Correctors and Potentiators Rescue Function of the Truncated W1282X-Cystic Fibrosis Transmembrane Regulator (CFTR) Translation Product*♦

W1282X is the fifth most common cystic fibrosis transmembrane regulator (CFTR) mutation that causes cystic fibrosis. Here, we investigated the utility of a small molecule corrector/potentiator strategy, as used for ΔF508-CFTR, to produce functional rescue of the truncated translation product of the W1282X mutation, CFTR1281, without the need for read-through. In transfected cell systems, certain potentiators and correctors, including VX-809 and VX-770, increased CFTR1281 activity. To identify novel correctors and potentiators with potentially greater efficacy on CFTR1281, functional screens were done of ∼30,000 synthetic small molecules and drugs/nutraceuticals in CFTR1281-transfected cells. Corrector scaffolds of 1-arylpyrazole-4-arylsulfonyl-piperazine and spiro-piperidine-quinazolinone classes were identified with up to ∼5-fold greater efficacy than VX-809, some of which were selective for CFTR1281, whereas others also corrected ΔF508-CFTR. Several novel potentiator scaffolds were identified with efficacy comparable with VX-770; remarkably, a phenylsulfonamide-pyrrolopyridine acted synergistically with VX-770 to increase CFTR1281 function ∼8-fold over that of VX-770 alone, normalizing CFTR1281 channel activity to that of wild type CFTR. Corrector and potentiator combinations were tested in primary cultures and conditionally reprogrammed cells generated from nasal brushings from one W1282X homozygous subject. Although robust chloride conductance was seen with correctors and potentiators in homozygous ΔF508 cells, increased chloride conductance was not found in W1282X cells despite the presence of adequate transcript levels. Notwithstanding the negative data in W1282X cells from one human subject, we speculate that corrector and potentiator combinations may have therapeutic efficacy in cystic fibrosis caused by the W1282X mutation, although additional studies are needed on human cells from W1282X subjects.

and conditionally reprogrammed cells generated from nasal brushings from one W1282X homozygous subject. Although robust chloride conductance was seen with correctors and potentiators in homozygous ⌬F508 cells, increased chloride conductance was not found in W1282X cells despite the presence of adequate transcript levels. Notwithstanding the negative data in W1282X cells from one human subject, we speculate that corrector and potentiator combinations may have therapeutic efficacy in cystic fibrosis caused by the W1282X mutation, although additional studies are needed on human cells from W1282X subjects.
Premature termination codons (PTCs, 5 or nonsense mutations) in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are found in nearly 7% of patients with CF (1). PTCs result in synthesis of truncated protein, generally without normal function, as well as reduced transcript levels caused by nonsense-mediated mRNA decay (NMD) (2,3). NMD suppressors that restore gene function by promoting incorporation of the correct or a near-cognate aminoacyl tRNA into nascent polypeptides represent attractive drug development candidates (2,4,5). The read-through candidate drug Ataluren (PTC124) is under evaluation for CF caused by PTCs, although initial phase III clinical trial data did not show efficacy (6,7), and some laboratories were unable to demonstrate Ataluren-mediated translation read-through in various systems (8). One limitation of therapeutics causing NMD suppression is insertion of incorrect amino acids at the PTC site.
The W1282X mutation, which truncates CFTR to remove ϳ60% of nucleotide binding domain 2 (NBD2; Fig. 1A, top), is the fifth most common CF-causing mutation worldwide with a prevalence of ϳ50% in Ashkenazi Jewish subjects with CF (1). Motivated by the fact that the W1282X mutation is predicted to generate a truncated protein (CFTR 1281 ) constituting most of the full-length wild type CFTR (1480 amino acids), and postulating that CFTR 1281 has defective cellular processing and gat-ing, we investigated whether functional rescue of CFTR 1281 is possible using correctors and/or potentiators, a strategy used for CF caused by the ⌬F508 mutation as well as G551D and other CFTR missense mutations (9 -11).
Several lines of evidence suggest multiple defects in W1282X-CFTR and support corrector, potentiator, and readthough strategies (12). Transcript encoding W1282X-CFTR was seen in nasal epithelial cells of homozygous W1282X CF subjects with 30 -80% abundance compared with that of wild type CFTR in non-CF subjects (13). In bronchial epithelial cells virally infected to express W1282X-CFTR cDNA, butyrate-induced up-regulation of truncated CFTR expression produced cAMP/genistein-stimulated chloride conductance (14), and in the same cell model, VX-770 produced an ϳ4-fold increase in CFTR activity despite the apparent absence of full-length protein (15). Patch clamp experiments showed that VX-770 in combination with curcumin produced significant activation of W1282X-CFTR despite evidence of impaired ATP-dependent NBD1-NBD2 dimerization (16). Biochemical and functional approaches using heterologous expression systems have demonstrated that NBD2 is not required for efficient cell-surface presentation or CFTR activity, albeit with reduced open probability (17,18). Partial deletion of NBD2 reduces channel biosynthetic processing and post-Golgi stability (18 -21), probably by interfering with NBD2 folding and cooperative CFTR domain assembly (18).
