ABCMdb

PMID: 23890012

Farinha CM, King-Underwood J, Sousa M, Correia AR, Henriques BJ, Roxo-Rosa M, Da Paula AC, Williams J, Hirst S, Gomes CM, Amaral MD
Revertants, low temperature, and correctors reveal the mechanism of F508del-CFTR rescue by VX-809 and suggest multiple agents for full correction.
Chem Biol. 2013 Jul 25;20(7):943-55. doi: 10.1016/j.chembiol.2013.06.004., [PubMed]

Sentences

No. Mutations Sentence Comment

16 ABCC7 p.Arg1070Trp ABCC7 p.Gly550Glu ABCC7 p.Val510Asp The second F508del-associated defect impairs CFTR interdomain folding, namely, (1) the NBD1-NBD2 dimerization interface (critical for channel activation and accounting for the F508del-CFTR gating defect; Dalemans et al., 1991), which can be rescued by the G550E revertant, and (2) the interaction of NBD1 with the fourth intracellular loop (ICL4) of TMD2 (Serohijos et al., 2008), shown to be reverted by either V510D (Loo et al., 2010) or R1070W, which both fill the pocket left empty by F508del (Thibodeau et al., 2010). Login to comment 23 ABCC7 p.Arg1070Trp ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys ABCC7 p.Val510Asp Herein, we explored the MoA of VX-809 by analyzing its synergistic/additive effect with those of previously characterized genetic revertants, which rescue F508del-CFTR by causing different effects: 4RK affecting traffic (Roxo-Rosa et al., 2006), G550E (Roxo-Rosa et al., 2006) and R555K increasing channel gating by strengthening the NBD1:NBD2 dimer interface, and R1070W (Serohijos et al., 2008) and V510D (Wang et al., 2007a; Loo et al., 2010) by filling the NBD1:ICL4 interface. Login to comment 36 ABCC7 p.Arg1070Trp ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys ABCC7 p.Val510Asp VX-809 Adds to VRT-325 and Corr-4a to Rescue F508del-CFTR but Exhibits Variable Effects on Genetic Revertants In order to characterize the rescue mechanism of VX-809 on F508del-CFTR, we then tested the effect of incubating it together with VRT-325 and Corr-4a on BHK cells stably expressing this mutant alone or in cis with the following genetic revertants: (1) 4RK (where the four AFTs were simultaneously mutated to lysines), (2) G550E, (3) R1070W, (4) V510D, or (5) R555K (Figures 1A-1E). Login to comment 41 ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys Interestingly, however, analysis of the effects of the three compounds upon the revertants showed that VX-809 (but neither VRT-325 nor Corr-4a) is additive to G550E or R555K in correcting F508del-CFTR (by 38% and 32%, respectively), strongly suggesting that VX-809 acts differently from these two revertants. Login to comment 42 ABCC7 p.Arg1070Trp Moreover, whereas both VRT-325 and VX-809 add significantly to the effect of R1070W by 17% (p = 0.011) and 28% (p = 1.6 10 5 ), respectively, Corr-4a does not. Login to comment 44 ABCC7 p.Val510Asp Curiously, all the three compounds further increase band C levels of F508del-V510D-CFTR, but VRT-325 produces the greatest effect. Login to comment 46 ABCC7 p.Arg1070Trp ABCC7 p.Gly550Glu These analyses were performed for three of the revertants with different F508del-CFTR effects (4RK, traffic; G550E, NBD1:NBD2 dimer interface; and R1070W, NBD1:ICL4 interface). Login to comment 48 ABCC7 p.Gly550Glu For example, an increase in F508del-G550E-CFTR processing is detected at 0.3 mM VX-809, whereas a 10-fold higher concentration is needed for significant rescue of F508del-CFTR (Figure 2C). Login to comment 57 ABCC7 p.Arg1070Trp ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys ABCC7 p.Val510Asp Effect of Small Molecule Correctors on F508del-CFTR and Genetic Revertants (A-F) BHK cell lines stably expressing CFTR bearing F508del alone (A) or in cis with 4RK (B), G550E (C), R1070W (D), V510D (E), and R555K (F) were incubated for 24 hr with 6.7 mM VRT-325, 10 mM Corr-4a, or 3 mM VX-809 alone or in combination. Login to comment 72 ABCC7 p.Arg1070Trp ABCC7 p.Gly550Glu ABCC7 