). Therefore, treatment of F508del CFTR homozygous patients should be addressed not only with correctors and potentiators but also with compounds stabilizing the rescued CFTR, by targeting both the CFTR anchoring proteins and the peripheral quality control machinery.

Dual-acting CFTR corrector and potentiator compounds

Consensus was reached that multiple defects of F508del CFTR protein should be addressed by combination of correctors and potentiators [for review see ( 130 )]. In order to avoid negative side effects due to multiple drug interactions, compounds able to act at the same time both as correctors and potentiators, i.e., dual-acting compounds, have been proposed ( 131 , 132 ). Several dual-acting compounds have been identified so far ( 99 , 103 , 104 , 133 - 135 ). In this regard, an interesting example has been given by 4,6,4'-trimethylangelicin (TMA) which besides correcting and potentiating CFTR activity displays anti-inflammatory properties ( 99 , 134 ). TMA exerts its dual action by interacting directly with the MSD1 on F508del CFTR protein ( 136 ).

The CFTR “amplifiers”

Beside the above mentioned molecular defects, F508del mutation produces a somewhat low amount of non-glycosylated immature Band B CFTR ( 44 ). Transcriptional inducers such as 4-phenylbutirrate were found to repair CFTR function by increasing band B CFTR protein that could escape at least in part the quality control systems ( 137 , 138 ). Therefore, forcing the production of band B CFTR protein in association with CFTR correctors could improve the overall efficacy of treatment ( 139 ). In this respect, new class of compounds called “CFTR amplifiers” seem to provide promising results in vitro ( 140 ). In particular, PTI-428 has been tested in a phase 1 clinical trial in CF patients under sponsorship of Proteostasis Therapeutics ( ). An alternative approach is to inhibit the degradative pathway of CFTR mRNA intervening on the epigenetic down regulation of CFTR expression, e.g., by microRNA miR-145, which inhibits CFTR translation by degrading CFTR mRNA and blocking CFTR protein translation. MiR-145-specific cell permeable peptide-nucleic acid chimera relevantly increased CFTR protein ( 141 ).

Effects of repairing mutated CFTR on lung infection and inflammation

It has been suggested that repairing of the ion transport defect by CFTR correctors and potentiators can by itself solve CF chronic lung infection and inflammation. In vitro evidence supports this idea as VX-809 abolished the exaggerated inflammatory pathways in F508del CFTR bronchial epithelial cells ( 142 , 143 ). A different F508del CFTR corrector, miglustat, was also found to have anti-inflammatory effects in CF bronchial epithelial cells, although not directly related to correction of mutated CFTR ( 144 ). On the contrary, derivatives of the angular furocoumarin angelicin already proved as correctors of F508del CFTR protein (TMA analogues) showed that rescue and anti-inflammatory activity can coexist or be separated in the same molecule as a function of structural changes ( 145 ). These findings provide evidence that CFTR rescue per se is not enough to reduce excessive inflammation. Despite different results in vitro indicate that F508del CFTR rescue could per se repairs excessive inflammation ( 142 , 144 ), no evidence of reduced lung inflammation after VX-809 has been presented so far in CF patients. Moreover, CFTR restoration for all individuals with CF is challenging because approximately 2000 CFTR variants have been reported, most of them are rare (see the Cystic Fibrosis Mutation Database of the Cystic Fibrosis Gene Analysis Consortium, ) and personalized medicine approaches based on each individual’s genetic profile may not be sufficiently efficacious in patients with irreversible lung damage. Thus, it appears that both combinations of novel CFTR potentiators and correctors as well as newer compounds for conventional therapies, such as inhaled antibiotics and anti-inflammatory agents, remain a cornerstone of treatment for CF lung disease [for review see ( 146 - 149 )].

Conclusions and open issues

The ability to repair CF defect by using personalized medicine based on each patient’s genetic profile represents a new challenge for CF research community. Despite exciting advances, several issues still remain open:

All this considered, a good therapeutic strategy should be based on more than one option.


Funding: This review was made possible by support of different research projects with grants from Telethon Foundation, CariVerona Foundation and Italian Cystic Fibrosis Research Foundation (FFC # 4/2004, 4/2005, 12/2008, 8/2010, 16/2010, 17/2010, 1/2011, 5/2011, 1/2012, 14/2012, 1/2013, 8/2014, 17/2014, 9/2015, 22/2015, 1/2016, 3/2016).

Conflicts of Interest: The authors have no conflicts of interest to declare.

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