In this study, we demonstrate the activity of available correctors and potentiators in transfected cells expressing the truncated protein product of the W1282X CFTR gene (CFTR 1281 ), identify improved correctors and potentiators by high throughput screening, and test compounds in human nasal epithelial cell cultures from a W1282X-homozygous CF subject. These studies were presented in part in abstract form at the 29th North American CF Conference in October, 2015 (22).

CFTR Correctors Stabilize W1282X-CFTR Translational
Products in Respiratory Epithelial Cells-To test whether the W1282X-CFTR gene product is susceptible to pharmacological modulation, initial biochemical assessment was done in a CF bronchial epithelial cell line (CFBE41oϪ, designated as CFBE) lacking endogenous CFTR expression (23). Lentiviral vectors were generated to express W1282X-CFTR translational products stably under the control of the tetracycline transactivator as described (24). Two expression cassettes were used. The first contained a stop codon inserted into the full-length cDNA (W1282X-CFTR), permitting expression of both the full-length channel and truncated protein product (CFTR 1281 ). The second encoded CFTR 1281 (Fig. 1A). Both constructs were engineered with a triplet hemagglutinin epitope tag (3HA) in the fourth extracellular loop of CFTR to monitor cell-surface expression in a live cell enzyme-linked immunosorbent assay format (25).
Immunoblot analysis in combination with endo H and PNGase F digestion showed the expression of core-glycosylated (band B, endo H-sensitive; black arrowhead) and complex-glycosylated (band C, PNGase F-sensitive; white arrowhead) forms of both W1282X-CFTR and CFTR 1281 (Fig. 1B). The apparent molecular mass of the core-and complex-glycosylated forms of CFTR 1281 and W1282X-CFTR was reduced compared with wild type CFTR. Assuming comparable transcript levels, these observations suggest that that the truncated CFTR 1281 has inefficient biosynthetic processing and/or impaired stability. Supporting inefficient processing, the known ⌬F508-CFTR correctors VX-809 and corr4a (C4) increased the complex-glycosylated forms of CFTR 1281 and W1282X-CFTR, as well as their plasma membrane expression as assessed by immunoblot analysis and live-cell ELISA (Fig. 1, C and D). As evidence for improved CFTR 1281 processing in the presence of VX-809, metabolic pulse-chase labeling with [ 35 S]methionine and [ 35 S]cysteine in polarized CFBE cell cultures showed ϳ75% increased incorporation of radiolabel into complex-glycosylated CFTR 1281 with VX-809 ( Fig. 1E).
High Throughput Assay Development for W1282X-CFTR Correctors and Potentiators-Motivated by the observations above, we developed a high throughput screen to identify correctors and potentiators of W1282X-CFTR. FRT cell lines were generated that stably express YFP-H148Q/I152L/F46L (YFP) and CFTR containing either the W1282X mutation (FRT-YFP-W1282X-CFTR) or CFTR 1281 (FRT-YFP-CFTR 1281 ); FRT cells were used because of their low intrinsic permeability to iodide and other CFTR-permeable ions and prior experience with these cells in various CFTR modulator screens (26,27). The corrector assay was done by 24-h incubation of cells with test compounds followed by brief (10 min) incubation with the cAMP agonist forskolin (fsk) and a potentiator prior to assay ( Fig. 2A, left). For potentiator assays, cells were incubated with a corrector to increase cell-surface expression of W1282X-CFTR and then briefly incubated (10 min) with test compounds and forskolin prior to assay ( Fig. 2A, right). CFTR activity was deduced from the initial rate of YFP fluorescence quenching in response to iodide addition to the extracellular solution.
Novel Small Molecule CFTR 1281 Correctors Identified by High Throughput Screening-Screening was done to identify novel CFTR 1281 correctors with improved efficacy over known ⌬F508-CFTR correctors. Screening of ϳ30,000 synthetic small molecules identified six compound classes that at 25 M nor- malized forskolin/VX-770-stimulated CFTR 1281 activity to Ͼ50% of that produced by 3 M VX-809. Fig. 3A shows chemical structures of the two most active compounds, W1282X corr -A23 and W1282X corr -B09, which are chemically distinct from VX-809. In YFP quenching assays, these compounds increased CFTR 1281 activity in a concentration-dependent manner producing ϳ5-fold greater maximal activity than VX-809 (Fig. 3B).
