Therapeutic approaches to repair defects in ΔF508 CFTR folding and cellular targeting

Advanced Drug Delivery Reviews 54 (2002) 13951408www.elsevier.com/ locate /drugdeliv

T herapeutic approaches to repair defects in DF508 CFTRfolding and cellular targeting

*Kristina Powell, Pamela L. ZeitlinThe Johns Hopkins University School of Medicine, 316 Johns Hopkins Hospital, 600 N. Wolfe St., Baltimore, MD 21287 USA

Received 28 February 2002; accepted 21 August 2002

Abstract

The DF508 mutation in the cystic fibrosis transmembrane regulator (CFTR) gene is the most common mutation in CF. Themutant CFTR protein is defective with respect to multiple functions including cAMP-regulated chloride conductance,nucleotide transport, and regulatory actions on other ion channels. Since the DF508 protein is also temperature-sensitive andunstable during translation and folding in the endoplasmic reticulum (ER), most of the nascent chains are targeted forpremature proteolysis from the ER. This paper focuses on the events that occur in the ER during folding and reviewspotential targets for therapeutic intervention. 2002 Elsevier Science B.V. All rights reserved.Keywords: Cystic fibrosis; Protein chaperone; Endoplasmic reticulum; Golgi apparatus; Ion channel; Butyrates; Xanthines; Phospho-diesterase inhibitors; Isoflavones

Contents

1 . Introduction ............................................................................................................................................................................ 13962 . Background ............................................................................................................................................................................ 1396

2 .1. The CF gene .................................................................................................................................................................... 13962 .2. CFTR.............................................................................................................................................................................. 13962 .3. Mutations in CFTR .......................................................................................................................................................... 1397

3 . Therapies directed at folding .................................................................................................................................................... 13983 .1. Kinetic therapies .............................................................................................................................................................. 13983 .2. Chemical chaperones........................................................................................................................................................ 13983 .3. Molecular chaperones....................................................................................................................................................... 13993 .4. Sodium-4-phenylbutyrate.................................................................................................................................................. 1400

4 . Therapies to augment conductance ........................................................................................................................................... 14024 .1. Xanthines ........................................................................................................................................................................ 14024 .2. Phosphodiesterase inhibitors ............................................................................................................................................. 14034 .3. Isoflavones (genistein, apigenin, kaempferol, quercetin) ...................................................................................................... 1403

5 . High throughput screening ....................................................................................................................................................... 14046 . Conclusions ............................................................................................................................................................................ 1404References .................................................................................................................................................................................. 1405

*Corresponding author. Tel.: 11-410-955-1167; fax: 11-410-955-1030.E-mail address: [email protected] (P.L. Zeitlin).

0169-409X/02/$ see front matter 2002 Elsevier Science B.V. All rights reserved.PI I : S0169-409X( 02 )00148-5

1396 K. Powell, P.L. Zeitlin / Advanced Drug Delivery Reviews 54 (2002) 13951408

1 . Introduction 2 . Background

Cystic fibrosis (CF), one of the most common 2 .1. The CF genelife-shortening inherited diseases in Caucasians, hasan incidence in the US of 1:3300 (carrier rate 1:29) In 1985, CF was linked to chromosome 7q31[1]. CF is an autosomal recessive monogenic disor- [12,13]. Later, Rommens et al. used chromosomeder resulting from over 900 mutations in the cystic walking and jumping to describe the CF locus andfibrosis transmembrance conductance regulator gene narrowed the search to an area of contiguous DNA(CFTR) on chromosome 7 (http: /www.genet.sic- consisting of 250 000 base pairs [14]. By mRNAkkids.on.ca /cftr). The CFTR is known to be a gel-blot hybridization, CF gene expression was de-temperature-sensitive, cyclic adenosine monophos- tected in the lung, colon, sweat glands, placenta,phate (cAMP)-mediated chloride channel [2] that is liver, parotid glands, pancreas, and in nasal polypfound in exocrine glands and secretory epithelia. The tissue [15]. Within the airways, expression is greatestCFTR directly regulates other ion channels, such as in the submucosal glands but present in the epithelialthe outwardly rectifying chloride channel (ORCC) layer as well [16,17]. Gene expression was not[3,4] and the amiloride-sensitive sodium channel detected in the adrenal gland, skin fibroblast or(ENaC) [5], thereby playing a major role in the lymphoblast cell lines [15].control of ion and water balance in body tissues.Elevated sweat chlorides, pancreatic exocrine insuf- 2 .2. CFTRficiency, and chronic sinopulmonary inflammationand infection are common features of this disease Riordan et al. described the product of chromo-[6]. some 7q31 as the cystic fibrosis transmembrane

Advances in molecular biology, cellular biology, conductance regulator protein (CFTR) in 1989 [15].and electrophysiology have furthered our knowledge It is a polypeptide of 1480 amino acids with aabout the consequences of mutations in different molecular mass of approximately 180 kDa whenregions of the CFTR. The most common mutation is fully glycosylated. The molecule consists of twothe DF508, which is found on 70% of CF alleles. repeated motifs, each containing six hydrophobicThe deletion of a phenylalanine at position 508 transmembrane domains and one hydrophilic in-results in an immature protein that is not fully tracellular nucleotide-binding fold (NBF) connectedglycosylated, and instead, is ubiquitinated and by a highly charged regulatory domain site. The twotargeted in the endoplasmic reticulum (ER) for motifs are not likely the result of exon duplication assubsequent proteolytic degradation [7]. there is only modest conservation of the sequences

Approximately 99% of the DF508 proteins are with the most conservation (27% identical) beingdegraded in a pre-Golgi compartment, thus never between the carboxy ends of the NBF. The structurereaching the cell surface [79]. However, a small is similar to others in the ABC protein family (ATPamount of DF508 CFTR has been shown to reach the binding cassette) except that it is unique in having aplasma membrane of certain tissues in vivo [10]. The regulatory domain and is thought to be an ionchloride channel is similar to wild type CFTR with channel and not a pump. The CFTR trafficks to therespect to anion selectivity, conductance and open apical surface of polarized epithelial cells. A shorttime kinetics, but the DF508 CFTR channel has a region between the 7th and 8th transmembranedecreased open probability [11]. These exciting segments is glycosylated and exposed to the exteriorfindings have led to the hope that CFTR processing milieu.and function can be manipulated. Several novel The physiology of the CFTR has been elucidated.chemicals have been developed that target molecular It is now known that the CFTR functions as atranscription, translation, and protein folding/ traf- chloride channel [2] as well as a regulatory proteinficking, as well as augmentation of CFTR channel [18]. In addition to chloride conductance, the mole-conductance. The purpose of this paper is to review cule transports ATP [18] from the intracellularthese pharmacologic developments and foreshadow compartment to the extracellular surface. At the cellthe advancements to come. surface, ATP can interact with purinergic P2Y2

K. Powell, P.L. Zeitlin / Advanced Drug Delivery Reviews 54 (2002) 13951408 1397

receptors and stimulate release of intracellular cal-cium and calcium-mediated chloride conductance,thus amplifying the chloride transport activity. Fur-ther amplification occurs by CFTR-mediated regula-tion of the ORCC channel [19,4]. CFTR, directly orindirectly, inhibits the amiloride-sensitive sodiumchannel [20,21] attenuating sodium transport in theairways. By controlling multiple conductance path-ways, CFTR is poised to maintain periciliary fluidhomeostasis. When CFTR is severely reduced orabsent, secretions in the pulmonary, gastrointestinal,and reproductive systems become dehydrated andviscous. In the airways, this leads to inflammationand chronic infection, and in the gastrointestinaltract, to fat malabsorption and obstruction.

