Target Identification with Live-Cell Photoaffinity Labeling and Mechanism of Action Elucidation of ARN23765, a Highly Potent 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. ARN23765 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 ARN23765's molecular mechanism in live cells. We used the photoaffinity labeling (PAL) approach to demonstrate the interaction of ARN23765-derived probes with CFTR in cells. We showed that ARN23765 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 ARN23765's mode of action and highlights the potential of studying the interactions between CFTR and its correctors in live cells.