Within the Task Force for Cystic Fibrosis project, our group discovered the corrector ARN23765. To better characterize the compound and strengthen its profile as a preclinical candidate for the treatment of CF, we aimed to identify at a molecular level its mechanism of action and the site of interaction with its biological target.
ARN23765 mechanism of action was studied in intact cells through the biochemical approach of the Photo-Affinity Labeling (PAL). To identify the regions of CFTR protein involved in the action of ARN23765, computational and functional studies were carried out along with mutagenesis experiments.
ARN23765 mechanism of action in intact cells was identified through PAL studies using chemical probes structurally related to our corrector. Furthermore, functional studies with specific CFTR domains were conducted to identify the protein portions involved in the correction induced by ARN23765. Finally, computational analyses together with mutagenesis experiments were carried out to elucidate the molecular basis of the interaction of ARN23765 with its biological target.
The interaction of ARN23765 with CFTR was studied in cells derived from people with CF overexpressing F508del-CFTR. Functional studies with CFTR domains identified the protein portion involved in the correction induced by ARN23765, while the binding site and amino acid residues implicated in the interaction with CFTR were clarified through computational analyses and mutagenesis studies.
The results of this project contribute to clarifying the molecular basis of the recovery of CFTR activity induced by ARN23765. The data show that in intact cells the corrector binds directly to CFTR, stabilizing it through specific interactions with amino acid residues in a defined protein region.