CHEST Recent Advances in Chest Medicine

Transcription

1 CHEST Recent Advances in Chest Medicine Cystic Fibrosis Transmembrane Conductance Regulator Intracellular Processing, Trafficking, and Opportunities for Mutation-Specific Treatment Mark P. Rogan, MD ; David A. Stoltz, MD, PhD ; and Douglas B. Hornick, MD, FCCP Recent advances in basic science have greatly expanded our understanding of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the chloride and bicarbonate channel that is encoded by the gene, which is mutated in patients with CF. We review the structure, function, biosynthetic processing, and intracellular trafficking of CFTR and discuss the five classes of mutations and their impact on the CF phenotype. The therapeutic discussion is focused on the significant progress toward CFTR mutation-specific therapies. We review the results of encouraging clinical trials examining orally administered therapeutics, including agents that promote read-through of class I mutations (premature termination codons); correctors, which overcome the CFTR misfolding that characterizes the common class II mutation F508del; and potentiators, which enhance the function of class III or IV mutated CFTR at the plasma membrane. Long-term outcomes from successful mutation-specific treatments could finally answer the question that has been lingering since and even before the CFTR gene discovery: Will therapies that specifically restore CFTRmediated chloride secretion slow or arrest the deleterious cascade of events leading to chronic infection, bronchiectasis, and end-stage lung disease? CHEST 2011; 139(6): Abbreviations: ABC 5 adenosine triphosphate-binding cassette; ATP 5 adenosine triphosphate; CF 5 cystic fibrosis; CFTR 5 cystic fibrosis transmembrane conductance regulator; COPII 5 coat protein complex II; ER 5 endoplasmic reticulum; ERAD 5 endoplasmic reticulum-associated degradation; HBE 5 human bronchial epithelium; HTS 5 high-throughput screening; MSD 5 membrane spanning domain; NBD 5 nucleotide-binding domain; NPD 5 nasal transepithelial potential difference; PKA 5 protein kinase A; PTC 5 premature termination codon; SERCA 5 sarcoplasmic/ endoplasmic reticulum calcium; UPS 5 ubiquitin proteasome system Manuscript received August 12, 2010; revision accepted December 12, Affiliations: From the Department of Respiratory Medicine (Dr Rogan), Waterford Regional Hospital, Waterford, Ireland; and Department of Internal Medicine (Drs Stoltz and Hornick), Division of Pulmonary, Critical Care, and Occupational Medicine, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA. Funding/Support: Dr Hornick is supported in part by the Cystic Fibrosis Foundation Translational Center Grant, which included funding to participate in industry-sponsored (eg, PTC Therapeutics, Vertex Pharmaceuticals) clinical trials. Correspondence to: Douglas B. Hornick, MD, FCCP, Division of Pulmonary, Critical Care, and Occupational Medicine, Department of Internal Medicine, C-33 GH UIHC, 200 Hawkins Dr, Iowa City, IA 52242; douglas-hornick@uiowa.edu Cystic fibrosis (CF) is an autosomal recessive inherited disorder characterized by elevated sweat chloride concentrations, abnormal epithelial chloride or bicarbonate transport, pancreatic insufficiency, recurrent lung infection and chronic bronchiectasis, and ultimately death from respiratory failure. 1 CF occurs in approximately one in 3,000 live births.2 The disease results from mutations in the CF transmembrane conductance regulator ( CFTR ) gene, which was first identified in Since this sentinel discovery, CF has benefited from an explosion in medical research. Among the many discoveries over that period, investigators have unraveled more precisely how specific CFTR gene mutations create varying intracellular consequences. Herein, we provide an update of the intracellular biology and the five classes of CFTR mutations currently recognized. This background sets the stage for describing a novel. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians ( site/misc/reprints.xhtml ). DOI: /chest Recent Advances in Chest Medicine

2 group of pharmacologic agents, some of which have shown remarkable preliminary success at enhancing mutant CFTR plasma membrane expression and function. Others show potential for rescuing the mutant CFTR protein from intracellular degradation. CFTR Molecular Biology and Cellular Quality Control The CFTR gene, comprising 180,000 base pairs, is located on the long arm of chromosome 7 and encodes a 1,480-amino acid membrane protein. The wild-type CFTR glycoprotein localizes in the apical plasma membrane and functions as a regulated chloride channel. CFTR might also affect bicarbonate-chloride exchange plus sodium and water transport in secretory and resorptive epithelium. 3,4 CFTR is a member of the large adenosine triphosphate (ATP)-binding cassette (ABC) transporter protein family. ABC transporters more commonly regulate transmembrane transport of small molecules. For example, in mammalian cells, they confer resistance to antineoplastic drugs, and in bacteria, they participate in nutrient uptake. In common with other ABC transporters, CFTR consists of two homologous cytoplasmic nucleotidebinding domains (NBDs) and two membrane spanning domains (MSDs). CFTR is unique among ABC transporters because it functions as an ion channel. CFTR also exhibits another distinctive feature because it contains a charged regulatory or R domain ( Fig 1 ) that opens the channel when phosphorylated by protein kinase A (PKA). 5 The journey followed by the CFTR glycoprotein from gene transcription to the cell membrane takes it through multiple interactions with cellular proteins within several cellular compartments where it must pass stringent quality control ( Fig 2 ). Within the nucleus, the DNA code is transcribed into mrna. The mrna leaves the nucleus and encounters ribosomes in the cytoplasm but mainly on the endoplasmic reticulum (ER) that unites with transfer RNA carrying specific amino acids, resulting in translation of nascent polypeptide within the ER. Further protein maturation within the ER involves a deliberate and complex protein folding process, which occurs within the ER lipid bilayer. 6 The multistep folding process is surprisingly inefficient, with misfolding occurring in more than one-half of wild-type CFTR. 7,8 Misfolded CFTR is tagged by a protein quality control system, the ER-associated degradation (ERAD) process. 