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Specific Rescue of Cystic Fibrosis Transmembrane Conductance Regulator Processing Mutants Using Pharmacological Chaperones

Department of Medicine and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada

Received March 2, 2006; accepted April 19, 2006

	Abstract

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Most mutants of the cystic fibrosis transmembrane conductance regulator (CFTR) that cause severe symptoms of cystic fibrosis do not reach the cell surface because they are defective in folding. Many CFTR folding mutants, however, including the F508 mutant found in more than 90% of cystic fibrosis patients, are potentially functional at the cell surface if they can be induced to fold correctly. In a previous study (Mol Pharm 2:407–413, 2005), we reported that a quinazoline derivative (CFcor-325) could rescue CFTR processing mutants. The corrector was not specific however, as it could also rescue a processing mutant of CFTR's sister protein, the multidrug resistance P-glycoprotein. The goal of this study was to test whether it was possible to specifically rescue CFTR processing mutants using a pharmacological chaperone. In this article, we report that two compounds, 4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)-phenol (CFpot-532) and 2-phenylamino-4-(4-ethylene-phenyl)-thiazole (corr-2b) could rescue CFTR processing mutants such as F508 CFTR but not a P-glycoprotein processing mutant. The compound CFpot-532 also acts as a potentiator of F508 CFTR channel activity. Therefore, the results suggest that the mechanism whereby CFpot-532 and corr-2b promote folding of CFTR processing mutants is through direct interaction with the CFTR mutant proteins. The compound CFpot-532 could be a particularly useful lead compound for treatment of cystic fibrosis because it is both a CFTR channel potentiator as well as a specific pharmacological chaperone.

One particular mutation, deletion of phenylalanine 508 (F508), is found on at least one chromosome in 90% of patients with cystic fibrosis (Bobadilla et al., 2002). The F508 CFTR protein is defective in folding, so it is retained in the endoplasmic reticulum and then rapidly degraded (Cheng et al., 1990; Ward and Kopito, 1994). Early studies showed that it was possible to increase the efficiency of folding of F508 CFTR to yield functional molecules at the cell surface through expression at low temperature (27°C) (Denning et al., 1992) or in the presence of nonspecific osmolytes, such as glycerol (Riordan et al., 1989; Brown et al., 1996; Sato et al., 1996) or in the presence of organic solutes such as myoinositol (Zhang et al., 2003). Studies on processing mutants of CFTR's sister protein, the multidrug resistance P-glycoprotein, showed that specific pharmacological chaperones could be used to rescue mutants defective in folding (Loo and Clarke, 1997; Loo et al., 2005). Therefore, high-throughput screening of chemical libraries was performed to identify pharmacological chaperones that could promote folding of CFTR processing mutants such as F508 (Pedemonte et al., 2005; Van Goor et al., 2006). One particularly efficient pharmacological chaperone that promotes folding of CFTR processing mutants is CFcor-325 (Loo and Clarke, 2005b; Loo et al., 2005, 2006; Van Goor et al., 2006). Expression of F508 CFTR in the presence of 1 to 10 µM CFcor-325 promoted maturation of the protein to yield functional channels at the cell surface. The chaperone was not specific, however, because it also promoted maturation of a P-glycoprotein processing mutant. Therefore, it was possible that CFcor-325 could be acting nonspecifically on a cellular pathway to cause a general effect on protein folding. In the current study, we tested whether it was possible to specifically rescue F508 CFTR with a pharmacological chaperone that did not promote maturation of P-glycoprotein. A variety of recently identified CFTR high-affinity pharmacological chaperones, such as corr-2b, corr-3a, corr-4a, and corr-4b (Pedemonte et al., 2005), were tested along with a new pyrazole-type CFTR channel activator, CFpot-532 (Van Goor et al., 2006).

	Materials and Methods

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Expression of Mutants. Wild-type, F508, and H1085R CFTR cDNAs were inserted into the pcDNA3 vector (Invitrogen Canada Inc., Burlington, ON, Canada) as described previously (Loo et al., 2006). Wild-type and mutant G268V P-glycoprotein cDNAs were inserted into the pMT21 vector (Genetics Institute, Boston, MA) as described previously (Loo and Clarke, 1994). Baby hamster kidney (BHK) cells stably expressing CFTR were generated by cotransfection of the cDNA with pWL-neo (Stratagene, La Jolla, CA) and selection with 1 mg/ml G418. The G418-resistant clones expressing CFTR or P-glycoprotein were identified by subjecting cell extracts to immunoblot analysis with rabbit polyclonal antibody against CFTR or P-glycoprotein (Chen et al., 2004; Loo and Clarke, 1995a). Transient expression of CFTR or P-glycoprotein was carried out by transfection of HEK 293 cells.

