Cell-Penetrating Peptide-Medicated Delivery of Insulin Degrading Enzyme as a Vehicle for Small Molecule Therapeutics
Disciplines
Amino Acids, Peptides, and Proteins | Biological Factors | Enzymes and Coenzymes
Abstract (300 words maximum)
Possible new therapeutic drugs often fail because they cannot cross cell membranes where the delivery is needed. A possible solution to this problem, and the source of this research project is using cell-penetrating peptides to deliver small molecules across membranes. Cell-penetrating peptides (CPPs) have the ability to cross membranes onto molecules to which they are attached. In this study, a cell-penetrating peptide-adaptor protein was used to deliver Insulin Degrading Enzyme (IDE) to mammalian cells. IDE is a homodimer with a cavity capable of containing small molecules. The IDE in this study has been engineered to have a disulfide bond between subunits which acts as a ‘clasp’ similar to a coin purse. The disulfide ‘clasp’ can be reduced to ‘open’ the protein, a drug molecule would then be loaded into the cavity, and then the ‘clasp’ can be oxidized to close it again. Once the IDE is loaded with the drug molecule, it can then be attached to a CPP-adaptor and delivered to cells where it would be transported across the membrane, and then released in the reducing environment of the cytoplasm. To test this theory, we designed, expressed, and purified IDE as a fusion to maltose-binding protein (MBP). The binding kinetics of the purified protein showed fast on, fast off kinetics, and high affinity in the presence of calcium, and negative affinity in its absence. Multiple different CPP-adaptors were added to the IDE, including GFP-calmodulin (GFP-CaM), TAT-naked mole rat calmodulin (NMR-CaM), and TAT-LAH4-CaM. Cell-penetration experiments were run with baby hamster kidney (BHK) cells. IDE was successfully delivered to cell interiors with all adaptors. Experiments are underway to deliver a fluorescently labeled drug molecule using the CPP-adaptor/IDE scheme. The results show hope for future success in delivering IDE with small-molecule therapeutics.
Academic department under which the project should be listed
CSM - Molecular and Cellular Biology
Primary Investigator (PI) Name
Jonathan McMurry
Cell-Penetrating Peptide-Medicated Delivery of Insulin Degrading Enzyme as a Vehicle for Small Molecule Therapeutics
Possible new therapeutic drugs often fail because they cannot cross cell membranes where the delivery is needed. A possible solution to this problem, and the source of this research project is using cell-penetrating peptides to deliver small molecules across membranes. Cell-penetrating peptides (CPPs) have the ability to cross membranes onto molecules to which they are attached. In this study, a cell-penetrating peptide-adaptor protein was used to deliver Insulin Degrading Enzyme (IDE) to mammalian cells. IDE is a homodimer with a cavity capable of containing small molecules. The IDE in this study has been engineered to have a disulfide bond between subunits which acts as a ‘clasp’ similar to a coin purse. The disulfide ‘clasp’ can be reduced to ‘open’ the protein, a drug molecule would then be loaded into the cavity, and then the ‘clasp’ can be oxidized to close it again. Once the IDE is loaded with the drug molecule, it can then be attached to a CPP-adaptor and delivered to cells where it would be transported across the membrane, and then released in the reducing environment of the cytoplasm. To test this theory, we designed, expressed, and purified IDE as a fusion to maltose-binding protein (MBP). The binding kinetics of the purified protein showed fast on, fast off kinetics, and high affinity in the presence of calcium, and negative affinity in its absence. Multiple different CPP-adaptors were added to the IDE, including GFP-calmodulin (GFP-CaM), TAT-naked mole rat calmodulin (NMR-CaM), and TAT-LAH4-CaM. Cell-penetration experiments were run with baby hamster kidney (BHK) cells. IDE was successfully delivered to cell interiors with all adaptors. Experiments are underway to deliver a fluorescently labeled drug molecule using the CPP-adaptor/IDE scheme. The results show hope for future success in delivering IDE with small-molecule therapeutics.