ABCMdb

PMID: 19846789

Van Goor F, Hadida S, Grootenhuis PD, Burton B, Cao D, Neuberger T, Turnbull A, Singh A, Joubran J, Hazlewood A, Zhou J, McCartney J, Arumugam V, Decker C, Yang J, Young C, Olson ER, Wine JJ, Frizzell RA, Ashlock M, Negulescu P
Rescue of CF airway epithelial cell function in vitro by a CFTR potentiator, VX-770.
Proc Natl Acad Sci U S A. 2009 Nov 3;106(44):18825-30. Epub 2009 Oct 21., 2009-11-03 [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC7 p.Gly551Asp In recombinant cells VX-770 increased CFTR channel open probability (Po) in both the F508del processing mutation and the G551D gating mutation. Login to comment 5 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp VX-770 also increased Cl-secretion in cultured human CF bronchial epithelia (HBE) carrying the G551D gating mutation on one allele and the F508del processing mutation on the other allele by Ϸ10-fold, to Ϸ50% of that observed in HBE isolated from individuals without CF. Login to comment 19 ABCC7 p.Gly551Asp ABCC7 p.Arg117His Although less common, CFTR mutations that primarily impair the ability of CFTR at the cell surface to open (e.g., G551D) or conduct Cl- or HCO3 - (e.g., R117H) are also found in CF patients (5, 18). Login to comment 22 ABCC7 p.Gly551Asp In a Phase 2 clinical study in CF patients carrying the G551D CFTR mutation, oral administration of VX-770 increased CFTR activity as determined by improvement in biomarkers of CFTR function and improved lung function (20). Login to comment 23 ABCC7 p.Gly551Asp The results shown here indicate that VX-770 increased the open probability (Po) of the CFTR channel only after its activation by PKA and increased the CFTR-mediated transepithelial current (IT) in cultured human bronchial epithelia (HBE) isolated from the bronchi of CF donor lungs carrying the G551D and/or F508del CFTR mutations. Login to comment 24 ABCC7 p.Gly551Asp In G551D/F508del HBE, potentiation of CFTR-mediated Cl-secretion by VX-770 reduced excessive ENaC-mediated Naϩ and fluid absorption, resulting in an increase in the surface fluid and cilia beat frequency (CBF). Login to comment 41 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp The effect of VX-770 on CFTR-mediated Cl-secretion was initially characterized by measuring the CFTR-mediated IT in Ussing chambers using recombinant Fisher rat thyroid (FRT) cells expressing either human wild-type, G551D, or F508del CFTR. Login to comment 42 ABCC7 p.Gly551Asp Consistent with the previously identified functional impairment of G551D CFTR (5), the addition of a maximally effective concentration of forskolin (10 ␮M) caused only a modest increase in the IT (Fig. 1B) compared to wild-type CFTR-FRT cells (6.7 Ϯ 0.9 to 50 Ϯ 1 ␮A/cm2 ; n ϭ 7). Login to comment 44 ABCC7 p.Gly551Asp In G551D-FRT, 10 ␮M VX-770 alone did not increase the IT (Fig. 1 B vs. E; unstimulated ϭ 0.45 Ϯ 0.03 ␮A/cm2 vs. 10 ␮M VX-770 ϭ 0.45 Ϯ 0.04 ␮A/cm2 ; n ϭ 3), and VX-770 did not increase cellular cAMP levels in FRT cells (Fig. 1F). Login to comment 48 ABCC7 p.Gly551Asp Taken together, the results indicate that VX-770 is a potentiator, not an activator, of G551D- and F508del CFTR-mediated Cl- secretion. Login to comment 49 ABCC7 p.Gly551Asp The biophysical basis for CFTR potentiation by VX-770 was investigated by measuring the Po of CFTR in excised membrane patches from recombinant cells expressing G551D-, F508del-, or wild-type CFTR. Login to comment 51 ABCC7 p.Gly551Asp Under these conditions, 10 ␮M VX-770 increased the Po of G551D CFTR by Ϸ6-fold (Fig. 2 A and C). Login to comment 53 ABCC7 p.Gly551Asp These data are consistent with the increases in the forskolin-stimulated IT observed