p.Gly550Glu We then assessed the effect of revertants G550E and R1070W by modeling (Figure 3F; Figures S2B and S2C), whereby G550E allows a salt bridge to form across the ATP binding site with Lys1250 and other residues from NBD2 (Figure 3F). Login to comment 73 ABCC7 p.Arg1070Trp By contrast, R1070W (Figure S2A) plausibly fills in the space left empty by absence of F508 at the NBD1:ICL4 interface as previously proposed (Thibodeau et al., 2010). Login to comment 87 ABCC7 p.Arg1070Trp ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys ABCC7 p.Val510Asp Rescue of F508del-CFTR by Low Temperature Is Additive to Genetic Revertants To learn more about how low temperature rescues F508del-CFTR, we assessed its combined effect with that of the above genetic revertants: G550E, R1070W, 4RK, V510D, and R555K (Figures 4A and 4B). Login to comment 88 ABCC7 p.Arg1070Trp ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys ABCC7 p.Val510Asp Results show that low temperature further increases processing levels of F508del-CFTR by the five genetic revertants, namely, V510D, G550E, R1070W, 4RK, and R555K, by an additional 35%, 65%, 38%, 27%, and 22%, respectively (compare gray and black bars in Figure 4B). Login to comment 89 ABCC7 p.Arg560Thr As a negative control, we also analyzed the effect of low temperature on CFTR bearing another class II disease-causing mutation R560T (Roxo-Rosa et al., 2006), confirming its previously described insensitivity to low temperature rescue (Figures 4A and 4B). Login to comment 96 ABCC7 p.Gly550Glu (F) Change in F508del NBD1-NBD2 heterodimer interface by the second-site mutation G550E. Login to comment 101 ABCC7 p.Arg1070Trp ABCC7 p.Gly550Glu ABCC7 p.Val510Asp Interestingly, these additive effects were observed not only for revertants promoting protein-autonomous folding (G550E, V510D, and R1070W) but also for the 4RK revertant, which bypasses the AFT-mediated ER retention. Login to comment 102 ABCC7 p.Arg1070Trp ABCC7 p.Gly550Glu ABCC7 p.Val510Asp Combination of Different Genetic Revertants Is Also Additive Next, to assess the full potential for F508del-CFTR rescue, we combined the effects of folding and traffic revertants by producing stable BHK cell lines expressing F508del-G550E-CFTR, where 4RK, V510D, or R1070W were also added in cis, and analyzed processing (Figures 4C and 4D). Login to comment 103 ABCC7 p.Arg1070Trp ABCC7 p.Gly550Glu ABCC7 p.Val510Asp Results in Figure 4C show that 4RK, V510D, and R1070W further increased processing of G550E-F508del-CFTR by another 12%, 59%, and 70%, respectively. Login to comment 104 ABCC7 p.Arg1070Trp ABCC7 p.Gly550Glu ABCC7 p.Val510Asp In fact, the combined effects of G550E with either V510D or R1070W bring F508del-CFTR processing to z80%, i.e., close to levels of WT-CFTR, which can be further increased at 26 C reaching 88%. Login to comment 105 ABCC7 p.Gly550Glu The combination of G550E with 4RK, although additive, has a more modest effect (32% in total) and is still additive to low temperature (Figure 4D, far right bar). Login to comment 106 ABCC7 p.Gly550Glu Low Temperature Kinetics of F508del-CFTR Alone or with 4RK/G550E The increased effect of combining correctors, revertants, and low temperature is strongly suggestive of different MoAs. Login to comment 107 ABCC7 p.Gly550Glu However, to further characterize the MoA of low temperature rescue, we studied the turnover rate and processing efficiency of F508del-CFTR or in cis with 4RK/G550E at 26 C in comparison to these variants at 37 C. Login to comment 123 ABCC7 p.Gly550Glu The dotted line corresponds to levels of band C in F508del-G550E- CFTR. Login to comment 124 ABCC7 p.Gly550Glu We then similarly assessed how low temperature affects the turnover and processing of F508del-CFTR bearing the two genetic revertants 4RK and G550E (Figure 6). Login to comment 125 ABCC7 p.Gly550Glu Results from pulse-chase experiments of F508del-G550E-CFTR