Synergistic Potentiation of Cystic Fibrosis Transmembrane Conductance Regulator Gating by Two Chemically Distinct Potentiators, Ivacaftor (VX-770) and 5-Nitro-2-(3-Phenylpropylamino) Benzoate

Abstract

Cystic fibrosis (CF) is caused by loss-of-function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding a phosphorylation-activated but ATPgated chloride channel. Previous studies suggested that VX-770 [ivacaftor, N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide], a CFTR potentiator now used in clinics, increases the open probability of CFTR by shifting the gating conformational changes to favor the open channel configuration. Recently the chloride channel blocker and CFTR potentiator 5-nitro-2-(3-phenylpropylamino) benzoate (NPPB) has been reported to enhance CFTR activity by a mechanism that exploits the ATP hydrolysis-driven, nonequilibrium gating mechanism unique to CFTR. Surprisingly however, NPPB increased the activity of nonhydrolytic G551D-CFTR, the third most common disease-associated mutation. Here, we further investigated the mechanism of NPPB's effects on CFTR gating by assessing its interaction with well-studied VX-770. Interestingly, once G551D-CFTR was maximally potentiated by VX-770, NPPB further increased its activity. However, quantitative analysis of this drug-drug interaction suggests that this pharmacologic synergism is not due to independent actions of NPPB and VX-770 on CFTR gating; instead, our data support a dependent mechanism involving two distinct binding sites. This latter idea is further supported by the observation that the locked-open time of a hydrolysis-deficient mutant K1250A was shortened by NPPB but prolonged by VX-770. In addition, the effectiveness of NPPB, but not of VX-770, was greatly diminished in a mutant whose second nucleotide-binding domain was completely removed. Interpreting these results under the framework of current understanding of CFTR gating not only reveals insights into the mechanism of action for different CFTR potentiators but also brings us one step forward to a more complete schematic for CFTR gating.

Potentiation of the cystic fibrosis transmembrane conductance regulator by VX‐770 involves stabilization of the pre‐hydrolytic, O1 state

“…Like K464A, the F508del mutation also destabilizes the pre-hydrolytic O 1 state (Jih et al, 2011). However, gating of F508del-CFTR is likely to be largely hydrolytic, as demonstrated by the strong potentiation by 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) (Csanády and Töröcsik, 2014 but also see Lin et al, 2016) and by almost 100-fold slowing of F508del-CFTR closing rate by the E1371S mutation (Kopeikin et al, 2014). The apparent discrepancy between the effect of VX-770 on WT-CFTR and the mutants investigated in this paper and its effects on F508del-CFTR might be related to the very severe defect in opening measured in the latter mutant (Miki et al, 2010;Cai et al, 2015).…”

Strategies for the etiological therapy of cystic fibrosis

“… 55 Recently, a synergistic effect of two distinct CFTR potentiators, VX-770 and a nitro-phenylpropylamino benzoate, has been reported. 56 Newly developed CFTR potentiators, as GLPG1837 (Galapagos, Mechelen, Belgium), Q8W251 (Novartis, Basel, Switzerland), FDL169 (Flatley Discovery Laboratory LCC, Charlestown, MA, USA), C-10355 and C-10358 (Concert, Lexington, MA, USA) are currently evaluated in clinical trials.…”

Structural stability of purified human CFTR is systematically improved by mutations in nucleotide binding domain 1

“…A subsequent 3.4 Å resolution structure of phosphorylated zebrafish CFTR showed two well-ordered NBDs in an asymmetric sandwich dimer conformation with two bound, non-hydrolyzed ATP molecules, with the channel open to the cytoplasm and the extracellular gate closed (PDB 5W81) [32]. The structure revealed insights into the gating mechanism and likely presents a “pre-open” closed channel state [33,34]. Despite these recent breakthroughs, higher resolution structures of human WT CFTR and of CFTR with at least some of the CF-causing mutations, and in conformationally distinct functional states, are still needed to better reveal the underlying structural defect for the many types of disease-causing mutations, and to advance therapeutic strategies.…”