with VX-770 in G551D- and F508del-FRT cells (Fig. 1), and suggest that VX-770 acts by increasing CFTR channel gating. Login to comment 54 ABCC7 p.Gly551Asp Effect of VX-770 in Primary Cultures of HBE Carrying G551D and F508del CFTR Mutations. Login to comment 56 ABCC7 p.Gly551Asp VX-770 acted as a potentiator, not an activator, of G551D- and F508del CFTR in recombinant cells. Login to comment 58 ABCC7 p.Gly551Asp (B) Representative IT recording from G551D-FRT showing the response to the sequential application of 10 ␮M forskolin (FSK), 10 ␮M VX-770, and 20 ␮M CFTRinh-172. Login to comment 59 ABCC7 p.Gly551Asp (C) Concentration-response curve of the net increase in forskolin-stimulated IT after sequential application of VX-770 at the concentrations indicated in G551D-FRT (filled circles; n ϭ 12) and temperature-corrected F508del-FRT (open circles; n ϭ 5). Login to comment 60 ABCC7 p.Gly551Asp (D) Mean (Ϯ SEM) of the IT response in G551D- (filled bars; n ϭ 6) and F508del- (openbars;nϭ5)FRTundertheconditionsindicatedinB.Singleasteriskindicates significant difference relative to unstimulated; double asterisk indicates signifi- cantdifferencerelativetoforskolinandunstimulated(PϽ0.05;one-wayANOVA followed by Tukey`s multiple comparison test). Login to comment 61 ABCC7 p.Gly551Asp (E) Representative IT recording from G551D-FRT showing the response to the sequential application of 10 ␮M VX-770, 10 ␮M FSK, and 20 ␮M CFTRinh-172. Login to comment 65 ABCC7 p.Gly551Asp Representative patch-clamp recording of the single channel current resulting from activation of G551D CFTR in FRT cells (A) and F508del CFTR in NIH 3T3 cells (B) by 1 mM ATP and 75 nM PKA before and during VX-770 application. Login to comment 68 ABCC7 p.Gly551Asp (C) Mean Po of F508del-, G551D-, and wild-type CFTR in the presence of PKA and ATP alone (open bars) and with VX-770 (filled bars). Login to comment 69 ABCC7 p.Gly551Asp For G551D CFTR, 10 ␮M VX-770 was added, whereas 1 ␮M VX-770 was added to F508del- and wild-type CFTR. Login to comment 73 ABCC7 p.Gly551Asp In HBE isolated from the bronchi of a single CF donor lung carrying the G551D and F508del CFTR mutations, VX-770 increased the forskolin-stimulated IT with an EC50 of 236 Ϯ 200 nM (n ϭ 16) that was blocked by the inhibitor CFTRinh-172 (Fig. 3 A and B). Login to comment 75 ABCC7 p.Gly551Asp To calibrate the increase in CFTR function by VX-770, the forskolin-stimulated IT in G551D/F508del HBE was compared to that in HBE isolated from four individuals without CF (non-CF HBE). Login to comment 76 ABCC7 p.Gly551Asp In G551D/F508del- and non-CF HBE, the peak IT response to 10 ␮M forskolin was 2.9 Ϯ 0.5 ␮A/cm2 and 56 Ϯ 6 ␮A/cm2 , respectively. Login to comment 77 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp This indicates that the maximum forskolin-stimulated IT in G551D/ F508del HBE is equivalent to 5 Ϯ 1% of non-CF HBE (n ϭ 16), consistent with the low CFTR function and severe CF disease observed in individuals carrying both the G551D and F508del mutations (24). Login to comment 78 ABCC7 p.Gly551Asp VX-770 increased the forskolin-stimulated IT in G551D/F508del HBE by Ϸ10-fold to 27 Ϯ 2 ␮A/cm2 (n ϭ 16), which is equivalent to 48 Ϯ 4% of non-CF HBE (Fig. 3B). Login to comment 79 ABCC7 p.Gly551Asp These data indicate that VX-770 is a potent and efficacious potentiator of CFTR in G551D/F508del HBE. Login to comment 85 ABCC7 p.Gly551Asp Thus, VX-770 was able to potentiate CFTR in some F508del HBE cultures, although the magnitude of the effect on the forskolin-stimulated IT was less than in G551D/F508del HBE (Fig. 3B). Login to comment 86 ABCC7 p.Gly551Asp Similar to the results in FRT cells, the EC50 