The project aims to identify the biological targets and mechanism of action of ARN23765, a CFTR corrector, discovered in our research group (Task Force for Cystic Fibrosis project), with a sub-nanomolar potency in rescuing the function of mutant CFTR in human bronchial epithelial cells from F508del/F508del CF patients. Despite the validated pharmacological effects, the mechanism of action of ARN23765 has not yet been conclusively defined. To pursue this challenging goal in living cells instead of purified proteins, the researchers decided to use a biochemical approach known as Photo-Affinity Labeling (PAL). This technique entails the synthesis of chemical probes structurally related to ARN23765, characterized by the presence of a photoreactive portion and a chemical group capable of binding to a reporter molecule. These photo-affinity probes were designed, synthesized and finally incubated with living cells expressing native or mutant (F508del-) CFTR. The cells were then exposed to UV light, thus allowing probe cross-linking to bio-molecules in close proximity. The reporter molecule was used to detect and/or isolate probe-protein adducts for the identification of the possible target(s) using protein gel electrophoresis, western blot and/or mass spectrometry analysis. The data achieved so far demonstrated ARN23765-like photo-affinity probes bind to CFTR in living cells, indicating that corrector ARN23765 may reasonably act directly on CFTR. To the best of our knowledge, this outcome is of great interest since it discloses the unprecedented interaction of a modulator to CFTR in an integral biological setting. By means of this PAL technology a set of interesting proteins (currently under evaluation), involved directly or indirectly with CFTR, were also identified and could reasonably represent additional targets of ARN23765.