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.