To quantify cell-surface expression, an FRT cell line expressing W1282X-CFTR containing a 3HA epitope tag in the fourth extracellular loop was generated. Cell-surface expression, as assayed in a live cell ELISA format, showed that the correctors VX-809, W1282X corr -A23, and W1282X corr -B09 (at 3 M) increased surface presentation of W1282X-CFTR in FRT cells with efficacy similar to that seen in the functional assays (Fig. 3C, top). To investigate whether CFTR 1281 or full-length (produced by read-through) CFTR was present at the cell surface in W1282X-CFTR-3HA-expressing cells, a luciferase-based W1282X read-through reporter was generated and expressed in FRT cells. As a positive control, the read-through compound geneticin (G418) produced a luciferase-dependent response, whereas W1282X corr -A23, W1282X corr -B09, and VX-809 did not (Fig. 3C, bottom). Together, these studies indicate that correctors identified by the screen, as well as VX-809, increase . Cells were treated with forskolin plus VX-770 for corrector assays, and cells were corrected with VX-809 and treated with forskolin for potentiator assays. Statistical analysis by ANOVA with Dunnett's post hoc test: for correctors, data were compared with cells treated with fsk/VX-770; for potentiators, data were compared with cells treated with fsk/VX-809 (*, p Ͻ 0.05; **, p Ͻ 0.001). D, original data (left) and summary (right; mean Ϯ S.E., n ϭ 3-6) for CFTR 1281 -mediated YFP quenching in FRT cells that are uncorrected or treated with correctors and/or potentiators (as in C). Statistical analysis as in C. Dashed lines in C and D represent unstimulated and maximal responses, respectively.

Pharmacological Correction of W1282X-CFTR
cell-surface CFTR 1281 in FRT cells rather than promoting read-through.

JOURNAL OF BIOLOGICAL CHEMISTRY 775
W1282X corr -A23, with near maximal activity at ϳ3 M. To investigate specificity of CFTR 1281 correctors, active analogs from three compound classes were compared at 3 M in CFTR 1281 and ⌬F508-CFTR-expressing cells. Whereas most known ⌬F508-CFTR correctors (VX-809, VX-661, C18, and Corr4a) were comparably active on both CFTR mutants, many of the CFTR 1281 correctors identified in the screen here were substantially more effective in CFTR 1281 -expressing cells ( Fig.  3G and Table 1). Although correctors with improved efficacy over VX-809 were identified by screening, subsequent assays were done with VX-809 as it is an approved drug.
Novel Small Molecule CFTR 1281 Potentiators Identified by High Throughput Screening-Initial experiments were done to characterize VX-770 as a potentiator of VX-809- corrected CFTR 1281 . Interestingly, increasing VX-770 increased CFTR 1281 activity without saturation at up to 200 M, as seen in both plate reader and short-circuit current assays (Fig. 4A, left, and data not shown). In contrast, ⌬F508-CFTR activity in VX-809-corrected cells was saturable, as expected (Fig. 4A, right). CFTR 1281 and ⌬F508-CFTR activities were fully blocked by CFTR inh -172. These data suggest that CFTR 1281 has very low open probably at clinically achievable concentrations of VX-770 but has the potential to be greatly activated.
A small molecule screen was done to identify novel CFTR 1281 potentiators as diagrammed in Fig. 2A (right), in which cells were incubated with VX-809 to increase cell-surface CFTR 1281 expression, and test compounds with forskolin were added 10 min prior to assay. Fig. 4B shows structures of five active compounds emerging from the screen, each of which is chemically distinct from VX-770. Concentration dependence measurements in VX-809-corrected CFTR 1281expressing cells showed that the new CFTR 1281 potentiators had similar or lower activity than VX-770 (Fig. 4C). Control experiments confirmed that the potentiators did not activate CFTR 1281 in the absence of forskolin and that YFP quenching did not occur in cells without CFTR 1281 . CFTR 1281 activity was confirmed by short-circuit current measurements in VX-809-treated cells, with data shown for W1282X pot -A15 and W1282X pot -C01 (Fig. 4D).
To estimate the CFTR 1281 channel activity in response to potentiators, cell-surface expression and channel activity were measured in FRT cells expressing epitope (3HA)-tagged wild type or W1282X-CFTR. Incubation of cells expressing CFTR- 3HA with forskolin produced robust current that was inhibited by CFTR inh -172 (Fig. 5C, top left). W1282X-CFTR-3HAexpressing cells corrected with W1282X corr -A23 produced CFTR channel activity in response to forskolin, VX-770, and W1282X pot -A15 that was inhibited by CFTR inh -172 (Fig. 5C, bottom left). Following normalization by cell-surface expression, deduced per-channel activity for CFTR 1281 relative to wild type CFTR (normalized to 1) is shown (Fig. 5C, right). Remarkably, the potentiator combination increased CFTR 1281 channel activity to that of wild type CFTR.
Structure-activity studies were done on 120 commercially available analogs of W1282X pot -A15 (Fig. 5D). Class A poten-tiators are composed of pyrrolo [2,3-b]pyridine with a phenylsulfonamide linked at the 3-position. Several alkyl groups (R 1 ) on the pyrrolo[2,3-b]pyridine were studied, and n-butyl and isobutyl groups gave active potentiators showing synergy with VX-770. Shorter alkyl group such as propyl and ethyl reduced activity. The R 2 substituent on the phenylsulfonamide ring also affected activity, with electron-neutral or donating groups such as ethyl, methyl, and methoxy giving the best activity, although halides such as fluoro and chloro reduced activity. The position of the substituent also affected activity, with para and ortho being more active than meta substitution, suggesting that electronic properties of the phenylsulfonamide ring affect activity.

