Please use this identifier to cite or link to this item:
|Title:||UNCONVENTIONAL HIGH-THROUGHPUT SCREENING TECHNIQUES FOR THE DISCOVERY OF CELL WALL ANTIBIOTICS|
|Department:||Biochemistry and Biomedical Sciences|
|Abstract:||The emergence of antibiotic resistance in recent years has radically reduced the clinical efficacy of many antibacterial treatments and now poses a significant threat to human health. One of the earliest studied and well validated targets for antimicrobial discovery is the bacterial cell wall. The essential nature of this pathway, its conservation among bacterial pathogens, and absence in human biology have made cell wall synthesis an attractive pathway for new antibiotic drug discovery. Indeed, nearly all cell wall active agents have been discovered using screens for growth inhibition, followed by cumbersome secondary screens to identify the cell wall target. What is lacking are selective primary screening assays for the sensitive detection of cell wall active compounds while avoiding off-target and nuisance compounds. The overarching objective of this work is to explore new and unconventional screening techniques for the discovery of new cell wall antibiotics. These approaches take an integrative approach merging the positive aspects of whole-cell phenotypic and target based screening while obviating many of their individual pitfalls. Two main approaches are taken herein. First we explore the validity of using the recently developed PywaC reporter based system in B. subtilis 168 to enrich for cell wall inhibitors. A pilot screen of 26,000 small molecules led to the discovery of 9 cell wall actives of which one had its direct target identified. Secondly we harness the powerful and complex dispensability pattern found within wall teichoic acid (WTA) in Gram-positives. Developed is a powerful antagonism screen to search for early step and substrate producing WTA inhibitors. This approach led to prolific enrichment for inhibitors of undecaprenyl-pyrophosphate synthase (UppS) through the interrogation of a small molecule library of 140,000 compounds. Overall, this thesis outlines effective and innovative approaches toward the identification of novel cell wall active compounds.|
|Appears in Collections:||Open Access Dissertations and Theses|
Files in This Item:
|PhD Thesis - tczarny.pdf||43.91 MB||Adobe PDF||View/Open|
Items in MacSphere are protected by copyright, with all rights reserved, unless otherwise indicated.