7-9 ERAD mainly involves the ubiquitin proteasome system (UPS), and CFTR is a substrate for the UPS. 10 The UPS asserts quality control over maturing CFTR polypeptide at a minimum of two checkpoints: in the ER membrane and, subsequently, in the Figure 1. Model of the proposed structural domains of cystic fibrosis transmembrane conductance regulator (CFTR). The entire protein encompasses 1,480 amino acids with both of the amino and carboxy termini within the cell cytoplasm. The two MSDs each contain six transmembrane segments, forming the channel through which the Cl 2 ion passes. Note that there are a total of six extracellular loops and that the final glycosylation modifications occur on asparagine residues in extracellular loop 4. Also shown are the two NBDs and the single regulatory domain. F508del mutation occurs within NBD1. Phosphorylation of the R domain is required for normal channel function. ATP 5 adenosine triphosphate; Cl 2 5 chloride; COOH 5 carboxyl-terminus; MSD 5 membrane spanning domain; NBD 5 nucleotide-binding domain; NH 2 5 amino terminus. cytoplasmic compartment Ubiquitinization entails stepwise covalent attachment of the ubiquitin monomeric 76-amino acid peptide by ubiquitinylating enzymes, which marks the defective protein for degradation. The UPS sorts, keeps soluble, and prevents toxic precipitation of nascent misfolded proteins then ultimately transfers them to the cytoplasmic 26S proteasome system for degradation Within the ER lumen, membrane-based calnexin, calreticulin, and other chaperones mediate correct CFTR cotranslational folding. 17,18 These chaperones also coordinate with specific proteins within the ERAD pathway, such as mannose-binding ER degradation enhancing and mannoside-like protein, Derlin-1, and Hsc70, to reverse translocate misfolded CFTR toward degradation. 19,20 Posttranslational transport in the ER and, subsequently, cytoplasmic transfer of CFTR to the Golgi apparatus also are coordinated by a list of chaperone compounds (early, Hsp70 and Hsc70; later, Hsp90 and Hdj-2) whose exact role in the forward translocation machinery continues to be defined In vitro data suggest that export of correctly folded CFTR from the ER involves another conformationdependent step: the binding of a diacidic acid moiety to the coat protein complex II (COPII). 25,26 This complex, highly regulated, and interactive process for maintaining proper cellular CFTR protein folding and location, conformation, and protein-protein binding interactions represents an example of a proteostasis CHEST / 139 / 6 / JUNE,

3 pathway or network that scientists have begun to also adapt for a vast array of other metabolic, degenerative, and oncologic diseases. 27,28 Final CFTR processing occurs in the Golgi with the conversion from mannose-enriched to a mature complex oligosaccharide side chain attached at asparagine residues in the fourth extracellular loop within MSD2 ( Fig 1 ). 7,8 Western blot analysis is a widely used laboratory technique where antibodies specific for CFTR can verify appropriate folding and maturation. The relative optical density and migration distance of specific bands correlate with the amount of CFTR that achieves the final glycosylation step. Migrating more slowly, band C represents the mature, fully glycosylated, 180-kDa CFTR complex, which is easily distinguished from band B representing the less complex mannose core-glycosylated form. 29,30 From the Golgi, clathrin-coated vesicles shuttle mature CFTR to the apical membrane. Once situated in the plasma membrane, CFTR turns over at a rate of 10% per minute, as do most of the plasma membrane proteins, and has a half-life of approximately Figure 2. Normal CFTR synthesis and trafficking in airway epithelium. On the left is a transmission electron micrograph image of human airway epithelia grown in culture at the air-liquid interface (scale bar 5 1 m m). On the right is a depiction of a single epithelial cell to illustrate CFTR synthesis, trafficking, quality control, and the approximate locations where the five classes of CFTR mutations occur (each CFTR mutation class is denoted by a corresponding roman numeral). mrna is transcribed from the CFTR gene. The bulk of CFTR protein translation occurs within the ER lumen where correct cotranslational folding is mediated by ER membrane-associated and other chaperone proteins. CFTR misfolding occurs frequently, and misfolded CFTR is bound by specific chaperone proteins that escort it through the ER-associated degradation process, ubiquitinization, and then degradation in the ubiquitin proteasome system. Properly folded CFTR leaves the ER through coat protein complex II, is carried by cytoplasmic chaperone proteins, and is transported to the Golgi apparatus for final conversion to the fully glycosylated protein complex. Subsequently, clathrin-coated vesicles shuttle CFTR to the apical plasma membrane. The quality control system that monitors membrane-associated CFTR includes clathrin-coated endosomal recycling and lysosomal degradation. Chr 5 chromosome; ER 5 endoplasmic reticulum; mrna 5 messenger RNA. See Figure 1 legend for expansion of the other abbreviation. 12 to 24 h. 8,31 Internalization of the mature CFTR glycoprotein appears to proceed through clathrin-coated endosomes. 32 Another layer of quality control exists within the membrane pool to clear senescent or poorly functioning CFTR. Plasma membrane CFTR quality control involves, sequentially, recognition by chaperone Hsc70; ubiquitinization; and internalization within endosomes, which commits CFTR to either recycling back to the plasma membrane or degradation within a lysosome. 33,34 The details of this process remain to be more definitively elucidated, but evidence indicates that when F508del-CFTR is successfully manipulated to the plasma membrane, it generally exhibits a shorter half-life than wild-type CFTR because of either more rapid shuttling to endosomes destined for lysosomal degradation through ubiquitin-dependent pathways or, in some cases, defective recycling. 31,34-36 Chloride transport by CFTR situated in the apical plasma membrane requires interaction among multiple domains. Opening the channel requires phosphorylation within the R domain by PKA, which alters the R domain interaction with other domains, particularly NBD Binding of ATP at the interface between NBD1 and NBD2 also promotes the openchannel conformation. 