Pharmacological Correction of W1282X-CFTR
Short-circuit current measurements were also done in VX-809-treated CFBE cells to confirm results from FRT cells in a human airway epithelial cell model (Fig. 5E). Little activation of CFTR 1281 was seen with VX-770 or W1282X pot -A15 alone; however, in combination these potentiators strongly increased channel activity (Fig. 5E).
Interestingly, we found that some of the new potentiators showed activity alone but limited synergy with VX-770, although others were relatively inactive alone but showed synergy with VX-770. Fig. 5F shows examples of each, with W1282X pot -B01 in the former category and W1282X pot -E01 in the latter.
Screen of Approved and Investigational Drugs for Synergy with VX-770 -Motivated by the marked synergy of VX-770 with some potentiators, we carried out a "synergy screen," reasoning that repurposing of an existing drug or bioactive molecule for use in combination with VX-809 and VX-770 might accelerate drug therapy for CF caused by the W1282X mutation. The screen might also identify bioactive compounds that are effective as potentiators when used alone. Screening was done using a collection of ϳ2600 bioactive compounds, natural products, and approved drugs that were added acutely with forskolin and VX-770 to VX-809-corrected CFTR 1281 -expressing cells (Fig. 6A). Screening identified several compounds that increased CFTR 1281 activity above that produced by VX-770 and forskolin, including the flavones apigenin, genistein, and kaempferol, and the flavone-like anti-inflammatory compound isoliquiritigenin (Fig. 6B). Fig. 6C shows original YFP fluorescence quenching data and concentration-activity curves. Compounds did not increase CFTR 1281 function when used alone (Fig. 6D). Apigenin and genistein had limited potentiator activity with forskolin, whereas isoliquiritigenin and kaempferol were inactive as potentiators. I sc data for apigenin, the most potent compound identified in the synergy screen, revealed limited CFTR 1281 activity when used alone but robust CFTR 1281 activity in the presence of VX-770 (Fig. 6E). CFTR 1281 activation by apigenin in the presence of VX-770 had an EC 50 of ϳ20 M (Fig. 6F), ϳ4-fold less potent than W1282X pot -A15. As found for W1282X pot -A15, activation of CFTR 1281 by VX-770 and apigenin was independent of the order of addition (data not shown). Comparing with cells incubated with W1282X pot -A15, the per-channel activity of CFTR 1281 relative to wild type CFTR in cells incubated with VX-770/apigenin is ϳ0.6 and with VX-770/genistein was ϳ0.2. Experiments in VX-809-treated CFBE cells confirmed synergy of VX-770 with apigenin (Fig. 6G).
Corrector and Potentiator Testing in W1282X Homozygous Human Airway Epithelial Cells-Compounds were tested in airway epithelial cells from a homozygous W1282X-CFTR subject. Initial experiments were done with airway epithelial cells from non-CF and homozygous ⌬F508-CFTR subjects to confirm the utility of conditionally reprogrammed cells (CRCs) for these studies. Primary and CRCs from a non-CF subject showed similar responses when treated with amiloride (ENaC channel blocker), forskolin, CFTR inh -172, and ATP (activator of calcium-activated chloride channels) (Fig. 7A). Also, primary and conditionally reprogrammed human bronchial epithelial cells from a homozygous ⌬F508-CFTR CF subject showed similar responses following correction with VX-809, with no activity in the absence of corrector (Fig. 7B). These studies support the use of CRCs to study pharmacology of CFTR modulators, which allows considerable expansion of cell cultures derived from rare patient specimens, as needed for the W1282X studies here.
Nasal epithelial cells were obtained by brushings from a single W1282X-CFTR homozygous CF subject. In primary cells, VX-770 and W1282X pot -A15 produced no significant CFTR current without or with VX-809 incubation (Fig. 7C). The amiloride and ATP responses were similar in these cells to those in non-CF and ⌬F508-CFTR P0 cultures and CRCs. No CFTR current was observed under the same experimental conditions using CRCs generated from nasal epithelial cells from the same CF subject (data not shown). Further experiments tested the activity of read-through compounds in CRCs generated from the W1282X-CFTR P0 cultures. As with P0 cultures, CRCs without and with VX-809 showed no response to CFTR modulators (Fig. 7D). VX-809-corrected W1282X-CFTR CRCs treated with the read-through agents PTC124/ataluren (6), amlexanox (30), and geneticin (14) produced no significant CFTR current in response to correctors and potentiators but showed robust amiloride and ATP responses.
