vx-770 and 5-nitro-2-(3-phenylpropylamino)benzoic-acid

VX-770 (Ivacaftor) has been approved for clinical usage in cystic fibrosis patients with several CFTR mutations. Yet the binding site(s) on CFTR for this compound and other small molecule potentiators are unknown. We hypothesize that insight into this question could be gained by comparing the effect of potentiators on CFTR channels from different origins, e.g., human, mouse, and Xenopus (frog). In the present study, we combined this comparative molecular pharmacology approach with that of computer-aided drug discovery to identify and characterize new potentiators of CFTR and to explore possible mechanism of action. Our results demonstrate that 1) VX-770, NPPB, GlyH-101, P1, P2, and P3 all exhibited ortholog-specific behavior in that they potentiated hCFTR, mCFTR, and xCFTR with different efficacies; 2) P1, P2, and P3 potentiated hCFTR in excised macropatches in a manner dependent on the degree of PKA-mediated stimulation; 3) P1 and P2 did not have additive effects, suggesting that these compounds might share binding sites. Also 4) using a pharmacophore modeling approach, we identified three new potentiators (IOWH-032, OSSK-2, and OSSK-3) that have structures similar to GlyH-101 and that also exhibit ortholog-specific potentiation of CFTR. These could potentially serve as lead compounds for development of new drugs for the treatment of cystic fibrosis. The ortholog-specific behavior of these compounds suggest that a comparative pharmacology approach, using cross-ortholog chimeras, may be useful for identification of binding sites on human CFTR.

Topics: Amino Acid Sequence; Aminophenols; Animals; Cells, Cultured; Chloride Channel Agonists; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Drug Evaluation, Preclinical; Glycine; Hydrazines; Membrane Potentials; Mice; Nitrobenzoates; Patch-Clamp Techniques; Quinolones; Sequence Deletion; Xenopus laevis

Cystic fibrosis (CF) is caused by loss-of-function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding a phosphorylation-activated but ATP-gated 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.

Topics: Adenosine Triphosphate; Aminophenols; Animals; CHO Cells; Cricetinae; Cricetulus; Cystic Fibrosis Transmembrane Conductance Regulator; Drug Synergism; Ion Channel Gating; Models, Biological; Mutation; Nitrobenzoates; Quinolones