40,41 ATP activity promotes dissociation of the NBDs, resulting in a return to the closed configuration More expansive discussion of CFTR cell biology has been recently published elsewhere. 5,14 The remarkable and persistent effort over the past 20 years has contributed much to our understanding of CFTR molecular biology. This work has allowed clinical investigators to begin realistically translating the findings into specific therapies targeting defects in CFTR biosynthesis, processing, and function. Correctors of Trafficking, Potentiators of Function, and Overcoming Premature Termination Codons F508del, the missense mutation causing a phenylalanine deletion at position 508 in the CFTR protein, accounts for approximately 70% of all CF alleles and is found in up to 90% of patients with CF in some populations. 45,46 In addition to the high frequency of the F508del mutation, two other facts make corrective and potentiating therapies strategically relevant. First, F508del-CFTR retains function (albeit reduced relative to wild-type CFTR) when delivered to the apical plasma membrane. 29,47,48 Second, several studies suggested that as little as 5% to 15% of native functional CFTR needs to be expressed to reinstate epithelial chloride transport Emerging mutation-targeted therapies carry descriptive names, which are based on the biosynthetic and cellular defects that they 1482 Recent Advances in Chest Medicine

4 overcome. For example, those that reroute misfolded F508del-CFTR to the plasma membrane are called correctors. F508del-CFTR and other specific mutant CFTR proteins once successfully inserted in the apical plasma membrane exhibit suboptimal gating and activation relative to wild-type CFTR. Potentiators refer to compounds that improve function and extend the half-life of apical membranesituated F508del-CFTR and other mutant CFTRs inserted in the apical membrane. Beyond therapies targeting F508del-CFTR, drugs aimed at other relatively common mutations also are emerging, such as one that overcomes premature termination codons (PTCs) in translation. To illustrate the impact of these mutation-specific therapies, we first review the five classes of CFTR mutations and the resultant aberrant CFTR processing. Five Classes of Defective CFTR Protein Processing Based on Gene Mutation Although. 1,500 mutations of CFTR have been identified, only four specific mutations besides F508del reach a frequency of 1% to 3%: G551D, W1282X, G542X, and N1303K. In fact, only about 20 specific mutations reach a threshold frequency. 0.1% (Cystic Fibrosis Genetic Analysis Consortium database, Specific mutations appear to be enriched within ethnic groups. 53 For example, the nonsense mutation, W1282X (a PTC in place of tryptophan residue 1282) accounts for slightly. 1% of worldwide mutations but for approximately 50% of the those in the Israeli Ashkenazi Jewish population where nonsense mutations nearly exceed the frequency of F508del. 54 Ultimately, the majority of CF mutations are rare and have not been functionally characterized. The most common CF mutations, representing about 80% of patients, may be divided into five different classes ( Table 1 ) based on their effects on CFTR transcription, cellular processing, concentration, and function. 53,55,56 In this classification system, although useful for organizing the various mutations, it is important to point out that many of the mutations can lead to more than one defect and extend across several classes. Class I Mutations Nonsense mutations and splice defects that interrupt CFTR biosynthesis characterize class I CF mutation. In particular, nonsense mutations, a single point alteration in DNA that creates an in-frame PTC (UAA, UGA, or UAG) in the protein-coding region, have been reported to cause approximately 30% of all human inherited diseases. 57 The designation for nonsense mutation ends with an X (eg, W1282X). The Table 1 Overview of the Five Classes of CFTR Mutations Relevant Agents in Clinical Trials Functional Consequence CFTR Protein Outcome Associated Phenotype Severity CFTR Domain Location Common Representative Mutations Predominant Mutation Type Approximate Worldwide Frequency Class High No CFTR No Cl 2 transport PTC read-through (eg, ataluren) NBD1, NBD2, MSD1 I? 10% (Ashkenazi, 50%) Nonsense, splice G542X, W1282X, G T II 70% Missense F508del, N1303K NBD1, NBD2 High Defective processing No Cl 2 transport Correctors (eg, VX-809) III 2%-3% Missense G551D, R560T NBD1, NBD1 High Defective regulation No Cl 2 transport Potentiators (eg, VX-770) Potentiators IV Uncertain,, 2% Missense R117H, R347P MSD1, MSD1 Reduced Altered conductance Minimal expression & Cl 2 transport Uncertain V Uncertain,, 1% Missense, splice kbC G Intron Reduced Reduced synthesis Reduced expression & Cl 2 transport CFTR 5 cystic fibrosis transmembrane conductance regulator; Cl 2 5 chloride; MSD 5 membrane spanning domain; NBD 5 nucleotide-binding domain; PTC 5 premature termination codon. CHEST / 139 / 6 / JUNE,

5 consequent premature cessation of translation produces unstable truncated and nonfunctional proteins that are typically degraded before they can reach the cell membrane. Nonsense mutations account for about 10% of all CF mutations worldwide. Class II Mutations F508del is the prime example of a class II mutation. Mutations in this class mostly give rise to misfolded CFTR, resulting in premature degradation as described earlier. However, F508del-CFTR can exemplify more than one classification. When F508del-CFTR is rescued and inserted in the plasma membrane, it exhibits defective regulation, characteristic of class III mutations. 58 Class III Mutations G551D is the third most common CFTR mutation (occurring in approximately 3% of patients with CF) and provides a representative example of class III mutations. The G551D missense mutation causes a glycine-to-aspartate substitution at residue 551. G551D-CFTR is adequately folded and inserts appropriately into the plasma membrane, but thereafter, it fails to open because of defective regulation. The defective site is localized in NBD1 ( Fig 1 ) and results in interference with NBD1 and NBD2 heterodimerization, ATP binding, and hydrolysis. 