Quantitative PCR was done to determine CFTR transcript levels in the human airway epithelial cell models (Fig. 7E). In nasal brushings, CFTR transcript was reduced only by ϳ20% in CF cells as compared with a non-CF subject (Fig. 7E, top left). In P0 cultures, non-CF nasal and bronchial airway epithelial cells had similar CFTR transcript levels (data not shown), and cells from the W1282X-CFTR subject had ϳ50% less CFTR transcript (Fig. 7E, top right). CRCs from non-CF and W1282X-CFTR subjects had similar levels of CFTR transcript (Fig. 7E,  bottom left). Finally, in P0 cultures and CRCs from non-CF or W1282X-CFTR subjects, CFTR transcript levels were similar, suggesting that reprogramming does not greatly reduce CFTR transcript. First passage CRC cultures were morphologically

Pharmacological Correction of W1282X-CFTR
indistinguishable from P0 cultures with both showing pseudostratified and ciliated differentiation (data not shown).

Discussion
Therapeutic approaches for CF caused by the W1282X mutation and other PTC mutations such as G542X have focused on promoting read-through to generate full-length CFTR protein (5). The limited efficiency of read-through and function of the translated full-length proteins, which generally contain non-native amino acids at the PTC, are major challenges in this approach. We demonstrate here that in the absence of read-through the truncated protein product generated by the W1282X mutation (CFTR 1281 ) has the potential to be efficiently processed and gated using combinations of small molecules. The major novel findings of this work include the application of correctors to facilitate processing of CFTR 1281 , the identification of correctors and potentiators with much greater efficacy than existing compounds, and the discovery of potentiator combinations that restore CFTR 1281 channel conductance to that of wild type CFTR. However, whether the findings from cell cultures models will translate to native human airway epithelial cells remains uncertain at this time, as corrector and potentiator combinations did not show efficacy in airway epithelial cell cultures from a single homozygous W1282X CF subject.
The results here suggest that pharmacological activation of CFTR 1281 may require triple drug combination therapy with a corrector and two potentiators acting in synergy. Of note, therapies for ⌬F508-CFTR in development include a single potentiator and two correctors, supporting the viability of triple drug CFTR modulator therapy for CF (25,(31)(32)(33). High throughput screening in this study produced novel chemical scaffolds that corrected CFTR 1281 trafficking with greater efficacy than existing ⌬F508-CFTR correctors. Perhaps second and third generation correctors under development for ⌬F508-CFTR may be active as CFTR 1281 correctors; and VX-770 together with newer potentiators with improved activity may act in synergy on CFTR 1281 .
Although this study was focused on the W1282X-CFTR mutation, the findings may have relevance to CF associated with a variety of different CF-causing CFTR mutations. Several PTC mutations, including W1274X, Q1281X, Q1291X, Y1307X, Q1313X, Q1412X and S1455X, and frameshift mutations, such as c.3855delC, c.3884_3885insT, c3890_3891insT, c3908dupA, c.4139delC, and c.4147_4148insA, result in deletion of various lengths of the carboxyl terminus of CFTR within NBD2. Corrector/potentiator strategies may be useful for CF patients with these CFTR mutations. Also, the activity of ⌬F508-CFTR modulators such as VX-809 and VX-770 on CFTR 1281 shown here may be of benefit in compound heterozygous CF patients having W1282X along with ⌬F508 or various gating mutations.
An important finding of this study is that potentiator combinations can activate CFTR 1281 gating to near-wild type levels, whereas single potentiators only produce limited channel opening. Although beyond the scope of this study, investigation of the mechanisms responsible for this observation will benefit from information on the structure of CFTR 1281 in the presence of potentiators. We speculate that binding of both potentiators is sufficient to induce CFTR 1281 conformational changes such that the channel assumes a structure with wild type characteristics. During completion of this manuscript, Wang et al. (16) reported synergistic stimulation of CFTR 1281 by VX-770 and the herbal supplement curcumin; patch clamp of FRT cells expressing W1282X-CFTR showed an increase in P o from Ͻ0.01 to ϳ0.2 with VX-770 or curcumin and both compounds increased P o to ϳ0.9 (16). Our study here identified flavones as additional bioactive compounds that activate CFTR 1281 in synergy with VX-770. Curcumin is unlikely to be useful clinically as a CFTR 1281 potentiator because of its low potency and poor bioavailability (34). In contrast, flavones represent potential candidates for drug development because of their slow metabolism, good bioavailability, and predominantly renal excretion (35,36).
A disappointing finding was that various combinations of potentiators and correctors, even together with read-through drugs, did not produce measurable chloride currents in primary and conditionally reprogrammed nasal epithelial cell cultures generated from a single homozygous W1282X CF subject. Quantitative PCR showed that transcript levels in CF cell models used in this study were not much lower than those in non-CF cells, suggesting that very low levels of CFTR transcript in the human cells tested here are not responsible for the absence of channel activity. Perhaps patient variability and subject-specific phenotypic factors might modulate drug responses, as seen for ⌬F508, mandating the need for drug testing in cells from other W1282X CF patients. It is well recognized that the efficacy of ⌬F508-CFTR correctors can be cell type-dependent (37,38), as correctors identified in transfected cells often are not effective in patient-derived human airway epithelial cells. However, potentiators tend to be cell context-independent as they likely target mutant CFTRs at the plasma membrane directly.
