Mechanistic Insightsinto CFTR Potentiation by theAntimicrobial Peptide Esc(1–21): Direct Interaction with theNBD1-NBD2 Interface

Abstract Cystic fibrosis (CF) is a genetic disorder caused by variants in the gene encoding the CFTR ion channel, leading to defective epithelial chloride transport, dehydrated airway mucus, chronic infections, and progressive lung damage. Although CFTR modulators have improved clinical outcomes, a subset of patients remains unresponsive to current therapies, highlighting the need for alternative strategies. The frog skin-derived antimicrobial peptide Esc(1–21) was recently identified as a CFTR potentiator, likely through direct interaction with the interface of the channel’s cytosolic nucleotide-binding domains (NBDs); yet its molecular mechanism is poorly understood. Here, we engineered a cysteine-less CFTR scaffold by introducing cysteine pairs within or outside the predicted peptide-binding region and used molecular dynamics simulation to define the putative interaction site. Disulfide cross-linking assays performed in cells expressing these constructs validated the computational predictions and showed that the peptide selectively prevents cross-link formation only when cysteines are positioned at the NBD interface, thereby supporting a direct and specific interaction with CFTR. Importantly, confocal microscopy provided evidence for peptide intracellular internalization, together with colocalization with the ion channel, indicating that Esc(1–21) can access the cytosolic side of CFTR to enhance its activity. Overall, these findings (i) provide the first mechanistic insights into the interaction of Esc(1–21) with CFTR in a cellular context; (ii) identify the NBD1-NBD2 interface as a suitable target for peptide-based potentiators; and (iii) support the development of Esc(1–21) and its derivatives as novel multifunctional therapeutic candidates for CF treatment.