To test for the effect of pharmacological chaperones on expression of CFTR- or P-glycoprotein-processing mutants, cells were grown to approximately 75% confluence in Dulbecco's modification of Eagle's medium (DMEM) with 2% (v/v) calf serum at 37°C. Various concentrations of CFcor-325, CFpot-532 (Vertex Pharmaceuticals, San Diego, CA), corr-2b, corr-3a, corr-4a or corr-4b (Chem Div, San Diego, CA) in Dulbecco's modified Eagle's medium with 2% (v/v) calf serum were then applied to the cells. The cells were harvested after 24 to 72 h and solubilized with 2x SDS sample buffer [125 mM Tris-HCl, pH 6.8, 20% (v/v) glycerol, 4% (w/v) SDS, and 4% (v/v) 2-mercaptoethanol] containing 50 mM EDTA. Samples were then subjected to SDS-PAGE (5.5% acrylamide gels) and immunoblot analysis with rabbit polyclonal antibody against CFTR or P-glycoprotein. The amount of mature CFTR relative to total (mature plus immature CFTR) was quantitated by scanning the gel lanes followed by analysis with the NIH Image program (http://rsb.info.nih.gov/nihimage1) using a Macintosh computer.

Treatment with Endoglycosidases. Cells expressing CFTR or P-glycoprotein were lysed with 2x SDS sample buffer containing 50 mM EDTA. For treatment with endoglycosidase H (endo H), a one-tenth volume of 0.5 M sodium citrate, pH 5.5, was added to cell extract followed by addition of 20,000 U/ml endo H (New England Biolabs, Mississauga, ON, Canada). The sample was treated for 15 min at 20°C. For treatment with peptide N-glycosidase F (PNGase F), a one-tenth volume of 0.5 M sodium phosphate, pH 7.5, and one-tenth volume of 10% (v/v) Nonidet P-40 were added to the cell extract followed by addition of 10,000 U/ml PNGase F (New England Biolabs). The sample was incubated for 15 min at 37°C. Samples were then subjected to immunoblot analysis with anti-CFTR monoclonal antibody M3A7 and enhanced chemiluminescence.

Iodide Efflux Assays. Measurement of cAMP-stimulated iodide efflux was performed on BHK cells stably expressing CFTR protein or COS-1 cells transiently transfected with CFTR cDNAs as described previously (Loo et al., 2005).

Isolation of P-glycoprotein and Measurement of Drug-stimulated ATPase Activity. Histidine-tagged P-glycoprotein was isolated by nickel-chelate chromatography as described previously (Loo and Clarke, 1995b). An aliquot of the isolated histidine-tagged P-glycoprotein was mixed with an equal volume of 10 mg/ml sheep brain phosphatidylethanolamine (type II-S; Sigma-Aldrich, Mississauga, ON, Canada) that had been washed and suspended in Tris-buffered saline, pH 7.4 (10 mM Tris-HCl, pH 7.4, and 150 mM NaCl) containing 2 mM dithiothreitol. The P-glycoprotein/lipid mixture was then sonicated for 45 s at 4°C. A sample of the mixture was then incubated in the presence of various concentrations (0–0.3 mM) of CFcor-325, CFpot-532, corr-2b, corr-3a, corr-4a, and corr-4b. ATPase activity was initiated by addition of an equal volume of ATPase buffer containing 100 mM Tris-HCl, pH 7.5, 100 mM NaCl, 20 mM MgCl₂, and 10 mM ATP with and without 0.6 mM verapamil. A final concentration of 0.3 mM verapamil was saturating and resulted in maximal activation of ATPase activity (Loo and Clarke, 2001). The samples were incubated at 37°C for 30 min followed by measurement of the amount of inorganic phosphate that was released.

