Synthesis and Characterization of a Photoaffinity Labelling Probe Based on the Structure of the Cystic Fibrosis Drug Ivacaftor

Cystic Fibrosis (CF) is a genetic disorder caused by loss-of-function mutations to the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Ivacaftor (1) was the first therapeutic approved for the treatment of CF that is able to restore gating activity to certain CFTR variants although the mechanism of action is poorly understood. Herein we describe the synthesis of a photoaffinity labelling (PAL) probe (2) based on the structure of ivacaftor incorporating a photoreactive diazirine moiety for use in labelling studies designed to identify the binding site for ivacaftor on mutant CFTR. The PAL probe 2 retained potentiation activity, with a potency similar to 1, using a Fluorescent Imaging Plate Reader (FLIPR®) assay measuring ion conductance potentiation of wild type (Wt)-CFTR. Photolabelling experiments with human serum albumin (HSA) as a model protein have shown that probe 2 can label HAS in a manner consistent with observed and predicted binding.