Neutrophil serine proteases (NSPs) play an important role in the innate immune system. However, when the balance between NSPs and their endogenous protease inhibitors (PIs) is disrupted, they also play a critical role in the pathogenesis of inflammatory lung disease. Excessive release of NSPs such as human neutrophil elastase (HNE), proteinase 3 (Pr3) and cathepsin G (CatG), leads to destruction of the lung matrix and continued propagation of acute inflammation. Under normal conditions, endogenous PIs counteract these effects by inactivating NSPs. In inflammatory lung diseases, including chronic obstructive pulmonary disease, cystic fibrosis, emphysema and acute lung injury, there are insufficient levels of endogenous PIs to mitigate damage. Therapeutic strategies are needed to modulate excessive NSP proteolytic activity in conditions of inflammatory lung disease, in order to restore the NSP-endogenous PI balance and decrease the inflammatory response.
The Kerns laboratory previously demonstrated that heparin derivatives substituted with structurally unique aromatic residues bind with high affinity and selectivity to select Glycosaminoglycan-binding proteins, and more recently, using the neomycin, kanamycin and apramycin aminoglycosides as chemical scaffolds a panel of N-arylacyl O-sulfonated aminoglycosides was prepared as novel structural mimics of heparin. The hypothesis guiding the study presented here is that structurally unique N-arylacyl O-sulfonated aminoglycoside derivatives will selectively bind and modulate the function of NSPs both in vitro and in vivo, thus representing novel lead structures for future development of a new class of therapeutic agents capable of modulating excessive NSP activity in the lung.
To this end, the first objective was to screen the recently synthesized panel of N-arylacyl O-sulfonated aminoglycosides for their ability of inhibit each of the NSPs. The inhibitory profile of each N-arylacyl O-sulfonated aminoglycoside with respect to HNE, CatG and Pr3 was characterized to determine if one N-arylacyl O-sulfonated aminoglycoside could inhibit multiple NSPs. Furthermore, the mechanism of protease inhibition of two lead N-arylacyl O-sulfonated aminoglycosides, identified in the initial screen, was elucidated using CatG as the representative NSP.
The N-arylacyl O-sulfonated aminoglycosides were also evaluated for their ability to inhibit the proteolytic activity of the three NSPs in a cell based assay that evaluates the ability of test compounds to inhibit NSP-mediated detachment of A549 lung epithelial cells from the surface of a 96-well plate. After concluding the in vitro analysis of the panel of N-arylacyl O-sulfonated aminoglycosides, one lead compound that inhibited all three of the NSPs was further evaluated in vivo to determine if this class of compounds exhibits any overt toxicity and if so at what concentrations, and secondly if our lead compound is able to decrease LPS-induced acute inflammation in the lung.
The results of these studies validate the approach of using N-arylacyl O-sulfonated aminoglycosides to target the three NSPs as a new therapeutic method for the treatment of inflammatory lung diseases.
| Identifer | oai:union.ndltd.org:uiowa.edu/oai:ir.uiowa.edu:etd-6927 |
| Date | 01 May 2016 |
| Creators | Craciun, Ioana |
| Contributors | Kerns, Robert J. |
| Publisher | University of Iowa |
| Source Sets | University of Iowa |
| Language | English |
| Detected Language | English |
| Type | dissertation |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | Copyright © 2016 Ioana Craciun |
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