	Results

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The pyrazole derivative CFpot-532 (Fig. 1A) was identified as a F508 CFTR channel potentiator during high-throughput screening for compounds that stimulate cAMP-mediated channel activity ("potentiators") using mouse NIH 3T3 cells (Van Goor et al., 2006). Because it is difficult to stimulate F508 CFTR channel activity after low temperature rescue in some cell lines with cAMP agonists such as forskolin (Bebok et al., 2005; Pedemonte et al., 2005), we tested whether CFpot-532 could enhance channel activity of F508 CFTR. BHK cells stably expressing F508 CFTR were used because they do not express detectable amounts of endogenous CFTR. F508 CFTR was expressed in BHK cells for 48 h at 27°C to promote maturation of the protein and delivery to the cell surface (Loo et al., 2004). CFTR channel activity was monitored using iodide efflux assays because few channels other than CFTR can conduct iodide ions (Gupta et al., 2001). Figure 2 shows that little iodide efflux could be detected after stimulation with forskolin. Iodide efflux, however, was greatly increased when 10 µM CFpot-532 was included in the stimulation buffer. No enhanced iodide efflux could be detected in BHK control cells or in F508 CFTR expressing BHK cells that were not incubated at low temperature. In the absence of forskolin, CFpot-532 did not increase channel activity, suggesting that it acts as a potentiator. The ability of CFpot-532 to enhance channel activity suggests that it directly binds to CFTR.

View larger version (21K): [in this window] [in a new window]   Fig. 1. Structures of pharmacological chaperones and CFTR. A, the structures of CFpot-532 and corr-2b are shown. B, the model of CFTR shows the transmembrane segments as numbered cylinders. The regions that make up the transmembrane domains (TMD) and nucleotide-binding domains (NBD) are shown. The branched lines represent glycosylation sites; R represents the regulatory domain. The positions of processing mutations are indicated.

View larger version (24K): [in this window] [in a new window]   Fig. 2. Measurement of iodide efflux activity of F508 CFTR after low temperature rescue. Iodide efflux assays were performed on BHK cells stably expressing F508 CFTR that had been incubated at 27°C for 24 h. Time 0 is the start of stimulation through addition of 10 µM CFpot-532, forskolin or forskolin and CFpot-532 together.

Compounds that can directly bind to an ABC transporter have the potential to act as specific pharmacological chaperones to promote folding of processing mutants (Loo and Clarke, 1997; Loo et al., 2006). Therefore, we tested whether expression of F508 CFTR in the presence of CFpot-532 would promote maturation of the protein. Because CFTR is N-glycosylated at two sites (Fig. 1B), maturation of the protein can be monitored by a difference in mobility on SDS-PAGE gels between the core-glycosylated immature protein and the mature protein containing complex carbohydrate. BHK cells stably expressing F508 CFTR were incubated for 48 h in the presence of various concentrations of CFpot-532. Whole-cell SDS extracts were then subjected to immunoblot analysis with an anti-CFTR antibody. Figure 3A shows that CFpot-532 seemed to promote maturation of F508 CFTR as there was a dose-dependent increase in a slower migrating species of immunoreactive protein that corresponded in size to mature CFTR.

View larger version (41K): [in this window] [in a new window]   Fig. 3. Rescue of F508 CFTR with pharmacological chaperones. BHK cells stably expressing F508 CFTR were incubated for 48 h in the presence of 0 to 20 µM CFpot-532 (A) or in the presence of 10 µM CFpot-532, CFcor-325 or corr-4b (B). Whole-cell extracts were subjected to immunoblot analysis with a rabbit polyclonal (A) or monoclonal (B) antibody against CFTR. In B, samples were treated with endo H (H) or PNGase F (F) before immunoblot analysis. The positions of mature (Mature), immature (Immature), and deglycosylated (Unglycos) CFTRs are indicated.

Next, we tested whether F508 was active at the cell surface after rescue with CFpot-532. BHK cells stably expressing F508 CFTR were treated for 48 h with or without 10 µM CFpot-532 and then used in iodide efflux assays. The cells were loaded with sodium iodide and cAMP-stimulated iodide efflux was measured. Figure 4 shows that BHK cells expressing F508 CFTR demonstrated iodide efflux activity only when expressed in the presence of CFpot-532 (Fig. 4). These results show that CFpot-532 induced maturation and trafficking of F508 CFTR to the cell surface in an active form.

View larger version (20K): [in this window] [in a new window]   Fig. 4. Iodide efflux activities of F508 CFTR expressed in the presence or absence of CFpot-532. BHK cells expressing wild-type or F508 CFTR were incubated at 37°C in the presence or absence of 10 µM CFpot-532 for 48 h. Time 0 is the start of stimulation through addition of 10 µM forskolin.