There are limited prior reports on the biological properties of the 1-arylpyrazole-4-arylsulfonyl-piperazine corrector scaffold identified in this study. Some naphthalene arylsulfonyl-piperazines were identified as inhibitors of activated coagulation factor X (39) and human 11␤-hydroxysteroid dehydrogenase type 1 (40). Limited biological data have been reported for the phenylsulfonamide-pyrrolopyridine class of W1282X potentiators. Some phenylsulfonamide-pyrrolopyridines were found as cannabinoid agonists for therapy for pain and nausea (41). Virtual screening also identified phenylsulfonamide-pyrrolopyridines as potential inhibitors of heat shock protein Hsp90 (42). The arylpyrazole-arylsulfonyl-piperazine and phenylsulfonamidepyrrolopyridine scaffolds have favorable drug-like properties, including the presence of multiple hydrogen bond acceptors, molecular masses of 396 and 357 Da, aLogP values of 3.2 and 3.8, and topological polar surface areas of 79.4 and 61.8 Å 2 , respectively (43,44). In addition, these scaffolds do not belong to promiscuous binder families known as pan-assay interference compounds (45). Further medicinal chemistry to generate targeted analogs may give compounds with improved potency.
In summary, the studies here support the potential utility of small molecule correctors and potentiators as a therapeutic approach for CF caused by the W1282X mutation and perhaps

Pharmacological Correction of W1282X-CFTR
for other CF-causing mutations in the carboxyl-terminal region of CFTR. Although compounds did not produce measurable current in nasal epithelial cells from a single homozygous W1282X CF subject, further testing of CFTR 1281 correctors and potentiators in cells from additional W1282X homozygous subjects may show benefit, as might testing in subjects with appropriate compound mutations such as W1282X/G542X.

Experimental Procedures
Chemicals-VX-809, VX-661, VX-770, and CFTR inh -172 were purchased from Selleck Chemicals (Boston, MA). The previously described potentiators and correctors (prefixed with P and C, respectively) P3, P8, P9, C3, C7, and C18 were obtained from the CFTR Compound Program, The Cystic Fibrosis Foundation Therapeutics Inc., which is administered at Rosalind Franklin University of Medicine and Science; Corr4a (C4), CoPo-22, PG01, A04, and P12 were obtained from an in-house repository of CFTR modulators. Geneticin (G418) was purchased from Gibco-Life Technologies, Inc. Screening was done using a collection of ϳ30,000 drug-like synthetic small molecules (Chem-Div, San Diego) and collections of approved and investigational drugs (ϳ2500 molecules; Microsource Spectrum Library, Gaylordsville, CT; the National Institutes of Health Clinical Collection, Biofocus, South San Francisco, CA). All other chemicals were purchased from Sigma unless otherwise stated.
Complementary DNA Constructs-Expression cassettes containing CFTR cDNA were generated in pcDNA3.1/Zeo(ϩ) (Invitrogen) that contains a cytomegalovirus intermediateearly (CMV) promoter. Constructs were generated in two steps. The 5Ј-region of CFTR (ϳ3.8 kb) was excised as a KpnI-BstXI fragment from a plasmid encoding enhanced GFP-CFTR (46). Subsequently, gBlock gene fragments (Integrated DNA Technology, Coralville, IA) were synthesized to regenerate the 3Ј-region of wild type and mutant CFTR cDNAs (Fig. 1A, bottom). Gene fragments were designed to contain an endogenous BstXI site and an engineered XhoI site for cloning, together with the following: CFTR cDNA with its endogenous stop codon for wild type CFTR (ϳ600 bp); CFTR cDNA, including the W1282X mutation and its endogenous stop codon for W1282X-CFTR (ϳ600 bp); or CFTR cDNA up to Gln 1281 and tandem stop codons (TGA TAG TAA) for CFTR 1281 (ϳ120 bp). To generate engineered CFTR constructs containing 3HA in the fourth extracellular loop, a cDNA region encompassing the 3HA tag was excised as an EcoRI-EcoRI fragment from a previously described CFTR-3HA construct (47) and ligated into wild type-and W1282X-CFTR cDNAs contained in pcDNA3.1/Zeo(ϩ). To measure W1282X read-through, a reporter (GFP-W1282X-luc) was generated in pcDNA3.1/ Zeo(ϩ) using gBlock gene fragments consisting of GFP, CFTR cDNA from residue Asp 1270 to Phe 1286 , including the W1282X PTC, and Photinus pyralis luciferase. Constructs were fully sequenced. Lentiviral particles expressing W1282X-CFTR and CFTR 1281 were generated using the Lenti-X TenON Advanced Inducible Expression System (Clontech) and the pLVX-tightpuro transfer vector as described (24).
