Structure-function analysis of tight junction-directed permeation enhancer PIP250

The intestinal paracellular route of absorption is modulated via tight junction (TJ) structures located at the apical neck of polarized intestinal epithelial cells to restrict solute movement through the intercellular space between them. Tight junctions open or close in response to changes in the phosphorylation status of light chain (MLC) at position Ser-19. This phosphorylation event is primarily controlled by MLC kinase (MLCK) and MLC phosphatase (MLCP), the latter being a holoenzyme that involves interaction between protein phosphatase 1 (PP1) and myosin targeting protein 1 (MYPT1). An entirely D-amino acid Permeant Inhibitor of Phosphatase (PIP) peptide sequence designed to disrupt PP1-MYPT1 interactions at the cytoplasmic surface of TJs, PIP250 (rrfkvktkkrk) localized at intracellular TJ structures, altered expression levels of specific TJ proteins, increased cellular phosphorylated MLC (pMLC) levels, binding to PP1, decreased epithelial barrier function, and significantly increased systemic uptake of the poorly absorbed antibiotic gentamicin in vivo. A series of PIP250 peptide analogues showed that positions phe3 and val5 were critical to its functional properties, with some providing opportunities to tune the dynamic actions of its TJ modulation properties. These data confirm the activity of PIP250 as a rationally designed oral permeation enhancer and validated key amino acids involved in its interaction with PP1 that define its overall actions; the magnitude and duration of these enhancing properties were associated with the MYPT1-mimetic properties of the PIP250 peptide analogues described.