Target Identificationwith Live-Cell PhotoaffinityLabeling and Mechanism of Action Elucidation of ARN23765, a HighlyPotent CFTR Corrector

Molecular-targeted therapies for the treatment of cystic fibrosis (CF) rely on small-molecule modulators that rescue the activity of the defective CF transmembrane conductance regulator (CFTR) anion channel. <b>ARN23765</b> is a small molecule with subnanomolar potency in rescuing the function of mutant CFTR in bronchial epithelial cells from CF patients carrying the F508del-CFTR mutation. Considering the multifaceted interactions of CFTR with the plasma membrane and the complexity of the protein network within the cellular compartments, here we report the investigation of <b>ARN23765</b>’s molecular mechanism in live cells. We used the photoaffinity labeling (PAL) approach to demonstrate the interaction of <b>ARN23765</b>-derived probes with CFTR in cells. We showed that <b>ARN23765</b> contributes to F508del-CFTR rescue by stabilizing the membrane-spanning domain-1 and interacting with CFTR at the same site as other type I CFTR correctors. Our study characterizes <b>ARN23765</b>’s mode of action and highlights the potential of studying the interactions between CFTR and its correctors in live cells.