%0 PDF %T The Design, Characterization, and Efficacy of Protease-Resistant Therapeutic Peptides. %A Heard, Kathryn. %D 2017-04-14T13:35:39.950Z %8 2017-04-14 %R http://localhost/files/70795m429 %X Abstract: ABSTRACT Peptides offer a number of advantages over small molecule and antibody-based drugs. These advantages, as well as advancements in methods of peptide production and formulation, have inspired a renewed interest in the development of peptides as novel therapeutics for the treatment of various diseases. The emergence of peptides as mainstream drug candidates has been severely hindered, however, by two major drawbacks. First, peptides are susceptible to degradation and inactivation by numerous enzymes expressed throughout the body. Second, due to their large size and hydrophilicity peptides are not readily orally available. We aimed to develop a simple and general method of stabilizing peptides against proteolytic degradation. We show here that introduction of an amino acid analogue containing a tertiary-substituted beta-carbon at the P1' position rendered peptides resistant to enzymatic degradation by the serine protease dipeptidyl peptidase IV (DPP IV), as well as by various other physiologically relevant serine proteases. Based on results from kinetic analyses, we suggest the modification interferes with the mechanism of cleavage rather than enzyme binding and recognition. Most importantly, receptor activation assays revealed the modified peptide analogues retained potent biological activity at their intended targets. The incretin hormone glucagon-like peptide 1 (GLP-1) lowers blood glucose levels by slowing gastric emptying and enhancing insulin synthesis and secretion. These glucose-lowering properties, in addition to the peptide's ability to preserve and restore beta-cell function and suppress appetite, make GLP-1 an ideal treatment for type 2 diabetes mellitus (T2DM). The application of GLP-1 as an antidiabetic agent is severely limited, however, by the peptide's low oral bioavailability and short in vivo half-life of only 1-2 minutes. The glucose-lowering activity of a protease-resistant analogue of GLP-1 (P1732) was tested in diabetic (db/db) mice and in oral glucose tolerance tests (OGTTs) in lean mice. P1732 consistently displayed more potent and prolonged antidiabetic activity than native GLP-1. Biotinylation of P1732 at Lys20 and Lys28 (DB-1732) rendered the peptide orally available by targeting it for transport through the intestinal epithelium by the sodium-dependent multivitamin transporter (SMVT). In a 2005 study, the neurotransmitter neuropeptide Y (NPY) was suggested to induce apoptosis of Ewing's sarcoma (ES) cancer cells through activation of the Y1 receptor (Y1R). DPP IV-mediated cleavage prevents NPY from activating the Y1R, and thus attenuates the peptide's pro-apoptotic activity. The anticancer activity of native NPY and the DPP IV-resistant analogues sNPY and ssNPY was tested in a mouse Ewing's sarcoma xenograft model. Neither native NPY nor the modified peptide analogues inhibited ES tumor growth. Since our analogue is metabolically stable and displays potent and selective activity at the Y1R, we now question the previously reported role of NPY in ES cell apoptosis. In conclusion, we present here a simple and highly adaptable method for designing enzymatically stable and biologically active therapeutic peptides. We believe this work will significantly impact the future development of peptide-based therapeutics.; Thesis (Ph.D.)--Tufts University, 2011.; Submitted to the Dept. of Biochemistry.; Advisors: William Bachovchin, and Andrew Bohm.; Committee: Joel Habener, Andrew Plaut, and Theoharis Theoharides.; Keywords: Biochemistry, Health sciences, and Pharmaceutical sciences. %[ 2022-10-11 %9 Text %~ Tufts Digital Library %W Institution