The characterization of SPX‐101, a new peptide promoter of ENaC internalization, revealed basic mechanistic understanding of ENaC internalization and downstream signaling events.

Cystic Fibrosis (CF) is a life‐altering disease that affects over 70,000 people worldwide. CF is defined by mutations in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) causing an improperly functioning protein or loss of protein expression. Loss of CFTR activity causes an imbalance in ion levels leading to defects in mucus clearance and acidification of the lung. A major effect of lung acidification is the loss SPUNC1 activity. SPLUNC1 is secreted to the airway surface liquid and regulates ENaC through direct binding to the extracellular region of the channels. This interaction is lost at low pH, resulting in ENaC accumulation on the membrane and dehydration of the lung. Restoring SPLUNC1 regulation of ENaC is an attractive target for CF therapy, as it will increase lung hydration allowing for increased mucus clearance and normalization of normal lung function. SPX‐101 is a SPLUNC1‐derived peptide that internalizes ENaC regardless of pH. SPX‐101 can be nebulized directly into the lung allowing for efficient drug delivery into the target organ while achieving low systemic circulation. SPX‐101 has shown significant efficacy in CF animal models and safety in pre‐clinical toxicology and phase 1 clinical trials. Mechanistically, SPX‐101 internalized ENaC in a dose dependent manner in CF‐derived human bronchial epithelial cell (HBEC), decreased amiloride sensitive current, and increased airway surface liquid. SPX‐101 induced ENaC ubiquitination causing a robust and prolonged decrease in the membrane levels of ENaC alpha, beta, and gamma. ENaC internalization was achieved through activation of a pro‐internalization kinase axis followed by a compensatory increase in signaling mediators responsible for regulating ENaC stability. This internalization and downstream signaling activation is markedly different than ENaC channel blockers such as amiloride or derivatives of the parent molecule. Amiloride and its derivatives do not induce ENaC internalization or regulate downstream kinases, suggesting that these downstream signaling events are caused by internalization and/or SPX‐101 induced changes in ENaC structure rather than changes in sodium secretion. Together this work provides the molecular basis for the activity of the first in class peptide promoter of ENaC internalization, SPX101, and provided valuable insight into the mechanisms by which ENaC proteins are regulated on the cell surface.