42,59 Class III CFTR mutations typically involve defects in CFTR regulation by ATP and phosphorylation. Class IV Mutations CFTR that results from a class IV mutation inserts into the plasma membrane but exhibits reduced single-channel chloride ion conductance because of reduced chloride permeation and open channel probability. R117H, among the most common class IV mutations, occurs at a worldwide frequency approaching 0.5% ( The R117H missense mutation causes an arginine-to-histidine substitution at residue 117. R117H-CFTR R domain is normally phosphorylated, and the NBD binds ATP, but channel open time and thus chloride transport are reduced.60 Additionally, the degree of R117H-CFTR function depends on the length of the polythymidine tract in intron 8 on the same chromosome (which influences splicing efficiency) such that the longer thymidine tracts (9T. 7T. 5T) produce more functional R117H-CFTR. Clinical disease typically requires the R117H mutation in cis with 5T. 61 Class V Mutations Found in, 1% of patients with CF, class V mutations produce normal plasma membrane CFTR. The quantity, however, generally is reduced as a result of transcriptional dysregulation. Class V mutations frequently influence the splicing machinery and generate both aberrantly and correctly spliced mrna, the levels of which vary among different patients and even among different organs of the same patients. Ultimately, the splice variants result in a reduced number of functioning CFTR in the plasma membrane. 62 Phenotypic Variation When considering a uniformly homozygous F508del population, in general, the phenotype is severe, exhibiting pancreatic insufficiency and relentless progressive bronchiectasis. However, significant numbers of outliers with variable clinical severity exist. Variation can be attributed to the environment, the quantity of retained CFTR function, adherence to therapies, and genetic modifiers. 63 Environmental variables include the advent of coordinated multidisciplinary CF care and enhanced mucous clearance, pancreatic enzyme replacement, nutrition supplementation, and antibiotic therapy, which all have been justifiably connected to the steady improvement in survival over the past 5 decades. 64 Data also support the hypothesis of a small subset of F508del homozygous individuals with milder pulmonary phenotype attributable to measurable levels of F508del-CFTR at the apical membrane. Investigators can detect low-level cyclic adenosine monophosphatedependent nasal epithelial chloride conductance consistent with limited functional F508del-CFTR at the epithelial surface that is not present in F508del- CFTR homozygotes with more severe phenotype, yet both groups demonstrate equal CFTR transcript levels by reverse transcription-polyermase chain reaction. 65 Confirming the existence of modifier genes and how they factor in the severity of the final phenotype is beyond the scope of this discussion and remains an enticing focus of study because better understanding holds promise for new treatments. Mutation Class and Prognostication Certain generalizations about variable organ dysfunction traditionally have been linked to genotype severity. For example, almost all men with CF are infertile and, thus, the vas deferens generally are believed to be the most sensitive human organ to the CF genotype. Similarly, CF-related liver disease, exocrine pancreatic insufficiency, and malnutrition are more common in nonfunctional CFTR mutations. The pancreas, however, is somewhat less sensitive to loss of CFTR function. Individuals with CF with milder genotypes exhibit pancreatic sufficiency (approximately 10%). The lung phenotype appears to 1484 Recent Advances in Chest Medicine

6 be more susceptible to CFTR-independent factors; however, many pancreatic-sufficient patients with CF have milder lung involvement. It is tempting for clinicians to use the five mutation classifications to more specifically prognosticate for individual patients. Mutations belonging to classes I to III when homozygous generally give rise to the classic CF phenotype. Meconium ileus affects about 15% of newborns with CF and is seen with higher frequency in class I to III mutations. 66 Similarly CF-related liver disease, exocrine pancreatic insufficiency, and malnutrition are more common. The degree of lung disease associated with class I to III mutations can be highly variable, although the overall tendency is for rapid decline in lung function. In contrast, patients whose genotype is homozygous for class IV or V mutations exhibit a less severe phenotype. Furthermore, class IV and V mutations are phenotypically dominant when occurring with class I to III mutations. Low-level CFTR function associated with these latter two classes contributes to the milder phenotypes, such as pancreatic sufficiency; less severe lung disease; and longevity. 55,56 Generally, clinicians should avoid prognostication for individuals. For patients with class IV and V CFTR mutations, however, one could provide some reassurance about the possibility of a milder course with the caveat that even within these classes substantial outcome variability occurs. 62,67 Progress Toward Mutation-Specific Modifier Treatments Most of the current therapeutic strategies in use for CF involve treatments that alleviate symptoms and complications that result from loss of CFTR function, and consensus statements detailing these therapies have been published recently. 68,69 On the cutting edge of CF treatment, gene therapy continues to be pursued aggressively as evidenced by. 200 gene therapy trials undertaken since the 1990s. To date, effective and meaningful clinical outcomes remain elusive, and no gene therapies have received US Food and Drug Administration approval. 