in F508del HBE was lower than that in G551D/F508del HBE. Login to comment 87 ABCC7 p.Gly551Asp Effect of VX-770 on Na؉ and Fluid Absorption in G551D/F508del HBE. Login to comment 89 ABCC7 p.Gly551Asp We monitored the baseline PD and amiloride response in cultured G551D/F508del HBE and wild-type HBE under open-circuit recording conditions that resemble the in vivo NPD assays commonly performed in individuals with CF (25). Login to comment 90 ABCC7 p.Gly551Asp As observed in CF subjects in vivo (23), the in vitro baseline PD was elevated and the amiloride response was larger in G551D/F508del HBE compared with non-CF HBE (Fig. 4 A and B). Login to comment 91 ABCC7 p.Gly551Asp Addition of VX-770 to G551D/F508del HBE decreased the PD with an IC50 of 43 Ϯ 38 nM (Fig. 4 B and C; n ϭ 6) and decreased the amiloride response (Fig. 4D; n ϭ 6). Login to comment 94 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp To determine the relative contributions of CFTR-mediated Cl-secretion and ENaC-mediated Naϩ absorption to the PD in non-CF HBE and G551D/F508del HBE, we monitored the response to amiloride and CFTRinh-172 in the presence or absence of VX-770 with or without forskolin (Fig. 4E). Login to comment 98 ABCC7 p.Gly551Asp To determine whether potentiation of CFTR by VX-770 was associated with an increase in the fluid levels on the apical surface of G551D/F508del HBE, we monitored the airway surface liquid (ASL) volume. Login to comment 102 ABCC7 p.Gly551Asp Although the initial rates of decline in the ASL volume were similar, the steady-state level was substantially less in G551D/F508del HBE compared with non-CF HBE (Fig. 5A). Login to comment 103 ABCC7 p.Gly551Asp In G551D/F508del HBE, the addition of VX-770 increased the ASL volume to approximately half of that observed in Fig. 3. Login to comment 104 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp VX-770 potentiated CFTR-mediated Cl-secretion in primary cultures of G551D/F508del HBE and F508del HBE. Login to comment 109 ABCC7 p.Gly551Asp (B)Theconcentration-responsecurveforVX-770inthe presence of FSK is shown for G551D/F508del HBE isolated from the bronchi of a single individual (filled circles; n ϭ 16) and F508del HBE isolated from the bronchi of the three individuals that responded to VX-770 (open circles; n ϭ 7-24). Login to comment 115 ABCC7 p.Gly551Asp These data show that VX-770 increased the amount of fluid on the apical surface of G551D/F508del HBE, indicating less absorption, more secretion, or both. Login to comment 117 ABCC7 p.Gly551Asp We next tested whether the increase in the apical fluid level by VX-770 in cultured G551D/F508del HBE was sufficient to increase the CBF, which was significantly reduced compared with that observed in non-CF HBE (Fig. 5 D and E). Login to comment 118 ABCC7 p.Gly551Asp Treatment of G551D/F508del HBE with 10 ␮M VX-770 for 5 days increased the CBF compared with vehicle-treated controls, and this response was augmented by VIP (Fig. 5 D and E). Login to comment 119 ABCC7 p.Gly551Asp In the presence of VIP and VX-770, the CBF in G551D/F508del HBE was similar to that observed for non-CF HBE (Fig. 5E). Login to comment 121 ABCC7 p.Gly551Asp These results suggest that the partial restoration of Cl- and Naϩ transport by VX-770 in G551D/F508del HBE is associated with an increase in the apical fluid level and an increase in cilia beating, and that these effects are further enhanced by augmenting CFTR activation by the addition of VIP to further stimulate the cAMP/PKA-signaling pathway. Login to comment 122 ABCC7 p.Gly551Asp Discussion We demonstrated in this study that VX-770 is a potentiator of human G551D-, F508del-, and wild-type