Peter van Mourik

54 Chapter 3 however, we also observed synergy between curcumin and VX-770, suggesting that the binding sites for these potentiators, or their mechanism of ATP-independent gating are not identical either. Data of rectal biopsies (Fig. 1) and organoids (Fig. 2h,i; 3g,h) indicate that at near-saturating concentrations of VX-770 the FIS of S1251N- and G551D-expressing cultures can be further enhanced by other potentiators, in particular by genistein. This supports previous observations in other G551D- expressing cell models 8,25 showing that the open probability ( Po ) of VX-770-treated G551D-CFTR remains far below the Po of wild-type CFTR channels. On the basis of these in vitro findings we have recently initiated clinical studies to investigate in vivo effects of genistein supplementation in Dutch CF subjects with the S1251N mutation that are treated with VX-770. Albeit that VX-770, genistein and curcumin are supposed to improve forskolin- induced swelling in organoids principally through the direct repair of defective CFTR channel gating, these compounds may influence the epithelial fluid transport via other mechanisms as well. Organoid swelling as a measure of CFTR activity is dependent on several other, potential rate-limiting steps in transepithelial fluid transport, e.g. the import of chloride and bicarbonate at the basolateral membrane, and the activity of hyperpolarizing basolateral potassium channels which, in concert, dictate the electrochemical driving force for anion exit across the apical CFTR channel. Therefore, though the fluid secretion is completely CFTR-dependent and proportional to CFTR activity within the dynamic range of the assay (Dekkers et al , manuscript submitted), compounds or conditions that affect the driving force for anion exit and/or co-determine the phosphorylation state of CFTR in the presence of forskolin (e.g. phosphodiesterase or protein phosphatase inhibitors), may also have an impact on the FIS assay. On one hand this consideration complicates the mechanistic interpretation of pharmacological stimulation of organoid swelling in case the stimulus is not just a pure CFTR potentiator. On the other hand the FIS assay measures restoration of net fuid secretion and luminal fluidity in primary epithelial organoids under quasi-physiological conditions, which is more relevant for CF patients than CFTR activity in isolation, as measured in patch clamp or iodide efflux studies. Previous studies report that the functional response of CFTR-wild-type and -F508del channels to genistein is bell-shaped, i.e. enhanced in a low concentration range and inhibited in a higher concentration range, suggesting the existence of a high affinity activatory binding site and a low affinity inhibitory binding site in CFTR 45,46 . The inhibitory effect of genistein was not observed for CFTR-G551D, most likely because the G551D mutation abolishes the low affinity inhibitory binding site 20,47 , which argues