The incidence of neurodegenerative disorders like Parkinson’s disease (PD) and Alzheimer’s disease (AD) are increasing as the population ages. Slowing the rate of neurological decline can have a huge impact on health care costs and quality of life for both the patients and those caring for them. Pituitary adenylate cyclase activating peptide (PACAP) is a Secretin family peptide that activates the PAC1, VPAC1 and VPAC2 receptors and is associated with neuroprotection and neuronal differentiation. PACAP administration protects neurons against toxic, hypoxic, traumatic or inflammatory insults. The receptors of the Secretin family are unique due to the large extracellular domain (ECD) necessary to bind the endogenous ligand prior to receptor activation. The Secretin family ligands are all peptides, this family of receptors being responsible for regulating and maintaining homeostasis within the organism. PACAP is a pleiotropic peptide acting both centrally and peripherally. Exogenously administered peptide is rapidly metabolized. For neuroprotective effects, PACAP must cross the blood brain barrier (BBB). Enhancing the transport across the BBB has been accomplished through peptide glycosylation. Here we design and synthesize a series of glycosylated PACAP agonists and antagonists to evaluate them for receptor activity and ability to cross the BBB. A homology model was constructed of the full length PAC1R based on the transmembrane portion of both the mu opioid receptor and the corticotropin releasing factor-1 receptor combined with the NMR derived solution structure of the PAC1R ECD bound with the receptor antagonist, PACAP6-38. Using this model to guide us, the decision was made to place the glycosylated residue at the C-terminus of the peptide. A series of PACAP based glycopeptide agonists and antagonists were prepared using solid phase peptide synthesis (SPPS). Synthesis of PACAP analogs is complicated by the inclusion of two sites of aspartimide formation, the D3-G4 and D8-S9 sequences. Initial SPPS trials resulted in very little desired peptide formation. Reagent adjustments and using an amino-group protection strategy improved peptide yield. Methionine sulfoxide formation occurs in PACAP analogs. Substitution of methionine with leucine avoids this oxidation issue. An initial screen of PACAP and two glycosylated analogs using PC12 cells for PAC1R activation indicated that all three promoted neurite-like process outgrowths indicating PAC1R activation. The diluent treated cells did not exhibit this morphological change. Quantification of cells for assessing antiproliferative effects was not performed. More PC12 experiments should be performed to assess antiproliferative action and to screen additional glycosylated PACAP analogs for PAC1R activation. One of the glycosylated PACAP analogs was detected in CSF after i.p. administration in a mouse. Microdialysis samples obtained in vivo were analyzed by a newly developed LC/MS² technique and found to contain the administered glycosylated PACAP still intact, demonstrating that the glycopeptide crosses the BBB. Additional experiments using other glycosylated PACAP analogs are planned.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/321595 |
| Date | January 2014 |
| Creators | Anglin, Bobbi Lynn |
| Contributors | Polt, Robin L., Polt, Robin L., Yang, Danzhou, Hruby, Victor J., Hulme, Christopher, Wondrak, Georg |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | en_US |
| Detected Language | English |
| Type | text, Electronic Dissertation |
| Rights | Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. |
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