CFTR Cl-channel function. Login to comment 132 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp The results reported here show that VX-770 increased CFTR-mediated Cl-secretion in G551D/ F508del HBE from Ϸ5% to a maximum level of Ϸ50% of that measured in non-CF HBE. Login to comment 133 ABCC7 p.Gly551Asp Based on these in vitro studies, VX-770 might be expected to result in clinical benefit in CF patients carrying the G551D mutation on at least one allele. Login to comment 136 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Nevertheless, this provocative result suggests that the efficacy of VX-770 in G551D/F508del HBE may be caused by potentiation of both G551Dand F508del CFTR and that consideration should be given to testing VX-770 in F508del-homozygous CF patients. Login to comment 138 ABCC7 p.Gly551Asp In both FRT cells and HBE, the EC50 for VX-770 was lower for F508del CFTR than for G551D CFTR, suggesting that VX-770 may have a higher affinity for F508del CFTR. Login to comment 139 ABCC7 p.Gly551Asp Other CFTR potentiators, including genistein, are also more potent against F508del CFTR compared with G551D CFTR (19). Login to comment 140 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp The lower potency of some CFTR potentiators against G551D CFTR along with homology modeling studies have suggested that these compounds bind at a site near the G551D mutation (35). Login to comment 143 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp VX-770 increased CFTR-mediated Cl-secretion and reduced ENaC-mediated Naϩ absorption in G551D/F508del HBE. Login to comment 144 ABCC7 p.Gly551Asp Representative recording of the PD in non-CF HBE (A) and G551D/F508del HBE (B) obtained with the Ussing chamber technique (open circuit mode) using equimolar Clon the basolateral and apical sides. Login to comment 145 ABCC7 p.Gly551Asp In G551D/F508del HBE, DMSO (vehicle; solid lines) or 10 ␮M VX-770 (dashed lines) was added to the apical surface before sequential addition of amiloride, low Cl- (5 mM) and 10 ␮M forskolin (FSK) to the apical surface. Login to comment 148 ABCC7 p.Gly551Asp (E) Representative PD recording in G551D/F508del HBE showing the response to DMSO (solid line) or 10 ␮M VX-770 without (dashed line) or with 10 ␮M FSK (dotted line) followed by sequential addition of 30 ␮M amiloride and 20 ␮M CFTRinh-172. Login to comment 149 ABCC7 p.Gly551Asp (F) The contribution of ENaC and CFTR to the PD in non-CF HBE and G551D/F508del HBE (bar) was determinedbymeasuringthePDchangeinresponsetoamiloride(openbars)and CFTRinh-172 (filled bars), respectively. Login to comment 150 ABCC7 p.Gly551Asp All data are from G551D/F508del HBE isolated from the bronchi of a single individual. Login to comment 153 ABCC7 p.Gly551Asp In this study, open-circuit recordings using cultured G551D/F508del HBE and non-CF HBE were performed in a way that resembles the in vivo assays of nasal PD that are commonly performed in CF patients (25). Login to comment 154 ABCC7 p.Gly551Asp As observed in vivo, the baseline PD was elevated and the amiloride response was larger in G551D/F508del HBE compared with that in non-CF HBE. Login to comment 156 ABCC7 p.Gly551Asp Addition of VX-770 decreased the baseline PD and amiloride response in G551D/ F508del HBE. Login to comment 163 ABCC7 p.Gly551Asp The increase in the fluid level on the apical surface of G551D/ F508del HBE after several hours of treatment with VX-770 can be explained by an increase in CFTR-mediated Cl-secretion and the concomitant decrease in excessive ENaC-mediated Naϩ absorption. Login to comment 164 ABCC7 p.Gly551Asp Although the amiloride response after addition of VX-770 to