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Investigating the mechanisms of inhibition of peptidoglycan glycosyltransferases in Staphylococcus aureus
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Deering, John (2023) Investigating the mechanisms of inhibition of peptidoglycan glycosyltransferases in Staphylococcus aureus. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b3984436~S1
Abstract
Antibiotics form an essential cornerstone of modern medicine, however the growing antimicrobial resistance crisis threatens to shake its foundations. Hospital-acquired infections caused by resistant strains of Staphylococcus aureus remain a leading cause of mortality and novel anti-infective agents are urgently required to combat this challenge. Drugs that target the biosynthesis of bacterial cell wall peptidoglycan have been at the forefront of antibiotic research since the field emerged, yet the enzymes that carry out the penultimate step of this pathway, the glycosyltransferases (GTs), have been relatively under-investigated. PBP2 and MGT, two GTs from S. aureus, have long held promise as antibiotic development targets. The discovery of compounds inhibiting these enzymes could pave the way to a novel class of antibiotics with the potential to save countless lives. To this end, this thesis reports the development of two assays designed for the identification of GT inhibitors. The first is a GT activity assay based on the fluorescence anisotropy of a dansyl-labelled substrate. This approach was shown to be continuous, quantitative and robust enough for high-throughput screening. The second is a binding assay that uses surface plasmon resonance (SPR) to measure binding interactions of PBP2 and MGT. Furthermore, this work describes the implementation of the SPR binding assay with a high-throughput fragment screen. The resulting hit-pool was refined by a second analogue screen, affording a number of high-affinity hits, including three fragments that bind to both targets. Subsequent investigation experiments revealed that several fragments appear to occupy the same site as the known inhibitor moenomycin – two of which even exhibit partial inhibition of GT activity. These findings support the hypothesis that GT inhibition might be best achieved by targeting the nascent peptidoglycan’s ‘exit tunnel’ as opposed to the active site itself. This research opens up new avenues for the discovery of novel GT-targeting inhibitors by increasing our arsenal of enzymological tools and by providing a strong starting point for a fragment-based lead development campaign.
Item Type: | Thesis (PhD) | ||||
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Subjects: | R Medicine > RM Therapeutics. Pharmacology | ||||
Library of Congress Subject Headings (LCSH): | Antibiotics, Drug resistance in microorganisms, Staphylococcus aureus, Glycosyltransferases, Peptidoglycans, Bacterial cell walls | ||||
Official Date: | October 2023 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | Warwick Medical School | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Roper, David I. , Kettleborough, Katy , | ||||
Sponsors: | Medical Research Council (Great Britain) ; Warwick Medical School.MRC Doctoral Training Partnership ; LifeArc | ||||
Language: | eng |
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