Immunoblot analysis of whole-cell extracts expressing P-glycoprotein mutant G268V in the presence or absence of pharmacological chaperone is shown in Fig. 5. In the absence of drugs, P-glycoprotein mutant G268V is expressed as a 150-kDa immature product (>95% of total P-glycoprotein). Expression of P-glycoprotein G268V in the presence of CFcor-325 or corr-3a caused a large increase in the level of mature 170 kDa protein (45 ± 7% and 73 ± 11%, respectively, of total P-glycoprotein). Reduced levels of mature P-glycoprotein were observed when the mutant was expressed in the presence of pharmacological chaperones corr-4a (17 ± 4% mature P-glycoprotein) or corr-4b (31 ± 7% mature P-glycoprotein). No detectable increase in expression of mature P-glycoprotein was observed, however, when the mutant was expressed in the presence of CFpot-532 or corr-2b (Fig. 5). Therefore, the pharmacological chaperones CFpot-532 and corr-2b were specific for correction of F508 CFTR.

View larger version (34K): [in this window] [in a new window]   Fig. 5. Effect of pharmacological chaperones on maturation of P-glycoprotein processing mutant G268V. HEK 293 cells were transiently transfected with P-glycoprotein G268V cDNA and then incubated for 24 h in the absence (–) or presence of 10 µM CFcor-325, CFpot-532, corr-2b, corr-3a, corr-4a, corr-4b, or cyclosporin A (Cyclo). Whole-cell extracts were then subjected to immunoblot analysis with a polyclonal antibody against P-glycoprotein. The positions of mature and immature P-glycoproteins are shown.

View larger version (19K): [in this window] [in a new window]   Fig. 6. Effect of pharmacological chaperones on P-glycoprotein ATPase activity. Histidine-tagged wild-type P-glycoprotein was expressed in HEK 293 cells, isolated by nickel-chelate chromatography and mixed with lipid. A, P-glycoprotein ATPase activity was then measured in the presence or absence of 0.3 mM CFcor-325, CFpot-532, corr-2b, corr-3a, corr-4a, or corr-4b. The -fold stimulation is the ratio of ATPase activity in the presence of compound to the ATPase activity in the absence of drug. B, P-glycoprotein ATPase activity was measured in the presence of 0.3 mM verapamil in the presence or absence (No drug) of 0.3 mM CFcor-325, CFpot-532, corr-2b, corr-3a, corr-4a, or corr-4b. The -fold stimulation is the ratio of ATPase activity in the presence of verapamil to the activity in the absence of verapamil.

It has been reported that it was possible to specifically rescue CFTR processing mutants containing a processing mutation in the front half of the protein (F508) by coexpression with a CFTR NH₂-half molecule whereas processing mutations located in the back half of the protein (H1085R) could be rescued by coexpression with a CFTR COOH-half molecule (Cormet-Boyaka et al., 2004). It seems that the location of a processing mutation can be important for rescue. Therefore, we tested whether a specific CFTR pharmacological chaperone such as CFpot-532 could rescue a CFTR processing mutant with a mutation in the COOH half of the molecule (H1085R). HEK 293 cells transiently expressing CFTR processing mutant H1085R were incubated in the presence or absence of 10 µM CFpot-532 for 48 h. Whole-cell extracts were subjected to immunoblot analysis. Figure 7 shows that expression of CFTR H1085R in the presence of 10 µM CFpot-532 promoted maturation of the protein. Therefore, CFpot-532 could rescue processing mutants with mutations in either half of the molecule.

View larger version (37K): [in this window] [in a new window]   Fig. 7. Effect of CFpot-532 on maturation of CFTR with a processing mutation in the COOH half of the molecule. HEK 293 cells were transiently transfected with CFTR wild-type or H1085R cDNAs and then expressed in the absence (–) or presence (+) of 10 µM CFpot-532 for 48 h. Whole-cell extracts were subjected to immunoblot analysis with a CFTR polyclonal antibody. The positions of mature (Mature) and immature (Immature) CFTRs are indicated.

