VX-770 and NPPB Modulate CFTR Gating via Different but Dependent Mechanisms

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Understanding how small molecules potentiate CFTR gating is critical for developing novel therapy for cystic fibrosis. Our previous studies have suggested that VX-770 (ivacaftor), a CFTR potentiator now used in clinics, increases the Po of CFTR by shifting the gating conformational changes in CFTR's transmembrane domains (TMDs) to favor the open channel configuration. Lately, another CFTR potentiator NPPB was reported to enhance CFTR activity through a modus operandi that exploits the ATP hydrolysis-driven, non-equilibrium gating mechanism unique to CFTR. It is however puzzling that NPPB can also increase the activity of non-hydrolytic G551D-CFTR, the third most common pathogenic mutation. We therefore set forth to unravel the mechanism of NPPB using VX-770 as a reference. Once the blocking effect of NPPB was corrected, we found that NPPB and VX-770, when applied alone, increased the Po of G551D-CFTR by 6.9 ± 0.8 (n = 12) and 9.3 ± 1.4 (n = 21) folds respectively. Once these two reagents were applied together, however, the Po was increased by 28.98 ± 7.6 fold (n= 10), less than the expected magnitude (∼ 60-fold) if NPPB and VX-770 work independently. Hence, NPPB only increases the Po of G551D-CFTR by 3.7 ± 0.4 fold (n = 9) in the presence of VX-770. Similarly, a much reduced enhancement (4.8 ± 1.8 fold, n = 5) of the Po by VX-770 in the presence of NPPB was seen. Interestingly, we also observed that VX-770 effectively potentiates a CFTR mutant with its second nucleotide-binding domain completely removed (i.e., dNBD2), whereas NPPB has virtually no effect (n = 12). These results can be explained by a gating model featuring an energetic coupling between opening/closing of the gate in TMDs where VX-770 binds and dimerization/dissociation of NBDs where NPPB acts on.