show a dramatic reduction in the turnover of the immature form (Figure 6A, left panel), which also corresponds to an increase in its processing efficiency (band C levels) when the chase was performed at 26 C (Figure 6A). Login to comment 128 ABCC7 p.Gly550Glu The differential behavior observed for F508del-G550E- and F508del-4RK-CFTR at 26 C becomes most evident when these data are plotted as the total amount of band B + band C against time (Figures 6B and 6C). Login to comment 129 ABCC7 p.Gly550Glu Indeed, whereas the total amount of F508del-G550E-CFTR at 26 C (Figure 6B, gray/dotted bars) onlybarelydecreasesincomparisontoitstotalat37 C(Figure6B, Figure 5. Login to comment 139 ABCC7 p.Gly550Glu Altogether they indicate that 4RK is not as additive to low temperature in stabilizing F508del-CFTR as G550E. Login to comment 158 ABCC7 p.Gly550Glu ABCC7 p.Gly550Glu ABCC7 p.Gly550Glu Low Temperature Stabilizes the Immature Form of G550E and 4RK Variants of F508del-CFTR and Acts Synergistically with G550E, but not 4RK, to Rescue F508del-CFTR (A-C) Pulse-chase experiments followed by IP were performed in BHK cells expressing (A) F508del-G550E (left panel) or F508del-4RK-CFTR (right panel) incubated at 26 C for 48 hr. After labeling with [35 S] methionine for 3 hr, chase was performed at 26 C (last five lanes in each panel). Login to comment 160 ABCC7 p.Gly550Glu ABCC7 p.Gly550Glu Percentage of bands B and C present at each time point of chase in F508del-G550E- (B) or F508del4RK-CFTR (C) incubated for the duration of the experiment (pulse + chase) at either 26 C or 37 C is shown in graph bars to illustrate the additive effect of low temperature and the genetic revertant G550E, but not 4RK, on F508del-CFTR rescue. Login to comment 164 ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys Indeed, VX-809 (in contrast to either VRT-325 or Corr-4a) adds to the rescue by G550E and R555K (both acting at the NBD1:NBD2 interface), indicating that the compound and these two revertants probably correct distinct conformational cues of F508del-CFTR. Login to comment 165 ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys ABCC7 p.Val510Asp ABCC7 p.Val510Asp In contrast, the additive effect of VX-809 to R1070W and V510D is rather modest, thus suggesting that this corrector acts more similarly to R1070W/V510D than to G550E/R555K. Login to comment 166 ABCC7 p.Arg1070Trp ABCC7 p.Val510Asp VRT-325 in turn (and comparatively to its modest effect alone) significantly increases (by z29% versus VRT-325 alone) the rescue efficiency of R1070W and also V510D, suggesting effects at distinct sites. Login to comment 168 ABCC7 p.Gly550Glu For example, F508del-G550E-CFTR required a 10-fold lower dose of VX-809 than F508del-CFTR for significant rescue. Login to comment 171 ABCC7 p.Asp567Ala CFTR with Mutated Diacidic Exit Code Is Rescued by Low Temperature, but Not by Correctors (A) BHK cell lines stably expressing WT-CFTR or bearing the DAA (D567A) mutation alone or in cis with 4RK were cultured at 37 C, incubated at 26 C for 48 hr or for 24hr with VRT-325, Corr-4a, or VX-809, as indicated. CFTR protein was analyzed by WB. Data are representative of n = 5 experiments. Login to comment 174 ABCC7 p.Asp567Ala ABCC7 p.Asp565Ala (C) BHK cell lines stably expressing WT-CFTR or bearing the DD/AA mutations (D565A, D567A) alone or in cis with 4RK were cultured at 37 C, incubated at 26 C for 48 hr or for 24 hr with VRT-325, Corr-4a, or VX-809, as indicated. CFTR protein was analyzed by WB. Data are representative of n = 6 experiments. Login to comment 193 ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp ABCC7 p.Gly550Glu ABCC7 p.Gly550Glu Furthermore, modeling also predicts that revertants G550E and R1070W act at different CFTR interdomain contacts disrupted by F508del: whereas G550E seems to restore the strength of the NBD1:NBD2 interface, R1070W rather promotes the NBD1:ICL4 