Peter van Mourik

49 Potentiator synergy in rectal organoids Incubation of VX-770, genistein and curcumin in rectal organoids derived from an F508del / G551D CF subject We next analyzed effects of VX-770, genistein, curcumin and their combinations on FIS of F508del / G551D organoids (Fig. 3) using an experimental setup comparable to Fig. 2. Because CFTR-S1251N has a higher residual activity than CFTR-G551D (Fig. 2f,g and 3e,f), different forskolin concentrations are required for optimal drug testing in F508del / S1251N and F508del / G551D organoids (Fig. 2 and Fig. 3). Titrations of VX-770, genistein and curcumin indicated a dose-dependent increase in FIS of F508del / G551D organoids, with highest potency for VX-770 and lowest potency for curcumin (Fig. 3a-c), similar as observed for F508del / S1251N organoids (Fig. 2c-e). VX-770 and genistein at near-saturating (Fig. 3d,e) or suboptimal (Fig. 3f) concentrations synergistically repaired FIS of F508del / G551D organoids at suboptimal forskolin levels. Compared to S1251N-expressing organoids (Fig. 2f,g), optimal detection of synergy between VX-770 and genistein in F508del / G551D organoids required somewhat higher forskolin levels, probably because of the low residual function associated with CFTR-G551D (Fig. 3e,f). Furthermore, both VX-770 and genistein induced FIS to a similar extent in S1251N-expressing organoids (Fig. 2f,g), while the effect of genistein was much lower compared to VX-770 in G551D- expressing cultures (Fig. 3e,f). This suggests that channel gating by genistein, but not by VX-770, is critically dependent on the site of the class III mutation within the multi- domain structure of the CFTR channel. We selected optimal forskolin concentrations based on Fig. 3e,f to study the effect of curcumin in addition to VX-770 and genistein using near-saturating (Fig. 3g) or suboptimal (Fig. 3h) potentiator concentrations and observed strong synergy for most double and triple combinations, except for genistein + curcumin at saturating dose (Fig. 3g,h). In contrast to F508del / S1251N organoids (Fig. 2i), curcumin, in combination with VX-770, was highly effective at suboptimal dose in organoids with G551D (Fig. 2h), suggesting that CFTR- G551D is more sensitive to curcumin than CFTR-S1251N. Chronic incubation of potentiators revealed that CFTR C-band expression was only reduced by genistein and curcumin (~70% of vehicle Fig. 3i,j). To conclude, organoids expressing CFTR- S1251N or -G551D responded differently to identical potentiator levels and CFTR phosphorylation conditions, suggesting that different molecular mechanisms underlie the gating defects of these mutants. 3