Fig. 1. CFTR biogenesis. Therapeutic targets in the DF508CFTR biogenesis is a complex process. Normally,biogenetic pathway include increasing mRNA production, proteinthe mRNA is transcribed at a low level under the folding, vesicular trafficking, and channel activation. Additional

control of a promoter that has features of a house- inhibitory targets include reduction in premature retrieval andkeeping gene [22]. The mRNA is translated on degradation from the ER and retardation of retrieval or recycling

from the plasma membrane.endoplasmic reticulum (ER)-associated ribosomes ina dynamic process. CFTR assumes a series ofpartially folded intermediates with the aid of protein ER-associated chaperones may trigger ubiquitination.chaperones. Core glycosylation begins during resi- Mutation of arginine-framed tripeptide motifs indence in the ER. Some of these structures are DF508 partially restores CFTR to the cell surfacequickly degraded; others move to the Golgi ap- [25]. The speed of translation may be anotherparatus [23]. The precise content of the vesicular- important variable, since full-length translation oftubular network that carries the CFTR to the Golgi CFTR occurs in 30 min; whereas ubiquitination ofcomplex for further glycosylation is not known [24]. CFTR can occur in 20 min, or cotranslationally [26].In the Golgi, additional glycosylation occurs. Trans- The CFTR chloride channel is a low conductance,port to the plasma membrane is regulated via a linear channel. Protein kinase A phosphorylates thenumber of additional soluble factors that interact CFTR and ATP is bound to the NBF-1 and thewith regions on the CFTR amino- and carboxy- channel opens. ATP is then hydrolyzed at the NBF-1termini, and this movement is subject to regulation site while the NBF-2 site binds ATP, attaining theby the cAMP-mediated pathway. One or more of most active state of the channel. This second ATP atthese steps along the CFTR biosynthetic pathway the NBF-2 is then hydrolyzed and the channel closes.might serve as a therapeutic target to restore DF508 The open probability of wild-type CFTR is about 0.3CFTR protein transport to and function at the cell [27], while the open probability of the mutant DF508surface (Fig. 1). CFTR is 0.1260.02 [27,28]. Wild-type CFTR func-

The production of mature wild type CFTR in cell tion is extremely efficient, and as a result, onlyculture models is inefficient. Approximately 75% of approximately 10% of the cells need to expressthe wild type version is degraded before reaching the normally functioning CFTR to prevent the clinicalplasma membrane [9]. One or more critical folding manifestations of CF [29].steps occur during translation in the ER. The attach-ment of multiple ubiquitin moieties to the nascent 2 .3. Mutations in CFTRchain marks the product as a substrate for proteolyticdegradation. Protein synthesis and ubiquitination Mutations in the CFTR can be classified accordingmay be linked or synchronized. Transient exposure to the fate of the final product [30]. Class I defectsof short sequence motifs during interaction with result in complete absence of CFTR. This occurs as a


result of a premature nonsense mutation, frameshift mature glycosylated form as the incubation tempera-mutation, or an abnormally spliced mRNA that leads ture for cell growth using fibroblast and mammaryto a premature stop in translation. Examples of carcinoma cells expressing DF508 cDNA was re-common mutations in this class are: W1282X, duced [34]. These authors [34] immunoprecipitatedG542X, R553X, 6211 1 G T, 1717-1 GA, the immature bands A and B of mutant DF508 CFTRand 3905insT. Class II mutations lead to defective in cells when grown at 37 8C. As the incubationCFTR folding and trafficking, and decreased func- temperature for cell growth was dropped, the per-tion. This class includes the most common mutation, centage of fully mature, band C increased. The effectthe DF508, as well as the N1303K, and P574H. on band C formation was reversible so that when theClass III mutations are defective in regulation of cells were placed back into culture at 37 8C, thechloride conductance through the CFTR at the amount of fully mature protein decreased with aplasma membrane and include the G551D and half-life of approximately 7 h (similar to wild type)G551S mutations. Class IV mutations (R117H, and was not replaced with mature protein. CyclicR334W, R347P) result in a CFTR that reaches the AMP activated chloride conductance was present forplasma membrane but has reduced chloride conduct- cells grown at 30 8C but not at 37 8C. In summary,ance. Class V defects (38491 10 kB CT, A455E, by lowering the incubation temperature for cell27891 5GA) lead to a reduction in the pro- growth, the CFTR may attain a conformation thatduction of CFTR, although residual product reaches allows it to escape from degradation in the ER andthe surface and is functionally operational. This class be fully processed and operational. It is important tooften involves mutations in the promoter or involves note that it is not the glycosylation itself that inducesalternative splicing. Some authors describe a Class trafficking, but instead, glycosylation is a marker ofVI, which results from defects in CFTR regulation of maturation beyond the ER [35]. Although loweringadjacent channels in the plasma membrane, such as the body temperature is not practical therapeutically,the ORCC and the ENaC [1]. New therapeutics are this study and other similar ones have given thenow being directed at selective classes. By knowing scientific community confidence that protein folding /a patients genotype and class of mutation, therapeu- trafficking can be manipulated therapeutically.tic approaches can be more specific.

3 .2. Chemical chaperones

3 . Therapies directed at folding Glycerol is a small, uncharged, polyhydric alcoholthat is highly permeable across plasma membranes.

3 .1. Kinetic therapies It has been shown to stabilize protein structure [36]and to augment the stability and kinetics of oligo-

It has long been recognized that protein folding meric microtubule assembly [37]. Based on theseand processing are temperature-sensitive. In 1990, findings, glycerol has been termed a chemicalLjunggren et al. found an increase in assembly and chaperone.surface expression of H-2b -microglobulin com- In a study by Sato et al. in 1996, high con-2plexes (class I major histocompatibility complexes) centrations (10%) of glycerol were found to promoteat temperatures of 1933 8C [31]. CFTR-mediated higher-molecular-weight DF508 CFTR in HEK 293chloride conductance through the DF508 CFTR and C127 cells [38]. These results were time depen-mutant channel occurred in Xenopus oocytes (aug- dent, fully reversible, and augmented by lowering themented with forskolin and 3-isobutyl-1-methylxan- temperature to 26 8C. Further, as seen through wholethine) [32] and in Sf9 insect cells, both of which are cell patch clamp techniques, HEK cells had in-typically maintained at lower temperatures [33] creased cAMP activated chloride currents, althoughThus, it was hypothesized that the DF508 mutant with slower kinetics and reduced amounts as com-might be temperature-sensitive. pared to wild-type CFTR. Maturation of the mutant

In 1992, Denning et al. observed increased pro- CFTR was not seen with other polyols such ascessing of the DF508 mutant protein to the more 1,2-propanediol, 1,3-propanediol, or 2% dimethylsul-


foxide (DMSO) possibly because of decreased cell intermediate forms of proteins, which transientlymembrane permeability to these agents. The results have exposed hydrophobic areas. These structuresof the study led investigators to believe that glycerol are stabilized through a number of different molecu-stabilizes the core-glycosylated intermediate form of lar chaperones that are found within the lumen of theCFTR, protecting it from rapid degradation and ER and in the cytosol.allowing it to be released from the ER. By studying Among the best-studied molecular chaperones areother CFTR mutations (D572A and S1251A), it was the 70-kDa heat shock proteins, including Hsp70shown that these mutations were not sensitive to [40] and Hsc70. They are highly homologous, how-glycerol and thus, the glycerol-mediated effect on the ever Hsc70 is constitutively expressed, and Hsp70,DF508 mutation is not due to a complete breakdown which lacks introns, is inducible. Hsc70 binds to thein the ERs quality control system. Although gly- N-terminus of the NBF-1 peptide during the inter-cerol was not toxic to the cell line in this study, due mediate stages of folding and stabilizes the protein,to the high concentrations that would be required in inhibiting aggregation [41]. However, Hsc70 was not

1 21vivo and potential for toxicity, glycerol and other effective in the presence of K and Mg -ATP,chemical chaperones such as deuterated water and suggesting that the environmental context is im-DMSO are not likely to be useful therapeutically. portant [41]. A prolonged association of Hsp70 with

Recently the naturally occurring chemical DF508 CFTR has been observed [42].chaperones, such as the intracellular small-molecu- The mammalian 90-kDa stress chaperones consti-lar-weight osmolytes myoinositol, betaine and tute an additional important family. Hsp90 is abun-taurine have caught the attention of investigators dant and involved in folding of many newly syn-working on CFTR folding [39]. These solutes are thesized proteins. It has strong links to the ubiquitinthought to function as osmoprotectants, protecting proteasome family. The antibiotic geldanamycinproteins from denaturation in hyperosmolar environ- binds Hsp90, but cannot protect DF508 CFTR fromments, such as in the renal medulla. They accumulate destruction [43].in cells through the sodium/potassium/chloride co- Another molecular chaperone is calnexin, a cal-transporter. Wang et al. studied a CF bronchial cium binding transmembrane protein found in theepithelial cell line containing one DF508 allele [39]. ER. Mutant CFTR undergoes a prolonged specificWhen the cells were treated with 10 mM myoinositol association with calnexin [44] and with Hsp70 [45].for 24 h, the CFTR collected by immunoprecipitation These associations may promote ubiquitination andcontained an increased percentage of Band C. There degradation [45]. Calnexin and Hsp70 also specifi-was an even greater increase in percentage of Band cally bind to wild-type CFTR in the ER but theC when the cells were treated for 48 h with association is transient and results in the release ofmyoinositol followed by taurine, betaine and then the wild-type CFTR [44].

