Peter van Mourik

55 Potentiator synergy in rectal organoids for the therapeutic use of genistein in G551D-expressing subjects. Remarkably, our results indicated that genistein (up to 200 μM) dose-dependently and uniformly increased FIS of organoids for all three mutants investigated, i.e. G551D, S1251N and F508del, without signs of inhibition at the highest dose (Fig. 2d, 3b and 4b). Possibly, the inhibitory effect of genistein on CFTR-F508del depends on the cell model used, or the genistein concentration that reaches CFTR in organoids is lower than the concentration in the surrounding medium. Compounds need to penetrate the matrigel that serves as 3D support, enter via the basolateral epithelial membrane and diffuse to the apical site to reach CFTR, which may differentially impact the efficacy of CFTR modulators. This may also explain why curcumin mono treatment of CFTR-G551D and –S1251N organoids was only effective at 25 μM and higher, while a higher CFTR-activating potency of curcumin was observed in other studies 21,28 . Aside the general similarities in the behavior of the S1251N and G551D gating mutants in the FIS assay and in their response to the potentiators noted above, pronounced differences were found in their basal activity (S1251N > G551D; Fig. 2f,g vs. 3e,f), in their maximal response to genistein (S1251N > G551D; Fig. 2f vs. 3e), and in their sensitivity to curcumin in the presence of VX-770 and genistein (G551D > S1251N; Fig. 3h vs. 2i). At a first glance these differences are rather unexpected because both mutated residues are situated inside the functionally important ATP binding pocket 2 (ABP2) of CFTR, and are predicted to disrupt ATP-induced head- to-tail dimerization of NBD1 and NBD2. However, residue G551 is part of the ABC signature sequence of NBD1, which is involved in ATP hydrolysis rather than ATP binding 48 , and the G551D mutation has been shown to abolish ATP hydrolysis and to convert ABP2 from a stimulatory into an inhibitory site 49 . Instead, residue S1251 is located in the Walker A sequence of NBD2 which is crucial for ATP binding 48 , suggesting that the S1251N mutation most plausibly impairs ATP binding at site 2 rather than affecting ATP hydrolysis or creating an ATP-inhibitory site. Such functional differences may have a strong impact on the open probability of the mutant CFTR channels and on their differential response to the potentiators. Clearly, additional studies, in particular of the poorly explored S1251N mutant channels, are needed to improve our mechanistic understanding of these differences. Previous studies indicated that chronic stimulation of potentiators may diminish expression and functionality of VX-809-repaired CFTR-F508del or wild-type CFTR, but not of CFTR-G551D, by yet undefined mechanisms 50,51 . In line with these studies, chronic stimulation with VX-770 did not affect protein expression of CFTR-G551D in organoids (Fig. 3i,j). While chronic single potentiator treatment had no effect on the protein expression of CFTR-G551D or CFTR-S1251N, some potentiatior 3