G551D/F508del HBE was similar to that in non-CF HBE, the apical fluid level at steady-state was approximately half of that observed in non-CF HBE (Fig. 5A). Login to comment 166 ABCC7 p.Gly551Asp Nevertheless, the increase in apical fluid levels in VX-770-treated G551D/F508del HBE was sufficient to increase cilia beating to levels observed in non-CF HBE. Login to comment 172 ABCC7 p.Gly551Asp Although transgenic mouse models of CF carrying the G551D or F508del mutation have been developed, we did not test VX-770 in these models because it does not potentiate mouse CFTR expressed in FRT cells (Fig. S4). Login to comment 176 ABCC7 p.Gly551Asp Potentiation of CFTR by VX-770 partially restored fluid regulation and cilia beating in G551D/ F508del HBE. Login to comment 177 ABCC7 p.Gly551Asp (A) The ASL volume in G551D/F508del HBE and wild-type HBE after up to 72 hours` incubation at 37 °C in the presence of 30 nM VIP with or without VX-770. Login to comment 179 ABCC7 p.Gly551Asp (B) Concentration-response curve (n ϭ 3-9) of the change in ASL volume in G551D/F508del HBE after VX-770 addition at the indicated concentrations in the absence(opencircles)orpresence(filledcircles)of30nM VIP. Login to comment 182 ABCC7 p.Gly551Asp (D)Representativetracingsofthelightinten- sity (y axis in relative units) derived from a single region of interest in G551D/F508del HBE monitored 5 days after adding 100 ␮l of fluid to the apical surface. Login to comment 183 ABCC7 p.Gly551Asp (E) Mean (Ϯ SEM; n ϭ 6) CBF wild-type HBE (filled bars) or G551D/ F508del HBE (open bars) after a 5-day treatment with DMSO, 30 nM VIP, 10 ␮M VX-770, or 30 nM VIP with 10 ␮M VX-770. Login to comment 184 ABCC7 p.Gly551Asp Single asterisk indicates significantly different (P Ͻ 0.05) from vehicle control in G551D/F508del HBE;doubleasteriskindicatessignificantlydifferent(PϽ 0.05) from vehicle control and VX-770 alone. Login to comment 185 ABCC7 p.Gly551Asp All data are from G551D/F508del HBE isolated from the bronchi of a single individual. Login to comment 187 ABCC7 p.Gly551Asp In G551D/F508del HBE, the increase in CFTR-mediated Cl-secretion by VX-770 was associated with a decrease in amiloride-sensitive Naϩ absorption and an increase in the apical fluid height and CBF. Login to comment 197 ABCC7 p.Gly551Asp The single-channel activity of G551D CFTR, wild-type CFTR, and temperature-corrected F508del CFTR was measured using excised inside-out membrane patch recordings as previously described (17). Login to comment 200 ABCC7 p.Gly551Asp To monitor changes in the ASL volume and CBF, the apical layer of G551D/F508del HBE was washed twice with absorption buffer followed by addition of 100 ␮l of absorption buffer to the apical layer. Login to comment 208 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp We also thank Dr. Joseph W. Pilewski for providing G551D/F508del HBE, Dr. L. J. V. Galietta for providing the G551D-FRT cells, Dr. Michael J. Welsh for providing the NIH 3T3 cells expressing F508del- and wild-type CFTR, and Dr. Mary Lang-Furr for providing the NIH 3T3 cells expressing ENaC. Login to comment 242 ABCC7 p.Gly551Asp Nature 362:160-164. 19. Pedemonte N, et al. (2005) Phenylglycine and sulfonamide correctors of defective delta F508 and G551D cystic fibrosis transmembrane conductance regulator chloride-channel gating. Login to comment 244 ABCC7 p.Gly551Asp Accurso FJ, et al. (2008) Interim results of phase 2a study of VX-770 to evaluate safety, pharmacokinetics, and biomarkers of CFTR activity in cystic fibrosis subjects with G551D. Login to comment 250 ABCC7 p.Gly551Asp Illek B, et al. (1999) Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein. Login to comment