myoinositol again. Chemical chaperones offer an Sodium-4-phenylbutyrate (Buphenyl ), a drugexciting look at how processing of the DF508 CFTR that was developed to treat elevated blood ammoniaprotein can be manipulated. in urea cycle disorders, is a histone deacetylase

inhibitor that promotes DF508 CFTR trafficking.3 .3. Molecular chaperones This activity appears to be secondary to down

regulation of Hsc70 [46,47] and upregulation ofProtein processing involves transforming a linear Hsp70 [48]. When Hsp70 production is induced with

string of amino acids into a complex three-dimen- either 4-PBA, transfection with an Hsp70 plasmid, orsional structure. Misfolded structures may expose with supplemental glutamine, there is increasedhydrophobic areas that could lead to the formation of maturation of CFTR as seen by increased percentageinsoluble aggregates or act as signals for proteolysis. of band C [48].The cell has a quality control system that degrades The immunosuppressant, deoxyspergualin (DSG),these aggregates, often through the process of is a competitive antagonist of Hsc70 and Hsp 90ubiquitination and proteolysis [7]. Further, during the [49]. Immortalized human CF airway cells (DF508)formation of a three-dimensional product, there are and intrahepatic biliary cells (DF508), as well as


C127DF508 (mouse mammary tumor cells), de- in the fully mature band C CFTR. This study alsoveloped functional CFTR channels at the plasma looked at immortalized human airway cells, JME/membrane in the presence of DSG [49]. Drugs that CF15 (homozygous DF508). In these cells, treatmentbind to Hsp90 in the cytosol (ansamysin drugs such with 1550 mM butyrate led to chloride efflux inas geldanamycin and herbimycin A) can inhibit 20% of the cells as detected by SPQ halide efflux.protein processing and promote degradation [50]. In Again, there was cellular toxicity at concentrationssummary, the role of these chaperone proteins is of 50 mM. This study showed that sodium butyratevery complex, dynamic, and likely multimeric. increases chloride efflux of the DF508 CFTR. Its

exact mechanism of action is unclear. It may be3 .4. Sodium-4-phenylbutyrate through overexpression of the DF508 protein and

thus overwhelming of the ERs quality controlButyrate is known to be a transcriptional regulator system. However, there was only a small increase in

[5153]. The oral form of butyrate, sodium-4- the percentage of mature band C protein. Thisphenylbutyrate (4-PBA, Buphenyl ) has been shown finding led to the hypothesis that butyrate may also

to induce fetal hemoglobin [54,55]. It has been used act through transcription of other proteins that eitherin clinical trials for sickle cell anemia [55] and rescue mutant CFTR from mislocalization or in-b-thalassemia [56]. Because gene expression profiles crease channel opening of the few mutant CFTRs atbecame more differentiated [5759], it is in phase I the plasma membrane.trials in several different malignant disorders [60 Rubenstein et al. [64] studied 4-PBA in IB3-162]. The potential for therapeutic benefit in CF cells (0.11 mM) and nasal polyp epithelial cellsresides in an additional mechanism, involving pro- from patients with CF (5 mM). The study showed antein folding and the ER environment. increase in the molecular mass of CFTR, suggesting

Butyrate has been utilized to activate the metal- the development of fully mature glycosylated pro-lothionein promoter of recombinant cDNA expres- tein. Further, by administering 0.1 and 2 mM ofsion systems. C127 cells stably transfected with 4-PBA for 7 days to IB3-1 cells and CF nasalDF508 under the control of this promoter can be epithelial cells, they found a dose-dependent increaseinduced to express cAMP-mediated chloride trans- in forskolin- (an adenylate cyclase activator) acti-port in 5 mM butyrate [63]. The control cells without vated chloride conductance by the CFTR channels.promoter also behaved as if there were functional This study provided evidence that 4-PBA allowsCFTR molecules in the presence of butyrate, sug- mutant CFTR to escape the ER, become glycosylatedgesting an effect on endogenous DF508. The same in the Golgi, and then be transported to the plasmaresults were seen for butyrate concentrations of 550 membrane where it has some intrinsic channelmM although there was cellular toxicity at the higher activity.concentration. Further, after adding 5 mM sodium 4-PBA has been tested in Phase I clinical trialsbutyrate with the metallothionein promoter to the and evidence of functional DF508 CFTR was ob-cells, there was a 1050-fold increase in mRNA and served in nasal epithelia [65]. A randomized, double46-fold increase in protein, although only a small blinded, placebo controlled study was carried out inincrease in the percentage of band C as seen through 18 CF patients homozygous for the DF508 mutation.

32the in vitro phosphorylation assay with (g- P)ATP. Patients received by mouth 19 g drug or placeboThe levels of mRNA peaked at 34 h postinduction divided three times a day for 7 days. Nasal potentialand were stable in the presence of butyrate for 24 h. differences (NPD) and sweat chloride tests were

When studying the endogenous CFTR promoter in performed at baseline and after treatment. Patients inimmortalized pancreatic adenocarcinoma cells the 4-PBA group had a small but statistically signifi-(CFPAC-1 cell line), there was also detectable chan- cant increase in nasal chloride transport, yet therenel activity in 1015% of the cells (as seen through was no significant change in sweat chloride con-the SPQ halide efflux assay) with the addition of centration. Importantly, there was no toxicity seen in1525 mM butyrate [63]. There was no detectable this study. These results suggested that tissue-spe-current in the monolayer, nor was there any increase cific differences in the response to 4-PBA might


predict the functional outcome. A dose escalation result of this study, it is thought that there may be atrial was recently completed that confirmed a dose- prolonged association of mutant CFTR and Hsc70dependent relationship for CFTR-mediated chloride signaling for its degradation and 4-PBA may act totransport in nasal epithelia [66]. A maximum toler- interrupt this process by downregulating Hsc70. Theated dose was defined based on NPD and side mechanism of this downregulation has recently beeneffects. In a small pilot clinical trial of 20 g of elucidated. Rubenstein et al. found that incubation of4-PBA (open-label) divided t.i.d. for 1 month, persis- 4-PBA in IB3-1 cells resulted in increased Hsc70tent induction of nasal chloride transport was ob- mRNA degradation by approximately 40% [47].served [personal communication, P Zeitlin]. One There was no effect on a Chinese hamster ovaryexample of an elevation of chloride transport in the Hsc70 promoter or a human Hsc70 promoter. Thus,nasal epithelia of a CF volunteer after 3 weeks of one mechanism by which 4-PBA improves DF5084-PBA, is shown in Fig. 2. Loffing et al. found that CFTR trafficking is through degrading Hsc70 mRNAconcentrations of 5 mM of PBA given chronically to which leads to decreased formation of the Hsc70Calu-3 cells (a human airway cell line that expresses CFTR complexes which would otherwise causewild type CFTR) reduced basal and cAMP stimu- mislocalization of the protein.lated chloride efflux secondary to downregulation of Upregulation of Hsp70 with 4-PBA [5759,68]

1 1 2the Na K Cl cotransporter, whereas clinically results in increased maturation of CFTR as seen byachievable concentrations were associated with in- increased percentage of band C [48]. In the lattercreased CFTR and chloride transport [67]. study, three independent strategies to elevate Hsp70