70 In this section, we review novel therapies that target specific mutation classes, arising out of the widening comprehension of the CFTR basic cell biology. These strategies represent a noteworthy paradigm shift in CF treatment. Along with the development of these new therapies has come the requirement to rely more heavily on functional CFTR measures, including the longstanding nasal transepithelial potential difference (NPD) and sweat chloride testing. For this class of therapeutics, NPD technology generally has been recruited to measure in vivo evidence for CFTRmediated chloride ion transport on nasal epithelial surfaces as a reliable surrogate for lower airway epithelium. High-Throughput Screening Technology High-throughput screening (HTS) has supplanted traditional screening methods where finding the most effective compound involves deriving structural analogs of a candidate compound and testing each individually in a model cell culture system. Automated HTS technology has the capacity to rapidly screen hundreds of thousands of candidate compounds. Several groups began evaluating whether HTS could more efficiently identify candidate small molecules that target CFTR mutations. HTS assays use multiwell plates containing select epithelial cell lines expressing mutant and wild-type CFTR and a detection system, such as anion flux, adapted to a mechanism for rapid identification (eg, fluorescence emission). The first-pass group of hits are further validated in secondary-level assays that more completely assess purity, toxicity, dose response, and electrophysiologic characteristics in epithelial cell culture systems. A handful of the most promising scaffold structures can be further optimized through reiterative medicinal chemistry then winnowed down on the basis of efficacy and toxicity in primary human bronchial epithelial (HBE) cell culture. Characteristics related to the predicted mechanism of action and anticipated pharmacokinetics can be clarified in these preclinical model systems in preparation for human trials.48,58,71-76 Each of the compounds highlighted in the following sections emerged from HTS technology and are progressing through early and late-phase clinical trials. Compounds Promoting Read-Through of PTCs Therapeutic options for patients carrying in-frame nonsense mutations (class 1) promote read-through of PTCs and enable translation of the entire sequence and synthesis of the full-length, mature CFTR protein. It was originally demonstrated from studies in bacteria that aminoglycoside antibiotics can suppress PTCs. 77 Gentamicin, in particular, showed promise in early cell culture, CF mouse models, and limited human clinical trials However, a recent randomized, double-blind, multicenter clinical trial of patients with CF with heterogeneous PTC mutations was unable to demonstrate significant alteration in NPD or nasal histology as evidence of CFTR plasma membrane expression after 28 days of nasally administered gentamicin or tobramycin. 83 Ultimately, HTS technology led to specific development of ataluren, a nonaminoglycoside, which reliably reads through PTCs but not normal stop codons, without evidence of renal toxicity. CHEST / 139 / 6 / JUNE,

7 Ataluren, developed by PTC Therapeutics, Inc (South Plainfield, New Jersey), is an orally bioavailable small molecule that proved superior to gentamicin in in vitro assays at inducing ribosomal read-through of in-frame nonsense PTCs to produce full-length proteins. 76 Ataluren does not exhibit any antibiotic activity, and studies in transgenic CF mouse possessing hg542x- CFTR revealed that ataluren is capable of enhancing production of the full-length CFTR protein. 84 Ataluren also passed requisite tolerability studies in healthy non-cf human volunteers apart from a mild elevation of hepatic transaminases with repeated dosing. 85 In a subsequent phase II prospective trial in Israel where nonsense CFTR mutations are prevalent (eg, W1282X), 23 adult patients with CF exhibiting mainly heterozygous nonsense mutations were administered ascending doses of ataluren in two 2-week cycles (with a 2-week interval washout). The study found that mean CFTR-dependent chloride transport, as assessed by NPD, increased in the first treatment phase, with a change of mv (SD, 7.0 mv; P,.0001), and in the second phase, with a change of mv (SD, 7.3 mv; P 5.032). Sixteen of the 23 patients in the first cycle s treatment phase demonstrated a response in total chloride transport ( P,.0001), which was defined as a change in NPD of 2 5 mv or more and was replicated in eight of the 21 patients in the second cycle ( P,.0001). Remarkably, chloride transport normalized in more than onehalf of the patients in the first-cycle treatment phase ( P ) and for nine of 21 in the second cycle ( P 5.02). Overall, the drug was found to be safe and well tolerated, and side effects generally were mild. Despite some recent concern regarding the validity of the initial screening assay used during the HTS identification of ataluren, 86 this study in adults with CF as well as a recently published pediatric (6-18 years) study 87 demonstrated that ataluren successfully reduces the epithelial electrophysiologic abnormalities caused by nonsense mutations. 88 A phase III trial of ataluren has been initiated, which will assess long-term efficacy and safety. Correctors of F508del-CFTR Intracellular Processing Correctors are believed to work by enhancing productive trafficking of the class II mutation F508del- CFTR.18,89 Correction of F508del-CFTR trafficking was initially achieved in vitro by culturing cells at a low temperature (27 C) 29 or by introducing an effective chemical chaperone, such as glycerol. 90 Such techniques, however, are not practical in patients. Clinically relevant correctors must be nontoxic compounds that can overcome misfolding or, alternatively, alter or bypass ERAD quality control steps. Prior in vitro studies showed that many ER lumen chaperones are calcium-dependent proteins and when the sarcoplasmic/endoplasmic reticulum calcium (SERCA) pump is inhibited, ER calcium concentrations are reduced, with substantially more F508del-CFTR diverted to the plasma membrane. 91 Curcumin, derived from turmeric spice, is a SERCA pump inhibitor and being nontoxic, appeared to be a good candidate corrector. Early studies showed it could correct NPD in a F508del mouse model. 