ABCMdb

PMID: 23924900

Ren HY, Grove DE, De La Rosa O, Houck SA, Sopha P, Van Goor F, Hoffman BJ, Cyr DM
VX-809 corrects folding defects in cystic fibrosis transmembrane conductance regulator protein through action on membrane-spanning domain 1.
Mol Biol Cell. 2013 Oct;24(19):3016-24. doi: 10.1091/mbc.E13-05-0240. Epub 2013 Aug 7., [PubMed]

Sentences

No. Mutations Sentence Comment

60 ABCC7 p.Leu206Trp ABCC7 p.Pro67Leu ABCC7 p.Val232Asp ABCC7 p.Glu92Lys ABCC7 p.Glu56Lys There are several CF-associated mutations in MSD1 that cause defects in CFTR processing and function: N-terminal tail (E56K and P67L), TM1 (E92K), TM2 (L206W), and TM4 (V232D) (Figure 4, A-E). Login to comment 61 ABCC7 p.Leu206Trp ABCC7 p.Pro67Leu ABCC7 p.Glu56Leu The severe folding (Figure 4, A-B) and functional (Figure 4E) defects exhibited by E56L, P67L and L206W were completely corrected by 5 bc;M VX-809. Login to comment 62 ABCC7 p.Val232Asp ABCC7 p.Glu92Lys In contrast, 5 bc;M VX-809 only partially restored folding and function to E92K and V232D (Figure 4, A and E). Login to comment 63 ABCC7 p.Glu92Lys ABCC7 p.Glu92Lys Interestingly, VX-809 demonstrated reduced potency for E92K-CFTR relative to F508del-CFTR, for both correcting folding and function (Figure 4, B and C), yet was able to fully restore E92K-CFTR at 30 bc;M. Login to comment 64 ABCC7 p.Glu92Lys However, Corr4a could not restore E92K-CFTR function (Figure 4D). Login to comment 65 ABCC7 p.Val232Asp V232D-CFTR was the least responsive to VX-809, and higher concentrations of the compound did not restore function beyond the 25% of normal CFTR observed in the presence of 5 bc;M VX-809. Login to comment 72 ABCC7 p.Leu375Ala ABCC7 p.Leu375Ala ABCC7 p.Leu375Ala ABCC7 p.Leu375Ala ABCC7 p.Phe374Ala ABCC7 p.Phe374Ala ABCC7 p.Phe374Ala ABCC7 p.Phe374Ala Despite the severe biogenic defects exhibited by F374A-CFTR, L375A-CFTR, the double mutation F374A/L375A-CFTR (Figure 3A), F374A-CFTR 380, and L375A-CFTR 380 (Figure 3B), VX-809 almost completely suppressed the biogenic defects caused by the F374A and L375A mutations at the 5-bc;M concentration that maximally suppressed folding defects in F508del-CFTR (Figure 3, A and B). Login to comment 93 ABCC7 p.Phe429Ser ABCC7 p.Phe429Ser ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg ABCC7 p.Gln637Arg These mutations are termed solubilizing (S) mutations and were introduced into NBD1 in different combinations (Figure 5A, S2 [F429S, Q637R] and S3 [F429S, F494N, and Q637R]). Login to comment 96 ABCC7 p.Phe374Ala ABCC7 p.Phe374Ala ABCC7 p.Phe374Ala Interestingly, the positive effect of S2 and S3 on F508del-CFTR biogenesis was abolished by the F374A mutation, as VX-809 could not drive high-level accumulation of the folded C-band of F374A/S2/F508del-CFTR or F374A/S3/F508del-CFTR. Login to comment 97 ABCC7 p.Phe374Ala In addition, the F374A mutation hindered VX-809 from suppressing folding defects in F508del-CFTR. Login to comment 98 ABCC7 p.Phe374Ala In experiments with CFTR, the S2 and S3 mutations by themselves increased C-band accumulation almost twofold, and this effect was blocked by F374A (Figure 5C). Login to comment 99 ABCC7 p.Phe374Ala ABCC7 p.Phe374Ala Yet, in contrast to results with F508del-CFTR, VX-809 restored accumulation of the C-form of F374A/S2-CFTR and F374A/S3-CFTR to levels of S2 CFTR and S3 CFTR under control conditions. Login to comment 104 ABCC7 p.Phe374Ala ABCC7 p.Phe374Ala The folding defect caused by F374A is efficaciously corrected by VX-809 (Figure 3), but the F374A mutation blocks VX-809 action on F508del-CFTR. Login to comment 105 ABCC7 p.Phe374Ala In addition, F374A also blocks the positive