Although 4-PBA has been correlated with func- levelsglutamine, pCMVHsp70 plasmid transfec-tional cell surface CFTR, its mechanism of action is tion, or 4-PBA, were associated with increasednot completely understood. It may be due to its Hsp70, increased Hsp70CFTR complexes, andeffect on transcription regulation and/or secondary increased band C CFTR.to its interactions with chaperone proteins. Rubens- Once DF508 CFTR escapes the ER and passestein and Zeitlin [46] looked at IB3-1 cells (genotype through the Golgi, there are additional barriers toDF508/W1282X) grown in 0.055 mM 4-PBA for 2 cross to reach the plasma membrane. Estimates ofdays. In this study, a dose dependent reduction in the half-life in vitro of DF508 CFTR on the cellHsc70 mRNA was seen [46]. There was also de- surface suggest that this residence time is shortenedcreased coprecipitation of Hsc70 and CFTR. As a compared to wildtype CFTR [69,70]. This defect

Fig. 2. Nasal epithelial potential difference measurements in a subject with cystic fibrosis at baseline and after 3 weeks of open-label4-PBA. (a) Baseline. Nasal epithelial potential difference (NPD) was measured as described by Zeitlin et al. [66]. Basal NPD is determinedduring nasal superfusion with Ringers solution (solid line) and is more negative in CF individuals than unaffected individuals. Amiloride isadded to block sodium reabsorption and depolarize the potential (dotted line). Superfusion with a chloride-free gluconate-substitutedRingers in the continued presence of amiloride provides the chemical driving force for chloride secretion (dashed line). Subsequent additionof isoproterenol (dash and dotted line) will increase intracellular cAMP and stimulate wildtype CFTR-mediated chloride secretion. Thissubject (homozygous DF508 CFTR) has no CFTR-mediated chloride transport. (b) 3 weeks 4-PBA. The CF subject in (a) ingested 20 g4-PBA divided t.i.d. for 4 weeks. This NPD tracing from the same naris as in (a) now shows a reduction in amiloride-inhibited sodiumtransport and stimulation of CFTR-mediated chloride transport. Arrows indicate a 1-ml superfusion with 1 mM ATP to activate thecalcium-mediated chloride conductance.


may be conformational and temperature sensitive and Although the mechanism of action of IBMX is notlead to accelerated retrieval and degradation [71]. completely understood, it is thought that it prolongsThus measures that stabilize the mutant in both the the phosphorylated state of the regulatory site andER and in the plasma membrane would be desirable. increases the open probability of the channel [28]. In

the study by Schulz et al. the open probability ofDF508 CFTR went from 0.1260.02 to 0.4160.06with IBMX (5 mM) and forskolin, although there4 . Therapies to augment conductancewas some reduction in amplitude [28].

Haws et al. studied the effect of IBMX and 8-The strategies mentioned thus far all act to pro-cyclopentyl-1,3-dipropylxanthine (CPX), another

mote protein folding/ trafficking. However, it hasnonspecific phosphodiesterase and an A1 adenosinebeen shown that if the mislocalized CFTR channelreceptor antagonist, on stably transfected cells with

reaches the plasma membrane, it has intrinsic chan-DF508 CFTR [27]. In this study, both IBMX (5

nel function, although with an increased closed timemM) and CPX potentiated forskolins effect on[11]. Thus, therapies aimed at augmenting conduct- 125CFTR-mediated efflux of I by 2.5-fold. There was

ance might act synergistically with therapies that aida 7-fold increase in cAMP levels associated within correcting protein folding/ trafficking. There are IBMX treatment, but not CPX treatment. CPX was

several drugs that are being studied in this capacityalso found to be 25 times more potent than IBMX inincluding xanthines, phosphodiesterase inhibitors,activating DF508 CFTR. These results led to the

and isoflavones. hypothesis that CPX may be working through adifferent pathway to IBMX.

4 .1. Xanthines Eidelman et al. showed that CPX stimulated36 2

chloride efflux ( Cl ) through CFTR in the DF508It is well known that the methylxanthines, found CFTR homozygous pancreatic cell line, CFPAC-1, in

naturally in tea, coffee and cocoa, stimulate the concentrations of 20100 nM [73]. Forskolin poten-central nervous system, relax bronchial smooth mus- tiated the effect of CPX on chloride efflux. CPX iscle, and stimulate cardiac muscle. These purine selective and did not affect cells with the wild typederivatives function as adenosine receptor antago- CFTR (HT-29 cells, T84 cells, and CFPAC-1 cellsnists and as phosphodiesterase inhibitors. Due to transfected with CFTR).impact on the cAMP pathway and activity in low IB3-1 and a mouse fibroblast cell line (NIH3T3)concentrations, studies have been done looking at expressing DF508 CFTR behaved in a similar man-their effect on the cAMP activated CFTR channel. ner [74]. In this study, CPX did not stimulate

The phosphodiesterase inhibitor, 3-isobutyl-1- chloride efflux in cells with the wild type CFTR. Themethylxanthine (IBMX) also functions as an adeno- CPX effect on DF508 cells was dose dependent fromsine receptor antagonist. In 1991, Drumm et al. concentrations of 130 mM. At concentrations offound that IBMX increases the CFTR chloride CPX greater than 30 mM, there was actually acurrent in Xenopus oocytes expressing the DF508 decrease in potency. This group also looked at sixCFTR [32]. In 1993, Grubb et al. found that IBMX analogues (2-thio-CPX, CPT, 3,4-dehydro-CPX, 3-F-(5 mM) with forskolin (0.01 mM) did not stimulate CPX, 3-I-CPX, KW-3902) and found that thesechloride efflux in vitro when studying CF nasal chemicals did not potentiate chloride efflux. As abronchial epithelial tissues with DF508 CFTR [72]. result, it was thought that CPXs action was notWhen looking at IBMX and isoproterenol in five CF through A1 adenosine receptor antagonism.patients homozygous for DF508, they found no The mechanism of CPXs action is now thought toeffect on the NPD. It was thought that the findings in be through direct binding to the NBF-1 [75,76].this study differed from Drumms earlier work, Through associationdisassociation kinetics usingpossibly because Xenopus oocytes are maintained at rapid membrane filtration assays, Cohen et al. found

3lower temperatures or possibly due to the different that [ H]CPX associates with greater affinity to thepromoters or different phosphorylation capabilities. NBF-1 of DF508 CFTR than that of the wild type


CFTR [76]. The binding potency was greater for not associated with a significant rise in cAMP levelsCPX than IBMX for the DF508 cells. but it was inhibited by protein kinase A inhibitors

The therapeutic potential of CPX stimulated a (H-8 and Rp-cAMPS), suggesting that it might workphase I clinical study for safety and phar- through a more distal signal.macokinetics [77]. It was a single-dose placebo Kelley et al. also looked at endogenous CFTR incontrolled study in 37 adult patients homozygous for transformed nasal polyp tissue of patients homo-the DF508 mutation. Doses of $ 30 mg resulted in zygous for DF508 (CF-T43) [80]. They found thatC levels that in vitro had been shown to augment milrinone and amrinone, at 100-mM concentrations,maxchloride conductance. A dose of 300 mg resulted in increased chloride efflux (1961% from baseline)C levels that were consistent with the amount while in the presence of a b-agonist (isoproterenol)maxrequired in vitro to improve trafficking. Although and protein kinase A activator. Mice homozygous forthere was variability in the kinetics in these patients, DF508 CFTR were administered a combination ofthere was no toxicity. A phase II multi-dose trial is milrinone (100 mM) and forskolin (10 mM) [81].being planned. This combination of drugs resulted in an increased

A more recent study investigated the effect of magnitude of the murine NPD, while there was noCPX after pretreatment with 4-PBA on a CF bron- significant change with either drug alone. The impli-chial epithelial cell line that has the DF508 allele cations of this study are exciting, however, one must(CFBE41o2) [78]. In this study, CPX alone, or use caution when extending the mouse model to4-PBA alone, stimulated chloride efflux, as measured humans.through X-ray microanalysis, but 4-PBA plus CPXdid not enhance chloride efflux above CPX alone.Instead, genistein, an isoflavone, acted synergistical- 4 .3. Isoflavones (genistein, apigenin, kaempferol,ly with 4-PBA. This suggests a dual role for CPX in quercetin)trafficking and channel activation, but single andcomplementary roles for 4-PBA and genistein. Isoflavones are naturally occurring substances

found in legumes. They have multiple cellular effects4 .2. Phosphodiesterase inhibitors including activation of p53, activation of estrogen

receptors, scavenging of free radicals, and antagon-Phosphodiesterase (PDE) inhibitors increase ism of the epithelium sodium channel and the

cAMP by inhibiting one or more enzymes involved basolateral potassium channel. In 1996, Illek et al.in the degradation of cAMP. Cyclic AMP-activated found that genistein, a tyrosine kinase inhibitor,protein kinase A mediates phosphorylation of CFTR stimulated CFTR in vitro (HT-29/B6 and T84and increases the open time of the CFTR channel. colonic epithelial cells) and inhibited a basolaterol KDrugs in this class include amrinone and milrinone. channel [82].These drugs also cause vasodilation, which may be A later study by Illek et al. used the isoflavones inbeneficial for the CF airways. human epithelial Calu-3 cells and Calu-3 monolayers