92 Subsequent trials of curcumin using human CF airway epithelial cells, however, failed to replicate the promising mouse data. 93,94 The butyrate class of compounds effectively correct F508del processing, transport, and function in vitro. Sodium-4-phenylbutyrate, an orally bioavailable short-chain fatty acid, specifically upregulates the key cytoplasmic chaperone hsp70, which restores maturation of F508del-CFTR in vitro and in vivo. Although a phase I/II trial showed favorable effects on nasal ion transport in young adult patients with CF, it was found subsequently that the high doses required produced intolerable adverse effects Experiences such as these with curcumin and phenylbutyrate increased the impetus to use HTS techniques to find more potent correctors with encouraging preclinical pharmacokinetic profiles. KM11096 (analog of sildenafil), corr-2b and corr-4a (thiazole compounds), VRT-325 (quinazoline compound), and VX-809 (Vertex Pharmaceuticals, Inc; Cambridge, Massachusetts) are all examples of correctors identified using HTS technology Among these, the orally bioavailable compound VX-809 has proceeded into phase II clinical trials in adult patients with CF. Western blot analysis of electrophoresed F508del HBE cell lysates after treatment with VX-809 demonstrated measurable accumulation of band C, providing direct evidence for maturation of F508del-CFTR. Additionally, Ussing chamber studies using the same cells treated with VX-809 showed increased chloride secretion to levels approximately 15% of that seen in normal HBE cells, achieving the minimal threshold (5%-15%) associated with less severe disease manifestations. 104 Preliminary results of the phase II clinical trial in adults with F508del homozygous CF showed that VX-809 was well tolerated when taken once daily over 28 days and showed statistically significant lowmagnitude reductions in sweat chloride in the two highest dose cohorts. Although NPD changes were not detected, the promising data for VX-809 provide support for more clinical trials. 105 Potentiators of Mutant CFTR Protein Potentiator compounds increase chloride ion flow of PKA-activated mutant CFTR proteins that are 1486 Recent Advances in Chest Medicine

8 present in the plasma membrane, such as those expressing class III and IV mutational defects. Alternatively, potentiators can increase the ion flow of phosphorylated F508del-CFTR (class II mutation) once it has become situated in the plasma membrane. A large number of compounds can function as potentiators of cell surface-expressed F508del-CFTR and include alkylxanthines such as 8-cyclopentyl-1, 3-dipropylxanthine, the flavanoid genistein, derivatives of pyrrolo[2,3-b]pryazines, sulfonamide, phenylglycine, and benzothiphene None of these compounds has progressed substantially toward realistic use in patients with CF. In contrast, VX-770 (Vertex Pharmaceuticals), a product of HTS technology and a potent potentiator of G551D-CFTR, recently has progressed into late-phase clinical trials. VX-770 is a dihydroquinoline-carboxamide compound with oral bioavailability. HBE cells expressing G551D-CFTR treated with VX-770 demonstrated increased open probability and transepithelial current. Additionally, VX-770 increased chloride secretion from 5% to 50% of levels achieved in wild-type CFTR HBE cells, decreased excessive amiloridesensitive short-circuit current, and increased ciliary beat frequency within G551D/F508del HBE cells. 110 Recently published results from a phase IIa randomized, double-blind, placebo-controlled study of twice daily oral VX-770 therapy in 19 adult patients with CF with G551D mutation revealed only a few mild or moderate adverse events and no serious adverse events. 111 The majority (84%) of patients enrolled were compound heterozygous (G551D/F508del-CFTR). For those treated with 28 days of VX-770, withinsubject comparisons showed statistically significant increases in FEV 1 (1 8.7%), reduction in sweat chloride ( 2 59 mmol/l), and increased NPD zero chloride/ isoproteronol response. 111 These very favorable results have prompted further evaluation in larger phase III trials that also include younger patients with CF. Combination of Potentiator and Corrector Therapies The positive outcomes of these investigations have allowed investigators to consider the impact of combining therapy to enhance mutant CFTR function. For example, in F508del HBE cells, the level of VX-809 corrected F508del-CFTR function can be doubled by adding the potentiator VX The latter observation provides preclinical proof of principal for the development of clinical trials that combine corrector and potentiator therapies to obtain enhanced effect. Concluding Perspectives Mutation-specific treatments hold promise for finally answering the question that has been lingering since and even before the CFTR gene discovery: Will therapies that specifically restore CFTR-mediated chloride secretion slow or arrest the deleterious cascade of events leading to chronic infection, bronchiectasis, and end-stage lung disease? The successes reported from early phase trials translating mutationspecific approaches into measurable patient outcomes have created much excitement, and we may be on the brink of an affirmative answer. Such treatments that repair a flawed protein due to specific gene mutations may realize the concept of personalized medicine: subsets of patients with CF specifically selected for targeted therapy based on CFTR genotype information (eg, G551D mutation). 112 Ultimately, enthusiasm for this novel class of treatments must be balanced by the reality that long-term efficacy and safety data have yet to be established, and it could take several more years before any of these agents become part of the therapeutic arsenal in CF. Furthermore, we hasten to point out that these interventions will not likely reverse the established lung disease in thousands of patients with CF and advanced bronchiectasis. Current treatments as well as new developments broadly aimed at alleviating established symptoms and complications will remain a necessary part of care for patients with CF. Acknowledgments Financial/nonfinancial disclosures: The authors have reported to CHEST the following conflicts of interest: Dr Hornick has participated in enrolling patients in early phase clinical trials of Vertex Pharmaceuticals products and has participated in the writing of manuscripts and abstracts describing results of studies. Drs Rogan and Stoltz have reported that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article. Role of sponsors: The sponsor had no role in the design of the study, the collection and analysis of the data, or in the preparation of the manuscript. Other contributions: We thank Lynda Ostedgaard, PhD, and Michael Welsh, MD, for discussions regarding the manuscript and Phil Karp, BS, and Thomas Moninger, BS, for preparation of the human airway electron microscopic image. References 1. Boat TF, Welsh MJ, Beaudet AL. Cystic fibrosis. In: Scriver CR, ed. The Metabolic Basis of Inherited Disease. New York, NY : McGraw-Hill ; 1989 : Davis PB, Drumm M, Konstan MW. Cystic fibrosis. Am J Respir Crit Care Med ;154(5): Riordan JR, Rommens JM, Kerem B, et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science ;245(4922): Quinton PM. Cystic fibrosis: impaired bicarbonate secretion and mucoviscidosis. Lancet ;372(9636): Riordan JR. CFTR function and prospects for therapy. Annu Rev Biochem ;77: Gelman MS, Kopito RR. Cystic fibrosis: premature degradation of mutant proteins as a molecular disease mechanism. Methods Mol Biol ;232: CHEST / 139 / 6 / JUNE,

