Cystic Fibrosis Transmembrane Conductance Regulator Intracellular Processing, Trafficking, and Opportunities for Mutation-Specific Treatment

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):1480–1490 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

fibrosis (CF) is an autosomal recessive Cystic inherited disorder characterized by elevated

sweat chloride concentrations, abnormal epithelial chloride or bicarbonate transport, pancreatic insufficiency, recurrent lung infection and chronic bronManuscript received August 12, 2010; revision accepted December 12, 2010. 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; e-mail: [email protected] 1480

chiectasis, 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 1989.3 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 © 2011 American College of Chest Physicians. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (http://www.chestpubs.org/ site/misc/reprints.xhtml). DOI: 10.1378/chest.10-2077 Recent Advances in Chest Medicine

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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 www.chestpubs.org

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 Cl2 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; Cl2 5 chloride; COOH 5 carboxyl-terminus; MSD 5 membrane spanning domain; NBD 5 nucleotide-binding domain; NH2 5 amino terminus.

cytoplasmic compartment.11-13 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.14-16 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.21-24 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, 2011

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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 mm). 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. 1482

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 NBD1.37-39 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.42-44 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.49-52 Emerging mutation-targeted therapies carry descriptive names, which are based on the biosynthetic and cellular defects that they Recent Advances in Chest Medicine

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Missense, splice Uncertain, , 1% V

CFTR 5 cystic fibrosis transmembrane conductance regulator; Cl2 5 chloride; MSD 5 membrane spanning domain; NBD 5 nucleotide-binding domain; PTC 5 premature termination codon.

Uncertain

Missense Uncertain, , 2% IV

3349110kbC→G

Intron

Reduced

Reduced synthesis

Minimal expression & Cl2 transport Reduced expression & Cl2 transport Altered conductance Reduced MSD1, MSD1

Missense Missense 70% 2%-3% II III

R117H, R347P

High High

Defective processing Defective regulation

No Cl2 transport No Cl2 transport

PTC read-through (eg, ataluren) Correctors (eg, VX-809) Potentiators (eg, VX-770) Potentiators No Cl2 transport No CFTR High

NBD1, NBD2, MSD1 NBD1, NBD2 NBD1, NBD1 Nonsense, splice ?10% (Ashkenazi, 50%) I

G542X, W1282X, 62111G→T F508del, N1303K G551D, R560T

Functional Consequence CFTR Protein Outcome Associated Phenotype Severity CFTR Domain Location Common Representative Mutations Class

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

Predominant Mutation Type

Class I Mutations

Approximate Worldwide Frequency

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, www.genet.sickkids.on.ca/cftr). 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.

Table 1—Overview of the Five Classes of CFTR Mutations

Five Classes of Defective CFTR Protein Processing Based on Gene Mutation

Relevant Agents in Clinical Trials

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.

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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% (www.genet.sickkids.on.ca). 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 1484

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 F508delCFTR 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 Recent Advances in Chest Medicine

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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 www.chestpubs.org

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.78-82 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, 2011

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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 hG542XCFTR 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 27.1 mV (SD, 7.0 mV; P , .0001), and in the second phase, with a change of 23.7 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 25 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 5 .0003) 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) study87 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 F508delCFTR.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. 1486

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.95-98 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.99-103 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 Recent Advances in Chest Medicine

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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.106-109 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 FEV1 (1 8.7%), reduction in sweat chloride (259 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-770.104 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 www.chestpubs.org

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.

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