In 1991, Drumm et al. showed that inhibiting PDE [83]. There was a dose-dependent activation ofhad a greater effect on CFTR activation than CFTR chloride channels with genistein, apigenin,adenylate cyclase stimulants [32]. Kelley et al. kaempferol and quercetin in single cells and airwaystudied Calu-3 cells and 16HBE both from airway epithelium. The chloride currents were further stimu-epithelium and expressing the wild-type CFTR [79]. lated by pretreatment with forskolin.Using these cells, they found that type III PDE These same investigators also studied the iso-(milrinone, amrinone) at 100-mM concentrations, flavones in vivo. The isoproterenol responses instimulated channel efflux 13.7-fold without adenylate nasal potential differences of non-CF patients werecyclase activators. They found no effect on channel further hyperpolarized by 27.8% of normal levelsefflux by IBMX (a nonspecific PDE), type IV PDE [82,83]. They found that the maximal stimulated(rolipram) or type V PDE (dipyridamole). The aug- currents in whole cells were greatest with apigenin,mentation of channel efflux by the type III PDE was then kaempferol, genistein and quercetin. In 1999,


Illek et al. found through patch clamp techniques, have different effects on processing CFTR, althoughthat genistein and forskolin could increase chloride none had negative effects on band C expression [94].conductance of CFTR in G551D HeLa cells but not The flavonoids have many functions that may behomozygous DF508 cells unless they were pretreated important in modulating mutant CFTR, and the usewith 4-PBA [84]. Thus, it was thought that the of them synergistically with drugs that improveisoflavones augment conduction through mutant trafficking would be advantageous. A recent study byCFTR that is already present on the cell surface, as Andersson et al. showed that by treating a CFwith the G551D mutation and the DF508 mutation bronchial epithelial cell line, a line that expresses theafter treatment with 4-PBA. DF508 allele (CFBE41o2), with cAMP, genistein,

The mechanism of action of genistein has recently and 4-PBA resulted in enhanced chloride conductionbeen elucidated. It has been shown that genistein as compared to just genistein and cAMP alone [78].works through a protein kinase A- and tyrosine Thus, the flavones appear promising and furtherkinase-independent mechanism [85,86]. It has been research needs to be done in this area. As theproposed that genistein works as a phosphatase flavones have multiple cellular effects, it would beinhibitor thus increasing the phosphorylated state and advantageous to develop similar chemicals that arethe channels open time [82,85]. Reenstra et al. more selective for the CFTR.showed that genistein and calyculin A (a serine /threonine protein phosphatase inhibitor) both in-

125crease I efflux through the CFTR [85]. They had 5 . High throughput screeningan additive effect but were found to act throughdifferent phosphatase sites. Neither stimulated pro- Clearly, one important goal in CF therapy is thetein kinase A activity. A more recent study showed identification of compounds that restore chloridethat genistein directly interacts with the NBF-2 of permeability in CF epithelial cells regardless of theCFTR and inhibits ATPase, GTPase, and adenylate mechanism. Quantitative high throughput screeningkinase in a partial noncompetitive manner [87]. of potential modulators of CFTR halide permeability

The flavonoids have been shown to have other requires a sensitive, rapid, reliable, and automatedbeneficial properties that may be important for the methodology.Verkman [95] has developed a series oftreatment of CF. In patients with CF, there is fluorescent probes that meet these standards. Ainflammation in many tissues of the body that can be yellow fluorescent protein (YFP)-based halide sensordestructive. One study showed that the submucosal is now capable of reliably sensing a 2% activation ofglands in human CF bronchial tissues (homozygous CFTR. Merely increasing chloride permeability mayfor DF508) have increased levels of interleukin-8 be only the first screen, since CFTR clearly amplifies(IL-8) thought to be secondary to a lack of an ion balance by regulating additional chloride andinhibitor factor (IkBa) and to high levels of nuclear sodium channels. Further screens against secondaryfactor kB [88,89]. Genistein was found to increase pathways may be necessary, and the cDNA mi-production of the IkBa and inhibit the nuclear factor croarray technology capable of testing thousands ofkB, leading to decreased IL-8 production [90,91]. downstream genes from a single compound exposure

Suaud et al. studied genistein activation of oocytes may be informative.expressing DF508 CFTR and ENaC [92]. Genisteinrestored CFTRENaC interactions presumably byactivating DF508 CFTR chloride conductance. 6 . Conclusions

The flavonoids have also been shown to regulatethe expression of the heat shock family of chaperone The scientific community has made great strides inproteins. Hsp27 and Hsp70 heat induced synthesis the understanding of the protein folding/ traffickingwas inhibited by quercetin in a human breast cancer defect associated with the DF508 mutation in CF.cell line [93]. A more recent study by Lim et al. Through this knowledge, many novel pharmacologicshowed that different flavonoids, more specifically compounds have been developed to correct proteingenistein, apigenin, kaempferol, and quercetin, all processing / trafficking. Further, advancements in


to cystic fibrosis is located on chromosome 7, Nature 318strategies to augment channel conductance have been(1985) 380382.made. It is likely that a combination of these [14] J.M. Rommens, M.C. Iannuzzi, B. Kerem, M.L. Drumm, G.

strategies will most likely be beneficial in treating Melmer, M. Dean, R. Rozmahel, J.L. Cole, D. Kennedy, N.this disease. Hidaka et al., Identification of the cystic fibrosis gene:

chromosome walking and jumping, Science 245 (1989)10591065.

[15] J.R. Riordan, J.M. Rommens, B. Kerem, N. Alon, R.Rozmahel, Z. Grzelczak, J. Zielenski, S. Lok, N. Plavsic, J.L.R eferences Chou et al., Identification of the cystic fibrosis gene: cloningand characterization of complementary DNA [published

[1] J.E. Mickle, G.R. Cutting, Clinical implications of cystic erratum appears in Science 1989 Sep. 29;245(4925):1437],fibrosis transmembrane conductance regulator mutations, Science 245 (1989) 10661073.Clin. Chest Med. 19 (1998) 443458. [16] Q. Jiang, J.F. Engelhardt, Cellular heterogeneity of CFTR

[2] M.P. Anderson, R.J. Gregory, S. Thompson, D.W. Souza expression and function in the lung: implications for geneS.Pa, S. Paul, R.C. Mulligan, A.E. Smith, M.J. Welsh, therapy of cystic fibrosis, Eur. J. Hum. Genet. 6 (1998)Demonstration that CFTR is a chloride channel by alteration 1231.of its anion selectivity, Science 253 (1991) 202205. [17] X. Wang, Y. Zhang, A. Amberson, J.F. Engelhardt, New

[3] M.E. Egan, E.M. Schweibert, W.B. Guggino, Differential models of the tracheal airway define the glandular contribu-expression of ORCC and CFTR induced by low temperature tion to airway surface fluid and electrolyte composition, Am.in CF airway epithelial cells, Am. J. Physiol. (Cell Physiol) J. Respir. Cell Mol. Biol. 24 (2001) 195202.268 (37) (1995) C243C251. [18] E.M. Schwiebert, M.E. Egan, T.H. Hwang, S.B. Fulmer, S.S.

[4] S.E. Gabriel, L.L. Clarke, R.C. Boucher, M.J. Stutts, CFTR Allen, G.R. Cutting, W.B. Guggino, CFTR regulates out-and outward rectifying chloride channels are distinct proteins wardly rectifying chloride channels through an autocrinewith a regulatory relationship, Nature 363 (1993) 263268. mechanism involving ATP, Cell 81 (1995) 10631073.