Cell Culture Models-Fischer rat thyroid (FRT) cells were cultured in Kaign's modified Ham's F-12 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 units/ml penicil-lin, 100 g/ml streptomycin, 18 g/ml myoinositol, and 45 g/ml ascorbic acid. For screening, clonal FRT cell lines were generated in two steps. Cells expressing EYFP-H148Q/I152L/ F46L (YFP) were generated using the FELIX third generation feline immunodeficiency lentiviral system (deposited at Addgene as a gift by Garry Nolan, plasmid 1728); subsequently, cells were transfected with pcDNA3.1/Zeo(ϩ) plasmids encoding W1282X-CFTR or CFTR 1281 , and clonal cell lines were selected using 0.25 g/ml Zeocin. To study CFTR cell-surface presentation, cell lines expressing CFTR and W1282X-CFTR containing extracellular 3HA tags were generated in a similar manner. A cell line expressing ⌬F508-CFTR was described previously (27). To generate an FRT cell model to probe W1282X readthrough, cells were transfected with plasmid encoding GFP-W1282X-luc and enriched for reporter expression by culture with 0.25 g/ml Zeocin. CFBE41oϪ (CFBE) cells expressing tetracycline-inducible W1282X-CFTR and CFTR 1281 with 3HA tags were generated and cultured as described (24).
Biochemistry-Protein expression of CFTR-3HA variants was monitored by Western blotting of CFBE cell lysates using anti-hemagglutinin (HA) antibody (clone 16B12; Biolegend, San Diego) and enhanced chemiluminescence detection, as described (48). To monitor N-linked glycans as an indicator of CFTR biosynthetic processing, high molecular weight mannose N-glycans or all N-glycans were cleaved by incubating cell lysates with 10 g/ml endo H or 40 g/ml PNGase F (New England Biolabs, Ipswich, MA), respectively, for 2 h at 35°C, followed by immunoblotting (49). CFTR 1281 maturation efficiency in CFBE cells was measured by metabolic pulse-chase as described (50). Briefly, CFBE cells expressing CFTR 1281 were pulse-labeled with 0.2 mCi/ml [ 35 S]methionine and [ 35 S]cysteine (EasyTag Express Protein Labeling Mix, PerkinElmer Life Sciences) in cysteine-and methionine-free medium for 1 h at 37°C and then chased for 2 h at 37°C in normal medium. Radioactivity incorporated into the core-and complex-glycosylated glycoproteins was visualized by fluorography and quantified by phosphorimage analysis using a Typhoon imaging platform (GE Healthcare). Relative maturation efficiency of CFTR 1281 in the presence of VX-809 was determined by comparison of radioactivity incorporated into the complex-and core-glycosylated forms of CFTR, with efficiency expressed as fold change relative to control measured in the presence of DMSO.
Primary and Conditionally Reprogrammed Cultures of Human Nasal Epithelia-Human tissues were acquired and used with approval from the University of California, San Francisco Committee on Human Research. Nasal brushings were obtained on two occasions from a single CF subject homozygous for the W1282X mutation in CFTR, with no additional mutations identified by sequencing. Tissue isolated by brushing, consisting primarily of small aggregates of nasal mucosa and mucus, was rinsed three times in PBS with antibiotics (penicillin, 100 units/ml; streptomycin, 100 g/ml; gentamicin, 100 g/ml; amphotericin B, 2.5 g/ml) and 5 mM dithiothreitol and one time in PBS with antibiotics prior to overnight incubation in PBS with antibiotics and 0.025% type XIV protease at 4°C. A 1:1 mixture of DMEM and Ham's F-12 (DF12) containing 5% FBS and antibiotics was used to neutralize proteases and, as needed, trypsin (0.05%), or agitation was used to disperse remaining cell clumps. For some experiments, isolated nasal epithelial cells were plated on permeable tissue culture inserts (12 mm diameter, 0.4-m polyester membrane; Corning Costar Transwell) for polarized growth (termed P0 cultures) as described (25). Conditional reprogramming of nasal epithelial cells was performed using a modification of reported methods (51,52). Briefly, 3T3-L1 fibroblasts (obtained from the UCSF Cell Culture Facility) were suspended in F-medium (3:1 Ham's F-12/DMEM, supplemented with 5% FBS, 5 g/ml bovine insulin, 8.4 ng/ml cholera toxin, 10 ng/ml recombinant human EGF (Atlanta Biologicals, Norcross, GA), 25 ng/ml hydrocortisone, 1 M Y27632-ROCK inhibitor (Enzo Life Sciences, Farmingdale, NY), penicillin/streptomycin (as above), 250 g/ml amphotericin B, and 10 g/ml gentamicin) and irradiated in a Gammacell 3000 Elan (Best Theratronics, Springfield, VA) at 30 gray. Irradiated cells were then plated (3 ϫ 10 6 per 10-cm tissue culture dish) and allowed to attach for 2 h. Nasal cells (1.5 ϫ 10 6 ) were added to each dish and allowed to proliferate for 4 -7 days and were subsequently harvested by differential trypsinization when they reached ϳ80% confluence. Conditionally reprogrammed cells were subsequently plated (5 ϫ 10 5 cells/cm 2 ) on tissue culture inserts coated with human placental collagen (20 g/cm 2 ) in DF12 containing 5% FBS and antibiotics (penicillin, 100 units/ml; streptomycin, 100 g/ml) to generate differentiated nasal epithelial cell sheets (termed P1 CRCs). After 24 h, the inserts were rinsed with PBS containing antibiotics and cultured with UNC-ALI media as described (25). CFTR transcript levels in human cells were measured by TaqMan quantitative RT-PCR using the Hs00357011_m1 probe set (Thermo Fisher Scientific, Waltham, MA) with data normalized to GAPDH transcript levels. Experiments were done at the UCSF Helen Diller Cancer Center Genome Analysis Core.
