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Alternative Antimicrobial Agents Against Multidrug-Resistant Gram-Negative Bacteria

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Abstract

The emergence of antibiotic-resistant bacteria is outpacing the discovery and development of new antibiotics, and this phenomenon poses a global health crisis and exemplifies the need to develop alternative antibacterial strategies. Oligothioetheramides (oligoTEAs) are a class of synthetic, sequence-defined oligomers that have demonstrated antibacterial activity against both gram-positive and gram-negative bacteria. Particularly, the oligoTEA BDT-4G has exhibited potent activity against a range of multidrug-resistant Pseudomonas aeruginosa isolates. In addition, BDT-4G can evade mechanisms conferring resistance to the last resort antibiotic, polymyxin B, and is active against P. aeruginosa clinical isolates with an acquired resistance to polymyxin B, as well as intrinsically-resistant gram-negative species, such as Burkholderia and Pandoraea spp. However, BDT-4G is toxic to mammalian cells at concentrations required for bactericidal activity, and this narrow therapeutic window precludes clinical translation. To mitigate cytotoxicity, we first investigated a bacteria-responsive prodrug methodology whereby BDT-4G is temporarily inactivated through coupling to a polyethylene glycol (PEG) promoiety via a triglycine peptide linker (Gly3). PEGylation of BDT-4G decreases the in vitro cytotoxicity by an order of magnitude, and antibacterial activity is recovered via site-specific cleavage of the triglycine linker by LasA, a virulence factor secreted by P. aeruginosa. To further improve localization to the infection site for systemic applications, we next explored an antibody-bactericide conjugate (ABC) platform for the targeted delivery of BDT-4G. In this design, BDT-4G is coupled via a cleavable linker to an antibody that targets P. aeruginosa. For proof-of-concept of this ABC platform, we first coupled the prodrug, PEG-Gly3-BDT-4G, to an anti-Pseudomonas polyclonal antibody, and we demonstrate LasA-mediated release of the payload and bactericidal activity against P. aeruginosa. Lastly, to improve the reactivity of the ABC, we conjugated BDT-4G to the monoclonal antibody Cam-003, which binds to the conserved P. aeruginosa exopolysaccharide Psl. This conjugation was achieved through a host-cleavable peptide linker, which was selected to offset the selection pressures that may accompany a therapeutic that is activated by bacterial proteases. Through flow cytometry, fluorescence resonance energy transfer, and time-kill curves, we have demonstrated that the ABC binds to the bacterial cell surface, serum cleaves the linker, and the released oligoTEA payload is bactericidal. Overall, these studies validate the ABC strategy in vitro and motivate the translation to in vivo experiments.

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231 pages

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Date Issued

2022-08

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Keywords

Antibiotic; Drug delivery; Drug discovery; Gram-negative; Multidrug-resistant

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Union Local

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Committee Chair

Alabi, Christopher A.

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Committee Member

Westblade, Lars F.
DeLisa, Matthew

Degree Discipline

Chemical Engineering

Degree Name

Ph. D., Chemical Engineering

Degree Level

Doctor of Philosophy

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Government Document

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dissertation or thesis

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