- Title
- Inhibiting the NusB-NusE protein-protein interaction - a novel target for antibiotic drug development
- Creator
- Cossar, Peter
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2018
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- The strain of antibiotic resistances on global health has led to a movement away from traditional approaches to iterative antibiotic development. Over the past decade, there has been a growing body of evidence indicating that targeting protein-protein interactions (PPIs) is an efficacious approach to drug development. The bacterial interactome is central to all microbial processes and modulation of PPI by small molecule compounds leads to disruption of homeostasis, resulting in arrestment of bacterial proliferation. One microbial PPI of particular interest to the McCluskey research group is the NusB-NusE heterodimer. This heterodimer is essential for nucleation of the processive antitermination complex, functioning to regulate ribosomal transcription. Disruption of this PPI inhibits bacterial proliferation. Through virtual screening of the mini-Maybridge chemical library (56,000 compounds) using a pharmacophore model based on the α1-helix of NusE, 25 hit compounds were identified; five of these compounds were deemed synthetically tractable. Synthesis and biological evaluation of these leads using a competitive ELISA identified (4,6-bis(2',4',3,4-tetramethoxyphenyl))pyrimidine-2-sulphonamido-N-4-acetamide 1 and N,N'-[1,4-butanediylbis(oxy-4,1-phenylene)]bis(N-ethyl)urea 3, which exhibited IC50 values of ~6 μM and ~20 μM against NusB-NusE PPI, respectively. Bacterial growth assays also identified that 1 and 3 elicited moderate activity against representative Gram-positive Bacillus subtilis and Gram-negative Escherichia coli. These results validated the NusB-NusE PPI as a potentially ‘druggable’ target for antibiotic development (Chapter 3). A Structure-Activity Relationship (SAR) library based upon 3 was developed, consisting of four focused sub-libraries; 34 compounds in total. In vitro assessment identified ten analogues with ≥ 50% inhibition of the NusB-NusE PPI at 25 μM. Screening of these ten compounds in a bacterial growth assay identified that 2-((Z)-4-(((Z)-4-(4-((E)-(carbamimidoylimino)methyl)phenoxy)but-2-en-1-yl)oxy)benzylidene)hydrazine-1-carboximidamide 22 elicited excellent activity against clinically relevant pathogens, with MIC values of ≤ 3 µg/mL for Gram positive Streptococcus pneumoniae and methicillin-resistant Staphylococcus aureus and ≤ 51 µg/mL for Gram-negative Pseudomonas aeruginosa and Acinetobacter baumannii. Further, using epifluorescence microscopy we demonstrated that the mode of action of 22 was consistent with it inhibiting NusB’s interaction with NusE, which would lead to a significant reduction in rRNA synthesis (Chapter 4).N, N-Alkylation of primary amines under epoxide coupling conditions proved to be a significant challenge during synthesis of lead 5; not satisfied with unacceptable losses in yield, a flow chemistry approach to mitigate by-product formation was developed. We report the development of the flow coupling of epichlorohydrin with substituted phenols (22 – 89%); the flow epoxidation of aromatic and aliphatic olefins using in situ generation of dimethyl dioxirane; and a regioselective flow-mediated aminolysis to afford a library of β-amino alcohols (21 – 80%) (Chapter 5). Flow hydrodehalogenation was a considerable limitation during SAR library development. In an attempt to mitigate this problem a series of catalysts, in the presence of halogenated aromatics, were screened for their ability to effect reductions. Halogen retention was obtained using 5% Pt/C (sulfided) under nitro reduction, reductive aminations and alkene reduction conditions (Chapter 6).
- Subject
- antibiotic drug; drug development; NusB-NusE protein
- Identifier
- http://hdl.handle.net/1959.13/1383709
- Identifier
- uon:31976
- Rights
- Copyright 2018 Peter Cossar
- Language
- eng
- Full Text
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