Studies of myosin light chain-dependent modulation of tight junction function through the actions of membrane-permeant peptides

Doctoral thesis English OPEN
Owens, Sian-Eleri
  • Subject: RM

Myosin light chain phosphorylation plays a central role in the regulation of paracellular permeability. The main objective of this study was to design and synthesise membrane-permeant peptide inhibitors of myosin light chain kinase and phosphatase that could potentially decrease and increase paracellular permeability, respectively. Elevated myosin light chain kinase activity, as observed in a variety of inflammatory disease, phosphorylates myosin light chain, to increase paracellular permeability. Initial studies showed a peptide inhibitor of myosin light chain kinase termed PIK could rectify increased paracellular permeability in two different cell-based models of inflammation. PIK was also, however, shown to be too labile for use in vivo. From a series of PIK analogues, two candidates prepared using D-amino acids were identified that showed sufficient stability, membrane-permeant properties and retention of specific function of PIK for future in vivo studies. Since the ratio of myosin light chain kinase to phosphatase activity regulates the degree of myosin light chain phosphorylation, it was hypothesised that inhibition of myosin light chain phosphatase would increase paracellular permeability. A membrane-permeant peptide designed to inhibit myosin light chain phosphatase activity termed PIP was identified in a screen of potential candidates through its capability to significantly decrease transepithelial electrical resistance in a polarized human intestinal epithelial cell system in vitro without any apparent cytotoxicity. Further studies will be required to determine the extent to which PIP increases paracellular permeability. Using these novel membrane-permeant peptide inhibitors, studies were performed using polarized monolayers of human intestinal epithelial cells to assess the action of previously identified regulators of paracellular permeability in vitro. Previous studies demonstrated that the non-specific myosin light chain kinase inhibitor ML-7 prevented the absorption enhancing properties of sodium caprate. Treatment with PIK and sodium caprate simultaneously resulted in significant increases in the permeability of inert fluorescent probes while pre- and post-incubation with PIK inhibited sodium caprate effects. These surprising findings suggest a potential application for combination treatment with sodium caprate and PIK to increase paracellular permeability of poorly absorbed drugs.
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