	Discussion

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The results from this study show that the pharmacological chaperones CFpot-532 and corr-2b could specifically rescue CFTR processing mutants without promoting maturation of a P-glycoprotein processing mutant. It is interesting to note that corr-2b and CFpot-532 share some structural similarities. Both compounds are small and consist of a central 5-member ring (pyrazole and thiazole for CFpot-532 and corr-2b, respectively) with phenyl rings linked to two sites (Fig. 1A). Perhaps they could interact with CFTR at similar sites to correct folding. Although there is no direct evidence that corr-2b binds directly to CFTR as it does not activate CFTR channel activity (Pedemonte et al., 2005), CFpot-532 has been demonstrated to increase F508 CFTR channel activity (this study) (Van Goor et al., 2006). CFpot-532 acts as a CFTR potentiator because it only affects channel gating after addition of forskolin. The major effect of CFpot-532 on F508 CFTR was to increase the open probability (P_o) of the channel (Van Goor et al., 2006). Treatment of F508 CFTR with CFpot-532 in the presence of ATP and protein kinase A increased P_o 4-fold so that it was similar to that of wild-type CFTR. It was recently shown that F508 CFTR shows a 3- to 4-fold lower P_o than wild-type CFTR because the Phe508 aromatic side chain plays a key role in determining the residency time in the closed state (Cui et al., 2006). Although CFpot-532 was not as efficient a corrector of F508 CFTR maturation compared with CFcor-325 or corr-4a (Fig. 3B), it has the ability to act as both a corrector and potentiator of CFTR processing mutants. Therefore it may be a good lead compound to try to make derivatives with better corrector abilities but retain its potentiator characteristics.

Examples of corrector compounds from two other classes of compounds, such as corr-3a (2-quinazolinyl-4-aminopyrimidinone) or corr-4a and corr-4b (bisaminomethylbithiazoles) were not specific for CFTR because they could also rescue the P-glycoprotein processing mutant. There was good correlation between compounds that promoted maturation of P-glycoprotein mutant G268V and those that affected its ATPase activity. Compounds CFcor-325, corr-3a, corr-4a, and corr-4b all rescued P-glycoprotein G268V and could also stimulate or inhibit P-glycoprotein ATPase activity. Therefore, it is likely that they all rescue P-glycoprotein G268V through a direct interaction with the protein. We have demonstrated previously that P-glycoprotein processing mutants are rescued by substrates and modulators through interactions with the transmembrane domains (Loo and Clarke, 1999b). Therefore, we predict that specific rescue of CFTR processing mutants with CFpot-532 or corr-2b is mediated through interactions with the transmembrane domains.

In a previous study, it had been postulated that bisaminomethylbithiazoles such as corr-4a and corr-4b were specific pharmacological chaperones for CFTR because they did not rescue a dopamine receptor (DRD4) processing mutant (M345T) (Pedemonte et al., 2005). Our results show, however, that these compounds are not specific for CFTR because they could also rescue a P-glycoprotein processing mutant (G268V, Fig. 5).

In summary, the results show that some correctors are specific for CFTR (CFpot-532, corr-2b), whereas others, such as CFcor-325 and corr-3a, could rescue both CFTR and P-glycoprotein and may act through an indirect pathway. The ability to directly rescue CFTR processing mutants would be beneficial in that they would have less effect on other cellular processes. In addition, compounds that are not substrates of P-glycoprotein would be better candidates to administer orally to cystic fibrosis patients because they could bypass P-glycoprotein in the gut, liver and kidney (Loo and Clarke, 2005a).

	Acknowledgements

 
We thank the Cystic Fibrosis Foundation (USA) and Vertex Corporation (San Diego, CA) for the generous gift of CFcor-325 and CFpot-532.

	Footnotes

 
This work was supported by a grant from the Canadian Institutes of Health Research. D.M.C. is the recipient of the Canada Research Chair in Membrane Biology.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.106.023994.

ABBREVIATIONS: CFTR, cystic fibrosis transmembrane conductance regulator; CFcor-325, 4-cyclohexyloxy-2-{1-[4-(4-methoxy-benzenesulfonyl)-piperazin-1-yl]-ethyl}-quinazoline; corr-2b, 2-phenylamino-4-(4-ethylene-phenyl)-thiazole; corr-3a, 2-quinazolinyl-4-aminopyrimidinone; corr-4a, bisaminobithiazole CFTR corrector-4a; corr-4b, bisaminobithiazole CFTR corrector-4b; CFpot-532, 4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)-phenol; BHK, Baby hamster kidney; endo H, endoglycosidase H; PNGase F, peptide N-glycosidase F; HEK, human embryonic kidney.

Address correspondence to: David M. Clarke, Department of Medicine, University of Toronto, Rm. 7342, 1 King's College Circle, Toronto, ON M5S 1A8, Canada. E-mail: david.clarke{at}utoronto.ca

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