Potentiators: How do they impact the fate of CFTR during biogenesis?

Defects in CFTR that result in cystic fibrosis can be broadly categorized into three processes: CFTR translation, folding/maturation, and function. To address the underlying causes affecting the folding/maturation and functional properties of CFTR, roughly two biomolecular activities are required: correctors to improve CFTR conformation and increase its levels at the cell surface, and potentiators to improve effective opening of the CFTR channel. Combined, these activities allow chloride ion transport yielding improved hydration of the lung surface and subsequent restoration of mucociliary clearance. In the past few years, major efforts on identification of correctors and potentiators yielded several different molecules with these biological activities. The potentiator, Kalydeco, is on the market and shows good efficacy in clinical trials of patients carrying G551D and similar variants. Several publications in the last two years demonstrated that Kalydeco may counteract the positive effects of a corrector on misfolded CFTR variants. Galapagos has developed novel potentiators that are superior in channel opening activity compared to Kalydeco in preclinical studies. In several assays such as the YFP-halide assays or TECC using primary patient cells the maximal opening of the G551D CFTR channel (and that of several other mutants) exceeded that of Kalydeco by >200%. A broad set of compounds was evaluated in parallel on low temperature-rescued F508del CFTR and G551D CFTR and we found a good correlation between potencies in these two assays. Here we report insight into the mechanism of action of potentiator molecules, either on their own or combined with corrector molecules. Studies using limited proteolysis and time-of-addition experiments on wild-type and mutant CFTR protein provide insight into when and where potentiator molecules affect the CFTR protein. The impact of potentiators on protein levels (band B/C data) and folding and stability of CFTR domains (limited proteolysis) will be presented showing subtle differences between chemical series. We will also present combinations of potentiator with corrector molecules. In summary, we used our standard limited proteolysis assays to gain more insight in the impact of potentiators on CFTR biosynthesis and provide additional insight on the impact of combination cocktails of corrector and potentiators on CFTR conformation.