effect of S mutations on CFTR and F508del-CFTR biogenesis. Login to comment 107 ABCC7 p.Val232Asp V232D-CFTR biogenesis and function to normal levels (Caldwell et al., 2011), these data suggest that correctors such as VX-809 and Corr4a can act to selectively suppress folding defects in CFTR caused by different disease-related mutations in MSD1. Login to comment 108 ABCC7 p.Glu92Lys ABCC7 p.Glu92Lys Because E92K-CFTR was corrected to normal levels of function but F508del was corrected to ~15% of normal function, the impact of VX-809 on the double mutation E92K/F508del-CFTR was tested. Login to comment 110 ABCC7 p.Glu92Lys ABCC7 p.Glu92Lys The effect of VX-809 on the C-band of E92K/F508del-CFTR was consistent with the dose response for E92K-CFTR, while the level of efficacy was consistent with that for F508del-CFTR. Login to comment 111 ABCC7 p.Glu92Lys ABCC7 p.Glu92Lys Thus the E92K mutation causes a folding defect in E92K/F508del-CFTR that requires a higher compound concentration, but the folding defects caused by F508del limit the efficacy of VX-809. Login to comment 114 ABCC7 p.Glu92Lys E92K is unique among these mutants, as it alters the potency of VX-809, yet the biogenic defects caused by this mutation are completely corrected by VX-809. Login to comment 131 ABCC7 p.Val510Asp Defective contacts between F508del-NBD1 and ICL4 that limit F508del-CFTR assembly have been partially restored by introduction of the V510D mutation into NBD1 by permitting the formation of a salt bridge between D510 and R1070 of ICL4 (Wang et al., 2007; Figure 6B). Login to comment 132 ABCC7 p.Val510Asp ABCC7 p.Val510Asp The V510D mutation partially corrects misfolding of F508del-CFTR, as shown by the C-band for V510D/F508del-CFTR (Figure 6B, lane 4), to levels similar to that for F508del-CFTR in the presence of VX-809. Login to comment 133 ABCC7 p.Val510Asp Remarkably, VX-809 stimulated the accumulation C-band of V510D/ F508del-CFTR to the levels observed for normal CFTR. Login to comment 134 ABCC7 p.Val510Asp ABCC7 p.Arg1070Ala To determine whether formation of a salt bridge between D510 and R1070 was important for this effect, we introduced the R1070A mutation into V510D/F508del-CFTR. Login to comment 135 ABCC7 p.Val510Asp ABCC7 p.Val510Asp ABCC7 p.Arg1070Ala In the presence of VX-809, the accumulation of the C-band of R1070A/V510D/ F508del-CFTR was reduced by 75% relative to V510D/F508 -CFTR (Figure 6B, lane 7). Login to comment 136 ABCC7 p.Val510Asp ABCC7 p.Arg1070Ala Yet VX-809 was still able to increase folded R1070A/V510D/F508-CFTR to levels that were significantly higher than those for VX-809-treated F508del-CFTR. Login to comment 137 ABCC7 p.Val510Asp ABCC7 p.Arg1070Ala Because the V510D mutation can modestly improve the thermodynamic stability of purified NBD1 (Lewis et al., 2010; Wang et al., 2010), the residual VX-809 corrector function on R1070A/V501D/F508del-CFTR could result from thermodynamic stabilization of NBD1 that would occur in the absence of salt-bridge formation between D510 and R1070. Login to comment 153 ABCC7 p.Leu375Ala ABCC7 p.Phe374Ala (D) F374A L375A CFTR exhibits a folding defect that is not efficaciously suppressed by VX-809. Login to comment 157 ABCC7 p.Phe374Ala CFTR 380 F374A ࢞371-375 % of Cont. Login to comment 159 ABCC7 p.Leu375Ala 100 520 14 123 CFTR 380 L375A % of Cont. Login to comment 162 ABCC7 p.Leu375Ala ABCC7 p.Leu375Ala ABCC7 p.Phe374Ala ABCC7 p.Phe374Ala 380- CFTR 380 Trypsin (ug/ml) 25 20 15- - - - + Vehicle +VX-809 * CFTR 370 CFTR 370 2520- 15- CFTR 380 * +Vehicle + VX-809 ** ** + _ Wt _ VX-809 + _ + _ F374A L375A F374A L375A + _ ࢞371-375 Tub- C- BC- 