br Methods br Results br Discussion However this novel

Methods
Results
Discussion However, this novel treatment only applies to 9 of the 1900 CFTR gene mutations reported to date (~5% of all CF patients). The CF community should thus work fast to determine whether ivacaftor, or the investigational drug lumacaftor (for Class II mutants) rescue other CFTR mutants and thus can be extended to more CF patients. The objective of this study was to evaluate the effect of lumacaftor on additional CFTR mutants which, similarly to the most frequent mutation F508del (Van Goor et al., 2011), also affect the traffic of the protein to the plasma membrane. To this end, we used the best known in vitro CF model of human airways, consisting in primary cultures of human bronchial epithelial nmda (HBE) grown as monolayers in porous filters and we used then for CFTR bioelectric measurements in perfused micro-Ussing chambers (Moniz et al., 2013). Our data show that the effect of lumacaftor on A561E/A561E HBE cells was equivalent to that of this investigational drug in F508del/F508del cells. Indeed, after the incubation of A561E/A561E cells with 3μM lumacaftor for 24h, responses obtained in the Ussing chamber were 7-fold higher than when cells were incubated with DMSO-vehicle, representing ~6% of rescue vs non-CF cells. For F508del/F508del cells responses of lumacaftor-treated cells were 8/14-fold higher than those under DMSO, representing 5–15% of rescue vs non-CF cells. These data seem to indicate that the previously characterized trafficking defect of the A561E-CFTR protein (Mendes et al., 2003) can be, as least partially, corrected by lumacaftor. Interestingly, a previous study showed that A561E-CFTR can be rescued to the cell surface by the same genetic revertants as F508del-CFTR (Roxo-Rosa et al., 2006). In another more recent study, the A561E-CFTR channel was also described to have similar mechanisms of dysfunction and response to potentiators as F508del-CFTR (Wang et al., 2014). Of note is the striking difference between the responses of the two F508del/F508del donors, which can potentially be a predictor of variable patients\' response to this investigational drug. Data presented here also show a positive effect of VX-809 on HBE cells with genotypes F508del/G542X (~4% vs non-CF) and F508del/Y1092X (~7% vs non-CF) but not on N1303K/G542X cells. Our data also lead to the conclusion that the A561E responses to lumacaftor and its analogue C18 do not totally overlap, as observed from the significantly lower Fsk+Gen response of A561E/A561E cells pre-incubated with C18 vs those under lumacaftor. In contrast, F508del-CFTR responds similarly to both correctors, similarly to what was previously reported (Eckford et al., 2014). Noticeably, however, the Fsk-response of C18-treated A561E/A561E cells is significantly higher than in the DMSO-treated cells (Fig. 4c). Therefore, the failure in C18-treated to significant respond to the further stimulation by potentiator Genistein, might be due to a possible dual activity (corrector and potentiator) of the C18 compound as suggested (Eckford et al., 2014), which likely would be overlapping with that of Gen. Nonetheless, C18 also failed to rescue A561E-CFTR as assessed by Western blot, while VX-809 induces a detectable levels of mature A561E-CFTR (Fig. S2). Although those authors have used a higher C18 concentration for a longer pre-incubation time (6μM/48h) (Eckford et al., 2014), the conditions we employed here (5μM/24h) were also used in another study (Holleran et al., 2012) and in fact correspond to the concentration range recommended by CFFT (3–6μM). In contrast to the effect on A561E/A561E HBE cells, the magnitude of the response of lumacaftor-treated N1303K/G542X cells was just slightly higher by ~2-fold (both under Fsk and Gen) and not statistically different from that in DMSO-treated cells. Moreover, the percentage of rescue vs non-CF cells was barely 0.5%, thus showing a lack of an effect by VX-809 on N1303K. Two hypotheses may account for this lack of a significant response. Firstly, N1303K located in the second nucleotide binding domain (NBD2) of CFTR protein, may cause a different structural defect from that of F508del or A561E, both located in NBD1. Indeed, recent studies have suggested that the putative binding site of VX-809/lumacaftor is a “structural pocket” between NBD1 and the fourth intracellular loop (ICL4) of the second transmembrane domain (Farinha et al., 2013; He et al., 2013). Plausibly, NBD2-located N1303K creates a distinct defect which unlikely would be corrected by the lumacaftor. Secondly, it is possible that the response of a single copy of N1303K (the other CFTR allele is G542X, a “null” variant) may be insufficient to observe an effect similar in magnitude to that of A561E/A561E or F508del/F508del cells. Contradicting the latter hypothesis are the positive responses of the F508del/G542X and F508del/Y1092X cells, showing that VX-809 can elicit a detectable effect on a single dose of F508del, in contrast to N1303K.