[5] M.J. Stutts, C.M. Canessa, J.C. Olsen, M. Hamrick, J.A. [19] M. Egan, T. Flotte, S. Afione, R. Solow, P.L. Zeitlin, B.J.Cohn, B.C. Rossier, R.C. Boucher, CFTR as a cAMP- Carter, W.B. Guggino, Defective regulation of outwardly

2dependent regulator of sodium channels, Science 269 (1995) rectifying Cl channels by protein kinase A corrected by847850. insertion of CFTR [see comments], Nature 358 (1992) 581

[6] B.J. Rosenstein, G.R. Cutting, Diagnosis of cystic fibrosis: a 584.consensus statement, J. Pediatr. 132 (1998) 589595. [20] I.I. Ismailov, M.S. Awayda, B. Jovov, B.K. Berdiev, C.M.

[7] C.L. Ward, S. Omura, R.R. Kopito, Degradation of CFTR by Fuller, J.R. Dedman, M. Kaetzel, D.J. Benos, Regulation ofthe ubiquitinproteasome pathway, Cell 83 (1995) 121127. epithelial sodium channels by the cystic fibrosis transmem-

[8] S.H. Cheng, R.J. Gregory, J. Marshall, S. Paul, D.W. Souza, brane conductance regulator, J. Biol. Chem. 271 (1996)G.A. White, C.R. ORiordan, A.E. Smith, Defective intracel- 47254732.lular transport and processing of CFTR is the molecular basis [21] M.J. Stutts, C.M. Canessa, J.C. Olsen, M. Hamrick, J.A.of most cystic fibrosis, Cell 63 (1990) 827834. Cohn, B.C. Rossier, R.C. Boucher, CFTR as a cAMP-

[9] C.L. Ward, R.R. Kopito, Intracellular turnover of cystic dependent regulator of sodium channels, Science 269 (1995)fibrosis transmembrane conductance regulator. Inefficient 847850.processing and rapid degradation of wild-type and mutant [22] K. Yoshimura, H. Nakamura, B.C. Trapnell, W. Dalemans, A.proteins, J. Biol. Chem. 269 (1994) 2571025718. Pavirani, J.P. Lecocq, R.G. Crystal, The cystic fibrosis gene

[10] N. Kalin, A. Claass, M. Sommer, E. Puchelle, B. Tummler, has a housekeeping-type promoter and is expressed at lowDF508 CFTR protein expression in tissues from patients with levels in cells of epithelial origin, J. Biol. Chem. 266 (1991)cystic fibrosis, J. Clin. Invest. 103 (1999) 13791389. 91409144.

[11] W. Dalemans, P. Barbry, G. Champigny, S. Jallat, K. Dott, D. [23] S.I. Bannykh, G.I. Bannykh, K.N. Fish, B.D. Moyer, J.R.Dreyer, R.G. Crystal, A. Pavirani, J.P. Lecocq, M. Lazdun- Riordan, W.E. Balch, Traffic pattern of cystic fibrosis trans-ski, Altered chloride ion channel kinetics associated with the membrane regulator through the early exocytic pathway,DF508 cystic fibrosis mutation [see comments], Nature 354 Traffic 1 (2000) 852870.(1991) 526528. [24] A. Gilbert, M. Jadot, E. Leontieva, S. Wattiaux-De Coninck,

[12] L.C. Tsui, M. Buchwald, D. Barker, J.C. Braman, R. R. Wattiaux, DF508 CFTR localizes in the endoplasmicKnowlton, J.W. Schumm, H. Eiberg, J. Mohr, D. Kennedy, reticulumGolgi intermediate compartment in cystic fibrosisN. Plavsic, Cystic fibrosis locus defined by a genetically cells, Exp. Cell Res. 242 (1998) 144152.linked polymorphic DNA marker, Science 230 (1985) 1054 [25] X.B. Chang, L. Cui, Y.X. Hou, T.J. Jensen, A.A. Aleksan-1057. drov, A. Mengos, J.R. Riordan, Removal of multiple ar-

[13] R.G. Knowlton, O. Cohen-Haguenauer, N. Van Cong, J. ginine-framed trafficking signals overcomes misprocessingFrezal, V.A. Brown, D. Barker, J.C. Braman, J.W. Schumm, of DF508 CFTR present in most patients with cystic fibrosis,L.C. Tsui, M. Buchwald, A polymorphic DNA marker linked Mol. Cell 4 (1999) 137142.


[26] S. Sato, C.L. Ward, R.R. Kopito, Cotranslational ubiquitina- terminal nucleotide-binding domain of the cystic fibrosistion of cystic fibrosis transmembrane conductance regulator transmembrane conductance regulator [in process citation], J.in vitro, J. Biol. Chem. 273 (1998) 71897192. Biol. Chem. 272 (1997) 2542125424.

[27] C.M. Haws, I.B. Nepomuceno, M.E. Krouse, H. Wakelee, T. [42] Y. Yang, S. Janich, J.A. Cohn, J.M. Wilson, The commonLaw, Y. Xia, H. Nguyen, J.J. Wine, DF508-CFTR channels: variant of cystic fibrosis transmembrane conductance reg-kinetics, activation by forskolin, and potentiation by xan- ulator is recognized by hsp70 and degraded in a pre-Golgithines, Am. J. Physiol 270 (1996) C1544C1555. nonlysosomal compartment, Proc. Natl. Acad. Sci. USA 90

(1993) 94809484.[28] B.D. Schultz, R.A. Frizzell, R.J. Bridges, Rescue of dysfunc-tional DF508-CFTR chloride channel activity by IBMX, J. [43] W. Fuller, A.W. Cuthbert, Post-translational disruption of theMembr. Biol. 170 (1999) 5166. DF508 cystic fibrosis transmembrane conductance regulator

(CFTR)molecular chaperone complex with geldanamycin[29] L.G. Johnson, J.C. Olsen, B. Sarkadi, K.L. Moore, R.stabilizes DF508 CFTR in the rabbit reticulocyte lysate, J.Swanstrom, R.C. Boucher, Efficiency of gene transfer forBiol. Chem. 275 (2000) 3746237468.restoration of normal airway epithelial function in cystic

fibrosis, Nat. Genet. 2 (1992) 2125. [44] S. Pind, J.R. Riordan, D.B. Williams, Participation of the[30] M.J. Welsh, A.E. Smith, Molecular mechanisms of CFTR endoplasmic reticulum chaperone calnexin (p88, IP90) in the

chloride channel dysfunction in cystic fibrosis, Cell 73 biogenesis of the cystic fibrosis transmembrane conductance(1993) 12511254. regulator, J. Biol. Chem. 269 (1994) 1278412788.

[31] H.G. Ljunggren, N.J. Stam, C. Ohlen, J.J. Neefjes, P. [45] Y. Yang, S. Janich, J.A. Cohn, J.M. Wilson, The commonHoglund, M.T. Heemels, J. Bastin, T.N. Schumacher, A. variant of cystic fibrosis transmembrane conductance reg-Townsend, K. Karre, Empty MHC class I molecules come ulator is recognized by hsp70 and degraded in a pre-Golgiout in the cold, Nature 346 (1990) 476480. nonlysosomal compartment, Proc. Natl. Acad. Sci. USA 90

(1993) 94809484.[32] M.L. Drumm, D.J. Wilkinson, L.S. Smit, R.T. Worrell, T.V.Strong, R.A. Frizzell, D.C. Dawson, F.S. Collins, Chloride [46] R.C. Rubenstein, P.L. Zeitlin, Sodium 4-phenylbutyrateconductance expressed by DF508 and other mutant CFTRs in downregulates Hsc70: implications for intracellular traffick-Xenopus oocytes, Science 254 (1991) 17971799. ing of DF508CFTR, Am. J. Physiol Cell Physiol. 278

(2000) C259C267.[33] C.E. Bear, C.H. Li, N. Kartner, R.J. Bridges, T.J. Jensen, M.Ramjeesingh, J.R. Riordan, Purification and functional re- [47] R.C. Rubenstein, B.M. Lyons, Sodium 4-phenylbutyrateconstitution of the cystic fibrosis transmembrane conduct- downregulates HSC70 expression by facilitating mRNAance regulator (CFTR), Cell 68 (1992) 809818. degradation, Am. J. Physiol. Lung Cell Mol. Physiol. 281

(2001) L43L51.[34] G.M. Denning, M.P. Anderson, J.F. Amara, J. Marshall, A.E.Smith, M.J. Welsh, Processing of mutant cystic fibrosis [48] L.R. Choo-Kang, P.L. Zeitlin, Induction of HSP70 promotestransmembrane conductance regulator is temperature-sensi- DF508 CFTR trafficking, Am. J. Physiol. Lung Cell Mol.tive [see comments], Nature 358 (1992) 761764. Physiol. 281 (2001) L58L68.

