The Design, Characterization, and Efficacy of Protease-Resistant Therapeutic Peptides.
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 h... read moreindered, 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
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.read less