A protocol for identifying the binding sites of small molecules on the cystic fibrosis transmembrane conductance regulator (CFTR) protein.

We describe a protocol to identify the binding site(s) for a drug called ivacaftor that potentiates the CFTR chloride channel. We use photoaffinity probes-based on the structure of ivacaftor-to covalently modify the CFTR protein at the region that constitutes the drug binding site(s). We define the methods for photo-labeling CFTR, its membrane extraction, and enzymatic digestion using trypsin. We then describe the experimental methods to identify the modified peptides by using mass spectrometry. For complete details on the use and execution of this protocol, please refer to Laselva et al. (2021).

Identification of binding sites for ivacaftor on the cystic fibrosis transmembrane conductance regulator.

Ivacaftor (VX-770) was the first cystic fibrosis transmembrane conductance regulator (CFTR) modulatory drug approved for the treatment of patients with cystic fibrosis. Electron cryomicroscopy (cryo-EM) studies of detergent-solubilized CFTR indicated that VX-770 bound to a site at the interface between solvent and a hinge region in the CFTR protein conferred by transmembrane (tm) helices: tm4, tm5, and tm8. We re-evaluated VX-770 binding to CFTR in biological membranes using photoactivatable VX-770 probes. One such probe covalently labeled CFTR at two sites as determined following trypsin digestion and analysis by tandem-mass spectrometry. One labeled peptide resides in the cytosolic loop 4 of CFTR and the other is located in tm8, proximal to the site identified by cryo-EM. Complementary data from functional and molecular dynamic simulation studies support a model, where VX-770 mediates potentiation via multiple sites in the CFTR protein.

Lipophilicity of the Cystic Fibrosis Drug, Ivacaftor (VX-770), and Its Destabilizing Effect on the Major CF-causing Mutation: F508del.

Deletion of phenylalanine at position 508 (F508del) in cystic fibrosis transmembrane conductance regulator (CFTR) is the most common cystic fibrosis (CF)-causing mutation. Recently, ORKAMBI, a combination therapy that includes a corrector of the processing defect of F508del-CFTR (lumacaftor or VX-809) and a potentiator of channel activity (ivacaftor or VX-770), was approved for CF patients homozygous for this mutation. However, clinical studies revealed that the effect of ORKAMBI on lung function is modest and it was proposed that this modest effect relates to a negative impact of VX-770 on the stability of F508del-CFTR. In the current studies, we showed that this negative effect of VX-770 at 10 µM correlated with its inhibitory effect on VX-809-mediated correction of the interface between the second membrane spanning domain and the first nucleotide binding domain bearing F508del. Interestingly, we found that VX-770 exerted a similar negative effect on the stability of other membrane localized solute carriers (SLC26A3, SLC26A9, and SLC6A14), suggesting that this negative effect is not specific for F508del-CFTR. We determined that the relative destabilizing effect of a panel of VX-770 derivatives on F508del-CFTR correlated with their predicted lipophilicity. Polarized total internal reflection fluorescence microscopy on a supported lipid bilayer model shows that VX-770, and not its less lipophilic derivative, increased the fluidity of and reorganized the membrane. In summary, our findings show that there is a potential for nonspecific effects of VX-770 on the lipid bilayer and suggest that this effect may account for its destabilizing effect on VX-809- rescued F508del-CFTR.