Screening Procedures-High throughput screening used a semi-automated screening platform (Beckman, Fullerton, CA) configured as described (53). FRT cells were plated in 96-well black-walled, clear-bottom tissue culture plates (Corning) at a density of 20,000 cells/well and cultured to confluence over 48 h prior to assays. For the corrector screen ( Fig. 2A, left), cells expressing W1282X-CFTR and YFP were treated with test compounds in culture medium for 1 day, then washed twice with PBS, and incubated for 10 min in 100 l of PBS containing forskolin (10 M) and VX-770 (5 M) prior to assay of CFTR activity. All plates in the corrector screen contained wells with positive (3 M VX-809, 5 M VX-770, and 10 M forskolin) and negative (5 M VX-770 and 10 M forskolin) controls. For the potentiator screen ( Fig. 2A, right), cells expressing CFTR 1281 were cultured in medium containing VX-809 (3 M) for 1 day, washed twice with PBS, and then incubated for 10 min in 100 l of PBS containing forskolin (10 M) and test compounds prior to assay. All plates in the potentiator screen contained positive (3 M VX-809, 5 M VX-770, and 10 M forskolin) and negative (3 M VX-809 and 10 M forskolin) controls. A "synergy" screen to investigate combined effects of VX-770 with bioactive molecules and approved drugs was done using cells expressing CFTR 1281 in a similar manner as the potentiator screen, with the exception that test wells contained VX-770 (5 M). Assays were performed using a BMG Labtech FLUOstar OMEGA (Cary, NC) over 12 s with initial fluorescence intensity recorded for 1 s prior to additional of 100 l of NaI-substituted PBS (137 mM NaCl replaced with NaI). Initial I Ϫ influx was computed from fluorescence intensity by single exponential regression (26). Control experiments were done using FRT cells expressing EYFP-H148Q/I152L/F46L alone.
I sc Measurements-Measurements of I sc were done with cells cultured on permeable supports as described (27). For FRT cells, the basolateral membrane was permeabilized with 250 g/ml amphotericin B, and experiments were done using a HCO 3 Ϫ -buffered system (in mM: 120 NaCl, 5 KCl, 1 MgCl 2 , 1 CaCl 2 , 5 Hepes, 25 NaHCO 3 , pH 7.4) with a basolateral to apical chloride gradient (generated by replacing 60 mM NaCl with sodium gluconate in the apical solution). For nasal epithelial cells, experiments were done using symmetrical HCO 3 Ϫ -buffered solutions (containing 120 mM NaCl). Modulators of ion conductance were added to both apical and basolateral bathing solutions, and cells were equilibrated with 95% O 2 , 5% CO 2 and maintained at 37°C.
CFTR Cell Surface Assays-FRT cells expressing 3HA-tagged CFTR constructs were washed in PBS (supplemented with 0.5% BSA) and incubated at 4°C for 10 min prior to incubation with anti-hemagglutinin antibody (2 g/ml, 1 h, 4°C; clone 16B12, Biolegend, San Diego, CA), HRP-conjugated secondary antibody (0.16 g/ml, 1 h, 4°C; goat anti-mouse IgG antibody, Thermo Scientific, Waltham, MA), and Western Bright Sirius luminescence detection reagent (Advansta Inc., Menlo Park, CA). Luminescence representing surface CFTR was detected using a Tecan Infinite F500 plate reader (San Jose, CA). CFTR activity was measured by I sc for computation of relative channel activity. A similar approach was used for CFBE cells but with Amplex-Red (Molecular Probes, Waltham, MA) HRP substrate to measure CFTR cell-surface density (50).
Author Contributions-P. M. H. conceived the study, designed experiments, generated cDNA constructs and cell models used in the study, conducted read-through and surface presentation assays, analyzed results, and wrote the paper. P. W. P. conducted structureactivity studies and electrophysiological experiments using FRT and reprogrammed cell models, analyzed results, and wrote aspects of the paper. J. A. T. conducted HTS and structure-activity studies, and analyzed data. H. X. participated in generation of lentiviral expression constructs and performed biochemical analysis. R. G. A. participated in generation of lentiviral expression constructs, performed electrophysiological studies on CFBE cultures, and was responsible for the initial observation of combined VX-770/genistein potentiation. D. P. performed pulse-chase studies. L. Z. cultured primary and conditionally reprogrammed human cell cultures. D. W. N. acquired patient samples. W. E. F. conceived certain studies, designed experiments, and wrote aspects of the paper. G. L. L. and A. S. V. conceived the study, designed experiments, and wrote the paper.