interaction, as suggested previously (He et al., 2010; Serohijos et al., 2008) and in Figure S2A. Login to comment 195 ABCC7 p.Arg1070Trp ABCC7 p.Gly550Glu ABCC7 p.Val510Asp Indeed, G550E, besides being able to promote rescue of F508del-CFTR (DeCarvalho et al., 2002), shows the largest combined effect with R1070W (or V510D). Login to comment 197 ABCC7 p.Arg1070Trp ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys Moreover, the observed synergy of G550E or R555K with VX-809, but not VRT-325, is consistent with this model in which VRT-325 acts on the NBD1:NBD2 dimerization interface (also supported by the synergy between R1070W and VRT-325). Login to comment 198 ABCC7 p.Arg1070Trp Further modeling is, however, required to examine the observation that R1070W is still additive with VX-809 (albeit modestly). Login to comment 210 ABCC7 p.Arg1070Trp ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys ABCC7 p.Val510Asp Our data also show that low temperature, similar to chemical correctors, further increases processing levels of F508del-CFTR by the five genetic revertants, although to variable levels: V510D (by an additional 35%), G550E (65%), R1070W (38%), 4RK (27%), and R555K (22%). Login to comment 211 ABCC7 p.Gly550Glu ABCC7 p.Gly550Glu The strong synergistic effect of low temperature and G550E on the rescue of F508del-CFTR-efficient processing, but only modestly for 4RK, supported by pulse-chase data, suggest that low temperature and 4RK effects are mechanistically closer to each other (and thus related to bypassing the ER quality control) than low temperature and G550E. Login to comment 212 ABCC7 p.Gly550Glu This is further supported by the additive effects of G550E and 4RK effects (Figure 4). Login to comment 214 ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys ABCC7 p.Arg516Lys Although 4RK can also be claimed to impact on F508del-CFTR folding (namely, through its two NBD1 changes: R516K and especially R555K), this is somewhat disproven by the additive effects of 4RK with G550E (Figure 4), the latter truly correcting F508del-NBD1 folding, as assessed by channel gating (Farinha and Amaral, 2005; Rosser et al., 2008; Roxo-Rosa et al., 2006). Login to comment 225 ABCC7 p.Arg1070Trp ABCC7 p.Gly550Glu Assessing the synergistic/additive effects of investigational drug VX-809, one of the most promising to rescue the F508del-CFTR-trafficking defect, with those of genetic revertants as well as other correctors (VRT-325 and Corr-4a) or low temperature pointed to major insights into its MoA: (1) VX-809 is additive to both VRT-325 and Corr-4a, suggesting that each compound operates by a different MoA; (2) VX-809 is additive to low temperature rescue of the mutant almost to WT-CFTR levels; (3) VX-809, VRT-325, and Corr-4a show variable additive effects with the genetic revertants tested (4RK, G550E, and R1070W), thus providing clues for their possible action being exerted at specific protein binding pockets: VX-809 at the NBD1:TMD2 interface (and VRT-325 at NBD1:NBD2) or acting unspecifically (Corr-4a); and (4) VX-809 does not rescue the diacidic code traffic mutant in contrast to low temperature, which seems to act at trafficking surveillance checkpoints. Login to comment 231 ABCC7 p.Arg560Thr ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp ABCC7 p.Gly550Glu ABCC7 p.Gly550Glu ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys ABCC7 p.Arg516Lys ABCC7 p.Arg29Lys ABCC7 p.Val510Asp ABCC7 p.Val510Asp ABCC7 p.Asp567Ala ABCC7 p.Asp567Ala ABCC7 p.Asp565Ala EXPERIMENTAL PROCEDURES Cells and Culture Conditions BHK cell lines expressing F508del-4RK (R29K/R516K/R555K/R716K)-, F508del-G550E-, F508del-R1070W-, F508del-V510D-, F508del-R555K-, F508del-V510D/G550E-, F508del-G550E/R1070W-, DAA (D567A)-, 4RK- DAA-, DD/AA (D565A, D567A)-, 4RK-DD/AA-, and R560T-CFTR were produced and cultured as previously described (Roxo-Rosa et al., 2006). Login to comment