100 4 108 6 115 2 12 8 7 B31 24 28 23 24 25 27 22 19 CFTR %-Wt C-band C. VX-809 0 5 10 15 _ ࢞371-375 -C -B CFTR Wt %-Wt 100 4 6 5 2 C-band 25 12 18 11 8 + CFTR 3800 25 2 1 7 3 0 25 2 1 7 3 D. Login to comment 175 ABCC7 p.Phe374Ala (B) Mutation of F374A hinders the ability of misfolding suppressor mutations S2 and S3 to increase the efficacy of VX-809 on F508del-CFTR. Login to comment 176 ABCC7 p.Phe374Ala (C) Mutation of F374A hinders the ability of S2 and S3 to increase accumulation of the folded C-band of CFTR. Login to comment 178 ABCC7 p.Phe429Ser ABCC7 p.Phe429Ser ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg ABCC7 p.Gln637Arg S2 (F429S, Q637R) and S3 (F429S, F494N, and Q637R) are mutations introduced into NBD1 to increase the thermodynamic stability of NBD1 and thereby increase CFTR and F508del-CFTR (B and C) folding efficiency (Pissarra et al., 2008; Teem et al., 1993). Login to comment 182 ABCC7 p.Phe374Ala ABCC7 p.Phe374Ala ABCC7 p.Phe374Ala ABCC7 p.Phe374Ala ABCC7 p.Phe374Ala Vehicle VX-809 + + _ _ + + _ _ + + _ _ + + _ _ + + _ _ + + _ _ F508 F508 F374 F508 F374A S2 F508 F374A S3 F508 S2 F508 S3 + _ CFTR Tub- -C -B Wt C-100 1 12 2 4 3 4 1 3 1 42 8 51 B40 31 60 40 61 32 64 33 43 28 49 25 43 Vehicle VX-809 + + _ _ + + _ _ + + _ _ + + _ _ + + _ _ + _ F374A F374A S2 F374A S3 S2 S3 Tub- CFTR -C -B Wt C-100 9 80 8 158 7 162 136 182 163 225 B28 14 18 25 19 26 15 11 5 13 5 A. Login to comment 184 ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg ABCC7 p.Gln637Arg %-Wt C-Band %-Wt C-Band C. F508 V510 R1070 ICL4 ICL2 F429 Q637 F494 NBD1 S2=F429S, Q637R S3=F429S, F494N Q637R FIGURE 4:ߒ Functional defects in CFTR caused by disease-related mutations in MSD1 are suppressed by VX-809. Login to comment 186 ABCC7 p.Glu92Lys (B) Dose-dependent correction of E92K-CFTR misfolding by VX-809. Login to comment 187 ABCC7 p.Glu92Lys (C) Dose-dependent correction of E92K-CFTR function by VX-809. Login to comment 188 ABCC7 p.Glu92Lys (D) VX-809 (30 bc;M) and Corr4a (15 bc;M) restore E92K-CFTR channel activity to different levels. Login to comment 193 ABCC7 p.Glu92Lys (E) VX-809 was 5 bc;M, except in the case of E92K-CFTR, for which it was 30 bc;M. Login to comment 194 ABCC7 p.Leu206Trp ABCC7 p.Pro67Leu ABCC7 p.Glu92Lys (C-D): n = 3 &#b1; SE. _ _ _ _ + + + _ + E56K P67L E92K L206W Wt _ _ + V232D Wt -C- -BC- BTub- CFTR . Login to comment 196 ABCC7 p.Leu206Trp ABCC7 p.Pro67Leu ABCC7 p.Glu92Lys ABCC7 p.Glu92Lys ABCC7 p.Glu92Lys ABCC7 p.Glu92Lys ABCC7 p.Glu92Lys ABCC7 p.Glu56Lys D. E92K F508 -B C -B C VX-809 0 3 10 30 M CFTR CFTR B17 15 14 17 C0 25 66 95 B18 30 28 27 C 1 8 9 8 % Norm VX-809 0 3 10 30 M C-100 1 81 1 83 N.D. 24 5 78 100 N.D. 15 B14 4 6 4 7 1 1 6 12 16 3 2 C. B11 10 11 12 C 1 1 1 7 VX-809 0 3 10 30 M -B C F508 CFTR E92K % Norm % Norm E. VX-809 0 50 100 150 200 250 -10 -9 -8 -7 -6 -5 -4 Chloride Transport ( A/cm 2 ) VX-809 (Log M) E92K-CFTR Normal 0 50 100 150 200 250 DMSO VX-809 Corr-4a Chloride Transport ( A/cm2) E92K-CFTR 0 100 200 300 400 E56K P67L E92K L206W V232V dF508 I SC ( A/cm 2 ) DMSO VX-809 Normal VX-809 on MSD1 has potential to promote high-level functional correction of CFTR in people with CF who harbor mutations other than F508del (Bobadilla et al., 2002). Login to comment 219 ABCC7 p.Leu375Ala ABCC7 p.Phe374Ala Soluble lysates were obtained by centrifugation at 20,000 &#d7; g combined mutation of F374A and L375A but efficaciously suppresses folding defects caused by each individual mutation. Login to comment 229 ABCC7 p.Val510Asp (B) Introduction of the V510D suppressor mutation into NBD1 permits VX-809 to drive high-level folding of F508del-CFTR. Login to comment