9 7. Lukacs GL, Mohamed A, Kartner N, Chang XB, Riordan JR, Grinstein S. Conformational maturation of CFTR but not its mutant counterpart (delta F508) occurs in the endoplasmic reticulum and requires ATP. EMBO J ; 13 ( 24 ): Ward CL, Kopito RR. Intracellular turnover of cystic fibrosis transmembrane conductance regulator. Inefficient processing and rapid degradation of wild-type and mutant proteins. J Biol Chem ;269 (41 ): Ward CL, Omura S, Kopito RR. Degradation of CFTR by the ubiquitin-proteasome pathway. Cell ;83 (1 ): Cheung JC, Deber CM. Misfolding of the cystic fibrosis transmembrane conductance regulator and disease. Biochemistry ;47 (6 ): Meacham GC, Patterson C, Zhang W, Younger JM, Cyr DM. The Hsc70 co-chaperone CHIP targets immature CFTR for proteasomal degradation. Nat Cell Biol ;3 (1 ): Younger JM, Ren HY, Chen L, et al. A foldable CFTRDeltaF508 biogenic intermediate accumulates upon inhibition of the Hsc70-CHIP E3 ubiquitin ligase. J Cell Biol ;167 (6 ): Younger JM, Chen L, Ren HY, et al. Sequential qualitycontrol checkpoints triage misfolded cystic fibrosis transmembrane conductance regulator. Cell ;126 (3 ): Turnbull EL, Rosser MF, Cyr DM. The role of the UPS in cystic fibrosis. BMC Biochem ;8 (suppl 1 ):S Gelman MS, Kannegaard ES, Kopito RR. A principal role for the proteasome in endoplasmic reticulum-associated degradation of misfolded intracellular cystic fibrosis transmembrane conductance regulator. J Biol Chem ;277 (14 ): Davies JE, Sarkar S, Rubinsztein DC. The ubiquitin proteasome system in Huntington s disease and the spinocerebellar ataxias. BMC Biochem ;8 (suppl 1 ):S Harada K, Okiyoneda T, Hashimoto Y, et al. Calreticulin negatively regulates the cell surface expression of cystic fibrosis transmembrane conductance regulator. J Biol Chem ;281 (18 ): Rosser MF, Grove DE, Chen L, Cyr DM. Assembly and misassembly of cystic fibrosis transmembrane conductance regulator: folding defects caused by deletion of F508 occur before and after the calnexin-dependent association of membrane spanning domain (MSD) 1 and MSD2. Mol Biol Cell ;19 (11 ): Gnann A, Riordan JR, Wolf DH. Cystic fibrosis transmembrane conductance regulator degradation depends on the lectins Htm1p/EDEM and the Cdc48 protein complex in yeast. Mol Biol Cell ;15 (9 ): Sun F, Zhang R, Gong X, Geng X, Drain PF, Frizzell RA. Derlin-1 promotes the efficient degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) and CFTR folding mutants. J Biol Chem ; 281 ( 48 ): Alberti S, Böhse K, Arndt V, Schmitz A, Höhfeld J. The cochaperone HspBP1 inhibits the CHIP ubiquitin ligase and stimulates the maturation of the cystic fibrosis transmembrane conductance regulator. Mol Biol Cell ; 15 ( 9 ): Wang X, Venable J, LaPointe P, et al. Hsp90 cochaperone Aha1 downregulation rescues misfolding of CFTR in cystic fibrosis. Cell ;127 (4 ): Sun F, Mi Z, Condliffe SB, et al. Chaperone displacement from mutant cystic fibrosis transmembrane conductance regulator restores its function in human airway epithelia. FASEB J ;22 (9 ): Glozman R, Okiyoneda T, Mulvihill CM, Rini JM, Barriere H, Lukacs GL. N-glycans are direct determinants of CFTR folding and stability in secretory and endocytic membrane traffic. J Cell Biol ;184 (6 ): Wang X, Matteson J, An Y, et al. COPII-dependent export of cystic fibrosis transmembrane conductance regulator from the ER uses a di-acidic exit code. J Cell Biol ;167 (1 ): Pagant S, Kung L, Dorrington M, Lee MC, Miller EA. Inhibiting endoplasmic reticulum (ER)-associated degradation of misfolded Yor1p does not permit ER export despite the presence of a diacidic sorting signal. Mol Biol Cell ;18 (9 ): Koulov AV, Lapointe P, Lu B, et al. Biological and structural basis for Aha1 regulation of Hsp90 ATPase activity in maintaining proteostasis in the human disease cystic fibrosis. Mol Biol Cell ;21 (6 ): Balch WE, Morimoto RI, Dillin A, Kelly JW. Adapting proteostasis for disease intervention. Science ;319 (5865 ): Denning GM, Anderson MP, Amara JF, Marshall J, Smith AE, Welsh MJ. Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive. Nature ;358 (6389 ): Cheng SH, Gregory RJ, Marshall J, et al. Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell ;63 (4 ): Lukacs GL, Chang XB, Bear C, et al. The delta F508 mutation decreases the stability of cystic fibrosis transmembrane conductance regulator in the plasma membrane. Determination of functional half-lives on transfected cells. J Biol Chem ;268 (29 ): Lukacs GL, Segal G, Kartner N, Grinstein S, Zhang F. Constitutive internalization of cystic fibrosis transmembrane conductance regulator occurs via clathrin-dependent endocytosis and is regulated by protein phosphorylation. Biochem J ;328 (pt 2 ): Sharma M, Pampinella F, Nemes C, et al. Misfolding diverts CFTR from recycling to degradation: quality control at early endosomes. J Cell Biol ;164 (6 ): Okiyoneda T, Barrière H, Bagdany M, et al. Peripheral protein quality control removes unfolded CFTR from the plasma membrane. Science :329 (5993 ): Chang XB, Mengos A, Hou YX, et al. Role of N-linked oligosaccharides in the biosynthetic processing of the cystic fibrosis membrane conductance regulator. J Cell Sci ;121 (pt 17 ): Swiatecka-Urban A, Brown A, Moreau-Marquis S, et al. The short apical membrane half-life of rescued DeltaF508-cystic fibrosis transmembrane conductance regulator (CFTR) results from accelerated endocytosis of DeltaF508-CFTR in polarized human airway epithelial cells. J Biol Chem ;280 (44 ): Winter MC, Welsh MJ. Stimulation of CFTR activity by its phosphorylated R domain. Nature ;389 (6648 ): Csanády L, Seto-Young D, Chan KW, et al. Preferential phosphorylation of R-domain Serine 768 dampens activation of CFTR channels by PKA. J Gen Physiol ;125 (2 ): Baker JM, Hudson RP, Kanelis V, et al. CFTR regulatory region interacts with NBD1 predominantly via multiple transient helices. Nat Struct Mol Biol ;14 (8 ): Anderson MP, Berger HA, Rich DP, Gregory RJ, Smith AE, Welsh MJ. Nucleoside triphosphates are required to open the CFTR chloride channel. Cell ;67 (4 ): Anderson MP, Welsh MJ. Regulation by ATP and ADP of CFTR chloride channels that contain mutant nucleotidebinding domains. Science ;257 (5077 ): Lewis HA, Buchanan SG, Burley SK, et al. Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductance regulator. EMBO J ;23 (2 ): Recent Advances in Chest Medicine