[35] R.J. Gregory, D.P. Rich, S.H. Cheng, D.W. Souza, S. Paul, P. [49] C. Jiang, S.L. Fang, Y.F. Xiao, S.P. OConnor, S.G. Nadler,Manavalan, M.P. Anderson, M.J. Welsh, A.E. Smith, Matura- D.W. Lee, D.M. Jefferson, J.M. Kaplan, A.E. Smith, S.H.tion and function of cystic fibrosis transmembrane conduct- Cheng, Partial restoration of cAMP-stimulated CFTR chlo-ance regulator variants bearing mutations in putative nucleo- ride channel activity in DF508 cells by deoxyspergualin, Am.tide-binding domains 1 and 2, Mol. Cell Biol. 11 (1991) J. Physiol. 275 (1998) C171C178.38863893. [50] M.A. Loo, T.J. Jensen, L. Cui, Y. Hou, X.B. Chang, J.R.

[36] K. Gekko, S.N. Timasheff, Mechanism of protein stabiliza- Riordan, Perturbation of Hsp90 interaction with nascenttion by glycerol: preferential hydration in glycerolwater CFTR prevents its maturation and accelerates its degradationmixtures, Biochemistry 20 (1981) 46674676. by the proteasome, EMBO J. 17 (1998) 68796887.

[37] M.L. Shelanski, F. Gaskin, C.R. Cantor, Microtubule assem- [51] L. Cuisset, L. Tichonicky, P. Jaffray, M. Delpech, Thebly in the absence of added nucleotides, Proc. Natl. Acad. effects of sodium butyrate on transcription are mediatedSci. USA 70 (1973) 765768. through activation of a protein phosphatase, J. Biol. Chem.

[38] S. Sato, C.L. Ward, M.E. Krouse, J.J. Wine, R.R. Kopito, 272 (1997) 2414824153.Glycerol reverses the misfolding phenotype of the most [52] J.A. DAnna, R.A. Tobey, L.R. Gurley, Concentration-de-common cystic fibrosis mutation, J. Biol. Chem. 271 (1996) pendent effects of sodium butyrate in Chinese hamster cells:635638. cell-cycle progression, inner-histone acetylation, histone H1

[39] X. Wang, S. Leung, S.E. Guggino, Organic solutes repair the dephosphorylation, and induction of an H1-like protein,processing defect of the dF508 cystic fibrosis transmembrane Biochemistry 19 (1980) 26562671.conductance regulator protein, Pediatr. Pulmonol. E19 [53] D. Klehr, T. Schlake, K. Maass, J. Bode, Scaffold-attached(1999) 169. regions (SAR elements) mediate transcriptional effects due

[40] F.U. Hartl, Molecular chaperones in cellular protein folding, to butyrate, Biochemistry 31 (1992) 32223229.Nature 381 (1996) 571579. [54] G.J. Dover, S.W. Brusilow, D. Samid, Increased fetal hemo-

[41] E. Strickland, B.H. Qu, L. Millen, P.J. Thomas, The molecu- globin in patients receiving sodium 4-phenylbutyrate, Newlar chaperone hsc70 assists the in vitro folding of the N- Engl. J. Med. 327 (1992) 569570.


[55] G.J. Dover, S.W. Brusilow, S. Charache, Induction of fetal [68] L. Garcia-Bermejo, N.E. Vilaboa, C. Perez, A. Galan, E. Dehemoglobin production in subjects with sickle cell anemia by Blas, P. Aller, Modulation of heat-shock protein 70 (HSP70)oral sodium phenylbutyrate, Blood 84 (1994) 339343. gene expression by sodium butyrate in U-937 promonocytic

[56] A.F. Collins, H.A. Pearson, P. Giardina, K.T. McDonagh, cells: relationships with differentiation and apoptosis, Exp.S.W. Brusilow, G.J. Dover, Oral sodium phenylbutyrate Cell Res. 236 (1997) 268274.therapy in homozygous b-thalassemia: a clinical trial, Blood [69] G.L. Lukacs, X.B. Chang, C. Bear, N. Kartner, A. Mohamed,85 (1995) 4349. J.R. Riordan, S. Grinstein, The DF508 mutation decreases

[57] L. Liu, S. Shack, W.G. Stetler-Stevenson, W.R. Hudgins, D. the stability of cystic fibrosis transmembrane conductanceSamid, Differentiation of cultured human melanoma cells regulator in the plasma membrane. Determination of func-induced by the aromatic fatty acids phenylacetate and tional half-lives on transfected cells, J. Biol. Chem. 268phenylbutyrate, J. Invest. Dermatol. 103 (1994) 335340. (1993) 2159221598.

[58] H.L. Newmark, C.W. Young, Butyrate and phenylacetate as [70] G.D. Heda, M. Tanwani, C.R. Marino, The DF508 mutationdifferentiating agents: practical problems and opportunities, shortens the biochemical half-life of plasma membraneJ. Cell Biochem. Suppl. 22 (1995) 247253. CFTR in polarized epithelial cells, Am. J. Physiol Cell

[59] C.G. Wood, C. Lee, J.T. Grayhack, J.M. Kozlowski, Physiol. 280 (2001) C166C174.Phenylacetate and phenylbutyrate promote cellular differen- [71] M. Sharma, M. Benharouga, W. Hu, G.L. Lukacs, Conforma-tiation in human prostate cancer systems (Meeting abstract), tional and temperature-sensitive stability defects of theProc. Annu. Meet. Am. Assoc. Cancer Res. 35 (1994) DF508 cystic fibrosis transmembrane conductance regulatorA2404. in postendoplasmic reticulum compartments, J. Biol. Chem.

[60] M. Carducci, M. Bowling, M. Eisenberger, V. Sinibaldi, J. 276 (2001) 89428950.Simons, T. Chen, D. Noe, L. Grochow, R. Donehower, [72] B. Grubb, E. Lazarowski, M. Knowles, R. Boucher, Iso-Phenylbutyrate (PB) for refractory solid tumors: a phase I butylmethylxanthine fails to stimulate chloride secretion inclinical and pharmacological evaluation, Proc. AACR 39 cystic fibrosis airway epithelia, Am. J. Respir. Cell Mol.(1998) 506. Biol. 8 (1993) 454460.

[61] S.D. Gore, L.J. Weng, S. Zhai, W.D. Figg, R.C. Donehower, [73] O. Eidelman, C. Guay-Broder, P.J. van Galen, K.A. Jacob-G.J. Dover, M. Grever, C.A. Griffin, L.B. Grochow, E.K. son, C. Fox, R.J. Turner, Z.I. Cabantchik, H.B. Pollard, A1Rowinsky, Y. Zabalena, A.L. Hawkins, K. Burks, C.B. adenosine-receptor antagonists activate chloride efflux fromMiller, Impact of the putative differentiating agent sodium cystic fibrosis cells, Proc. Natl. Acad. Sci. USA 89 (1992)phenylbutyrate on myelodysplastic syndromes and acute 55625566.myeloid leukemia, Clin. Cancer Res. 7 (2001) 23302339. [74] C. Guay-Broder, K.A. Jacobson, S. Barnoy, Z.I. Cabantchik,

[62] J. Gilbert, S.D. Baker, M.K. Bowling, L. Grochow, W.D. W.B. Guggino, P.L. Zeitlin, R.J. Turner, L. Vergara, O.Figg, Y. Zabelina, R.C. Donehower, M.A. Carducci, A phase Eidelman, H.B. Pollard, A1 receptor antagonist 8-I dose escalation and bioavailability study of oral sodium cyclopentyl-1,3-dipropylxanthine selectively activates chlo-phenylbutyrate in patients with refractory solid tumor malig- ride efflux from human epithelial and mouse fibroblast cellnancies, Clin. Cancer Res. 7 (2001) 22922300. lines expressing the cystic fibrosis transmembrane regulator

[63] S.H. Cheng, S.L. Fang, J. Zabner, J. Marshall, S. Piraino, DF508 mutation, Biochemistry 34 (1995) 90799087.S.C. Schiavi, D.M. Jefferson, M.J. Welsh, A.E. Smith, [75] N. Arispe, J. Ma, K.A. Jacobson, H.B. Pollard, DirectFunctional activation of the cystic fibrosis trafficking mutant activation of cystic fibrosis transmembrane conductanceDF508CFTR by overexpression, Am. J. Physiol. 268 regulator channels by 8-cyclopentyl-1,3-dipropylxanthine(1995) L615L624. (CPX) and 1,3-diallyl-8-cyclohexylzanthine (DAX), J. Biol.

