AbstractAn increasing number of drugs are protein and peptide therapeutics. Proteins are involved in almost all cell pathways and tend to have a very specific function, making them attractive candidates as drugs. One of the main drawbacks to their use is they typically have very low oral bioavailability. The GI tract represents a chemical and physical barrier to molecules entering the body. The chemical barrier consists of enzymatic degradation and harsh pH conditions. The physical barrier primarily consists of the epithelium which restricts absorption of molecules from the intestinal lumen. Drug molecules can cross via the transcellular route (across cells) or the paracellular route (between cells). Epithelial cells are connected to adjacent cells by tight junctions (TJs), multi-protein complexes anchored to the cytoskeleton which form a semipermeable barrier in the paracellular space. TJ permeability is regulated by the specific proteins present and by phosphorylation of myosin light chain (MLC). MLC is phosphorylated by MLC kinase (MLCK) to open TJs and dephosphorylated by MLC phosphatase (MLCP) to close them. Here, a series of permeable inhibitor of phosphatase (PIP) peptides have been designed to inhibit MLCP to open TJs and an increase in permeability to biopharmaceuticals. Caco-2 monolayers and in vivo intestinal injections were used to assess the increase in permeability. Western blotting was used to assess PIP effect on MLC and tight junction proteins. Fluorescent microscopy was used to visualise the transport of drugs and distribution of tight junction proteins. Binding assays were used to assess the interaction of PIP peptides with MLCP.
Two lead peptides (PIP250 and PIP640) increased permeability of Caco-2 monolayers to 4 kDa dextran and intestinal epithelia to insulin, exenatide, salmon calcitonin and gentamicin. And increased pMLC over the same time course. In vitro, the PIP-induced permeation enhancement was charge-selective, with cations being more permeable than anions or molecules with no net charge. The charge selectivity was greater following PIP640 treatment. Both peptides induced a remodelling of TJ proteins. Both increased cation pore-forming claudin-2 and PIP250 reduced occludin. Claudin-2 increase was greater following PIP640 treatment. The level of claudin-2 increase correlates with the level of charge selectivity. Co-treatment with a myosin light chain kinase inhibitor suggested that the claudin-2 increase is dependent on pMLC whereas the occludin decrease is not. Single amino acid mutations to PIP250 identified residues that are key to binding to PP1. Mutations were identified that retained the permeation enhancer effect but had a faster recovery rate back to pre-treatment barrier function.
|Date of Award||27 Apr 2022|
|Supervisor||Randy Mrsny (Supervisor)|