10 43. Vergani P, Basso C, Mense M, Nairn AC, Gadsby DC. Control of the CFTR channel s gates. Biochem Soc Trans ;33 (pt 5 ): Aleksandrov AA, Aleksandrov LA, Riordan JR Jr. CFTR (ABCC7) is a hydrolyzable-ligand-gated channel. Pflugers Arch ;453 (5 ): Bobadilla JL, Macek M Jr, Fine JP, Farrell PM. Cystic fibrosis: a worldwide analysis of CFTR mutations correlation with incidence data and application to screening. Hum Mutat ;19 (6 ): Worldwide survey of the delta F508 mutation report from the Cystic Fibrosis Genetic Analysis Consortium. Am J Hum Genet ;47 (2 ): Brown CR, Hong-Brown LQ, Biwersi J, Verkman AS, Welch WJ. Chemical chaperones correct the mutant phenotype of the delta F508 cystic fibrosis transmembrane conductance regulator protein. Cell Stress Chaperones ;1 (2 ): Pedemonte N, Lukacs GL, Du K, et al. Small-molecule correctors of defective DeltaF508-CFTR cellular processing identified by high-throughput screening. J Clin Invest ;115 (9 ): Dorin JR, Farley R, Webb S, et al. A demonstration using mouse models that successful gene therapy for cystic fibrosis requires only partial gene correction. Gene Ther ; 3 (9 ): Johnson LG, Olsen JC, Sarkadi B, Moore KL, Swanstrom R, Boucher RC. Efficiency of gene transfer for restoration of normal airway epithelial function in cystic fibrosis. Nat Genet ;2 (1 ): Ramalho AS, Beck S, Meyer M, Penque D, Cutting GR, Amaral MD. Five percent of normal cystic fibrosis transmembrane conductance regulator mrna ameliorates the severity of pulmonary disease in cystic fibrosis. Am J Respir Cell Mol Biol ;27 (5 ): Farmen SL, Karp PH, Ng P, et al. Gene transfer of CFTR to airway epithelia: low levels of expression are sufficient to correct Cl- transport and overexpression can generate basolateral CFTR. Am J Physiol Lung Cell Mol Physiol ; 289 (6 ):L1123-L Castellani C, Cuppens H, Macek M Jr, et al. Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice. J Cyst Fibros ;7 (3 ): Kerem B, Chiba-Falek O, Kerem E. Cystic fibrosis in Jews: frequency and mutation distribution. Genet Test ; 1 (1 ): Welsh MJ, Smith AE. Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis. Cell ; 73 (7 ): Wilschanski M, Zielenski J, Markiewicz D, et al. Correlation of sweat chloride concentration with classes of the cystic fibrosis transmembrane conductance regulator gene mutations. J Pediatr ;127 (5 ): Frischmeyer PA, Dietz HC. Nonsense-mediated mrna decay in health and disease. Hum Mol Genet ; 8 ( 10 ): Van Goor F, Straley KS, Cao D, et al. Rescue of DeltaF508- CFTR trafficking and gating in human cystic fibrosis airway primary cultures by small molecules. Am J Physiol Lung Cell Mol Physiol ;290 (6 ):L1117-L Hopfner KP, Karcher A, Shin DS, et al. Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily. Cell ;101 (7 ): Sheppard DN, Rich DP, Ostedgaard LS, Gregory RJ, Smith AE, Welsh MJ. Mutations in CFTR associated with mild-diseaseform Cl- channels with altered pore properties. Nature ;362 (6416 ): Kiesewetter S, Macek M Jr, Davis C, et al. A mutation in CFTR produces different phenotypes depending on chromosomal background. Nat Genet ;5 (3 ): Kerem E. Pharmacological induction of CFTR function in patients with cystic fibrosis: mutation-specific therapy. Pediatr Pulmonol ;40 (3 ): Kerem E, Corey M, Kerem BS, et al. The relation between genotype and phenotype in cystic fibrosis analysis of the most common mutation (delta F508). N Engl J Med ; 323 (22 ): Davis PB. Cystic fibrosis since Am J Respir Crit Care Med ;173 (5 ): Sermet-Gaudelus I, Vallée B, Urbin I, et al. Normal function of the cystic fibrosis conductance regulator protein can be associated with homozygous (Delta)F508 mutation. Pediatr Res ;52 (5 ): Allan JL, Robbie M, Phelan PD, Danks DM. Familial occurrence of meconium ileus. Eur J Pediatr ;135 (3 ): McKone EF, Goss CH, Aitken ML. CFTR genotype as a predictor of prognosis in cystic fibrosis. Chest ;130 (5 ): Yankaskas JR, Marshall BC, Sufian B, Simon RH, Rodman D. Cystic fibrosis adult care: consensus conference report. Chest ;125 (1 suppl ):1S-39S. 69. Flume PA, O Sullivan BP, Robinson KA, et al ; Cystic Fibrosis Foundation, Pulmonary Therapies Committee. Cystic fibrosis pulmonary guidelines: chronic medications for maintenance of lung health. Am J Respir Crit Care Med ;176 (10 ): Flotte TR, Laube BL. Gene therapy in cystic fibrosis. Chest ;120 (3 suppl ):124S-131S. 71. Carlile GW, Robert R, Zhang D, et al. Correctors of protein trafficking defects identified by a novel high-throughput screening assay. ChemBioChem ;8 (9 ): Galietta LJ, Haggie PM, Verkman AS. Green fluorescent protein-based halide indicators with improved chloride and iodide affinities. FEBS Lett ;499 (3 ): Galietta LV, Jayaraman S, Verkman AS. Cell-based assay for high-throughput quantitative screening of CFTR chloride transport agonists. Am J Physiol Cell Physiol ; 281 (5 ):C1734-C Galietta LJ, Springsteel MF, Eda M, et al. Novel CFTR chloride channel activators identified by screening of combinatorial libraries based on flavone and benzoquinolizinium lead compounds. J Biol Chem ;276 (23 ): Trzcinska-Daneluti A, Ly D, Huynh L, Jiang C, Fladd C, Rotin D. High-content functional screen to identify proteins that correct F508del-CFTR function. Mol Cell Proteomics ; 2008 :8 (4 ): Welch EM, Barton ER, Zhuo J, et al. PTC124 targets genetic disorders caused by nonsense mutations. Nature ;447 (7140 ): Gorini L, Kataja E. Phenotypic repair by streptomycin of defective genotypes in E. coli. Proc Natl Acad Sci U S A ; 51 : Burke JF, Mogg AE. Suppression of a nonsense mutation in mammalian cells in vivo by the aminoglycoside antibiotics G-418 and paromomycin. Nucleic Acids Res ; 13 (17 ): Bedwell DM, Kaenjak A, Benos DJ, et al. Suppression of a CFTR premature stop mutation in a bronchial epithelial cell line. Nat Med ;3 (11 ): Du M, Jones JR, Lanier J, et al. Aminoglycoside suppression of a premature stop mutation in a Cftr-/- mouse carrying a human CFTR-G542X transgene. J Mol Med ; 80 ( 9 ): CHEST / 139 / 6 / JUNE,