[64] R.C. Rubenstein, M.E. Egan, P.L. Zeitlin, In vitro pharmaco- Chem. 273 (1997) 57275734.logic restoration of CFTR-mediated chloride transport with [76] B.E. Cohen, G. Lee, K.A. Jacobson, Y.C. Kim, Z. Huang,sodium 4-phenylbutyrate in cystic fibrosis epithelial cells E.J. Sorscher, H.B. Pollard, 8-Cyclopentyl-1,3-dipropylxan-containing DF508CFTR, J. Clin. Invest. 100 (1997) 2457 thine and other xanthines differentially bind to the wild-type2465. and DF508 first nucleotide binding fold (NBF- (1) domains

[65] R.C. Rubenstein, P.L. Zeitlin, A pilot clinical trial of sodium of the cystic fibrosis transmembrane conductance regulator,4-phenylbutyrate (buphenyl) in DF508-homozygous cystic Biochemistry 36 (1997) 64556461.fibrosis patients: evidence of restoration of nasal epithelial [77] N.A. McCarty, T.A. Standaert, M. Teresi, C. Tuthill, J.CFTR function, Am. J. Resp. Crit. Care Med. 157 (1998) Launspach, T.J. Kelley, L.J. Milgram, K.A. Hilliard, W.E.484490. Regelmann, M.R. Weatherly, M.L. Aitken, M.W. Konstan,

[66] P.L. Zeitlin, M. Diener-West, R.C. Rubenstein, M.P. Boyle, R.C. Ahrens, A phase I randomized, multicenter trial of CPXC.K. Lee, L. Brass-Ernst, Evidence of CFTR function in in adult subjects with mild cystic fibrosis, Pediatr. Pulmonol.cystic fibrosis after systemic administration of 4- 33 (2002) 9098.phenylbutyrate, Mol. Ther. 6 (2002) 119126. [78] C. Andersson, G.M. Roomans, Activation of DF508 CFTR

[67] J. Loffing, B.D. Moyer, D. Reynolds, B.A. Stanton, PBA in a cystic fibrosis respiratory epithelial cell line by 4-2increases CFTR expression but at high doses inhibits Cl phenylbutyrate, genistein and CPX, Eur. Respir. J. 15 (2000)

secretion in Calu-3 airway epithelial cells, Am. J. Physiol. 937941.277 (1999) L700L708. [79] T.J. Kelley, L. Al-Nakkash, M.L. Drumm, CFTR-mediated


chloride permeability is regulated by type III phosphodies- [88] O. Tabary, S. Escotte, J.P. Couetil, D. Hubert, D. Dusser, E.terases in airway epithelial cells, Am. J. Respir. Cell Mol. Puchelle, J. Jacquot, High susceptibility for cystic fibrosisBiol. 13 (1995) 657664. human airway gland cells to produce IL-8 through the I kB

[80] T.J. Kelley, L. Al Nakkash, C.U. Cotton, M.L. Drumm, kinase a pathway in response to extracellular NaCl content,Activation of endogenous DF508 cystic fibrosis transmem- J. Immunol. 164 (2000) 33773384.brane conductance regulator by phosphodiesterase inhibition, [89] O. Tabary, J.M. Zahm, J. Hinnrasky, J.P. Couetil, P. Cornil-J. Clin. Invest. 98 (1996) 513520. let, M. Guenounou, D. Gaillard, E. Puchelle, J. Jacquot,

[81] T.J. Kelley, K. Thomas, L.J. Milgram, M.L. Drumm, In vivo Selective upregulation of chemokine IL-8 expression inactivation of the cystic fibrosis transmembrane conductance cystic fibrosis bronchial gland cells in vivo and in vitro, Am.regulator mutant DF508 in murine nasal epithelium, Proc. J. Pathol. 153 (1998) 921930.Natl. Acad. Sci. USA 94 (1997) 26042608. [90] O. Tabary, S. Escotte, J.P. Couetil, D. Hubert, D. Dusser, E.

[82] B. Illek, H. Fischer, T.E. Machen, Alternate stimulation of Puchelle, J. Jacquot, Relationship between IkBa deficiency,apical CFTR by genistein in epithelia, Am. J. Physiol. 270 NFkB activity and interleukin-8 production in CF human(1996) C265275. airway epithelial cells, Pflugers Arch. 443 (Suppl 1) (2001)

[83] B. Illek, H. Fischer, Flavonoids stimulate Cl conductance of S40S44.human airway epithelium in vitro and in vivo, Am. J. [91] O. Tabary, S. Escotte, J.P. Couetil, D. Hubert, D. Dusser, E.Physiol. 275 (1998) L902L910. Puchelle, J. Jacquot, Genistein inhibits constitutive and

[84] B. Illek, L. Zhang, N.C. Lewis, R.B. Moss, J.Y. Dong, H. inducible NFkB activation and decreases IL-8 production byFischer, Defective function of the cystic fibrosis-causing human cystic fibrosis bronchial gland cells, Am. J. Pathol.missense mutation G551D is recovered by genistein, Am. J. 155 (1999) 473481.Physiol. 277 (1999) C833C839. [92] L. Suaud, J. Li, Q. Jiang, R.C. Rubenstein, T.R. Kleyman,

[85] W.W. Reenstra, K. Yurko-Mauro, A. Dam, S. Raman, S. Genistein restores functional interactions between DF508-Shorten, CFTR chloride channel activation by genistein: the CFTR and ENaC in Xenopus oocytes, J. Biol. Chem. 277role of serine / threonine protein phosphatases, Am. J. Phy- (2002) 89288933.siol. 271 (1996) C650657. [93] R.K. Hansen, S. Oesterreich, P. Lemieux, K.D. Sarge, S.A.

[86] C.E. Chiang, S.A. Chen, M.S. Chang, C.I. Lin, H.N. Luk, Fuqua, Quercetin inhibits heat shock protein induction butGenistein directly induces cardiac CFTR chloride current by not heat shock factor DNA-binding in human breast car-a tyrosine kinase-independent and protein kinase A-indepen- cinoma cells, Biochem. Biophys. Res. Commun. 239 (1997)dent pathway in guinea pig ventricular myocytes, Biochem. 851856.Biophys. Res. Commun. 235 (1997) 7478. [94] M. Lim, A. Floyd, P.L. Zeitlin, The effects of flavonoids in

[87] C. Randak, E.A. Auerswald, I. Assfalg-Machleidt, W.W. dF508 CFTR processing (2002).Reenstra, W. Machleidt, Inhibition of ATPase, GTPase and [95] L.V. Galietta, S. Jayaraman, A.S. Verkman, Cell-based assayadenylate kinase activities of the second nucleotide-binding for high-throughput quantitative screening of CFTR chloridefold of the cystic fibrosis transmembrane conductance reg- transport agonists, Am. J. Physiol. Cell Physiol. 281 (2001)ulator by genistein, Biochem. J. 340 (Pt 1) (1999) 227235. C1734C1742.

Therapeutic approaches to repair defects in Delta F508 CFTR folding and cellular targetingIntroductionBackgroundThe CF geneCFTRMutations in CFTR

Therapies directed at foldingKinetic therapiesChemical chaperonesMolecular chaperonesSodium-4-phenylbutyrate

Therapies to augment conductanceXanthinesPhosphodiesterase inhibitorsIsoflavones (genistein, apigenin, kaempferol, quercetin)

High throughput screeningConclusionsReferences

Documents

Therapeutic approaches to repair defects in ΔF508 CFTR folding and cellular targeting