Structure-guided drug design of novel antibiotics targeting DNA gyrase

Morgan, Harry 2026. Structure-guided drug design of novel antibiotics targeting DNA gyrase. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

The prevalence of multi-drug resistant strains of bacteria on a global scale demands the development and implementation of novel antibacterial therapeutics. DNA gyrase is a type IIA topoisomerase enzyme involved in the regulation and maintenance of DNA topology in bacteria. Targeting DNA gyrase as inhibitors, the fluoroquinolones have become one of the most prescribed antibiotic classes globally. However, due to the emergence of fluoroquinolone resistant bacterial strains, numerous resistance mechanisms to these antibiotics have been observed. Two novel, first-in-class antibiotics have recently achieved U.S Food and Drug Administration (FDA) approval; zoliflodacin, a spiropyrimidinetrione (SPT), and gepotidacin, a Novel Bacterial Topoisomerase Inhibitor (NBTI). These approvals mark a significant shift in antibacterial drug development, as they are the first new classes of antibiotics targeting DNA gyrase approved in decades. Protein X-ray crystallography played a vital role in the lead-compound development of gepotidacin, with six crystal structures published in the Protein Data Bank (PDB). Protocols detailing DNA gyrase crystallisation in complex with DNA and antibiotics favour the microbatch under-oil method, and no structure-based fragment screening programs had been done on the S. aureus GyrB27:A56 fusion truncateCORE construct for the discovery of novel compounds. Furthermore, DNA gyrase crystals are often twinned resulting in complications in structure solution and molecular refinement. In this thesis, a 2.78 Å resolution crystal structure (PDB ID 8BP2) showed two molecules of zoliflodacin binding to an S. aureus DNA gyrase - DNA cleavage complex. Structural analysis showed zoliflodacin binds more directly with conserved GyrB residues, rather than through the water-metal ion bridge to highly mutated GyrA residues, observed in fluoroquinolone structures. Furthermore, a 2.58 Å resolution crystal structure was determined (PDB ID 9FZ6), whereby anomalous difference Fourier maps enabled the modelling of three novel manganese binding sites. Investigations into crystal twinning using the nanofocus beamline VMXm at Diamond Light Source (DLS) demonstrated that multiple complete datasets can be solved from a single, large macromolecular crystal. Extensive crystallisation optimisation saw the development of a new crystallisation protocol for the S. aureus DNA gyrase - DNA complex, through sitting drop vapour diffusion, enhancing the reliability of growing highly diffracting crystals. By achieving a robust, high-throughput crystallisation protocol, the first structure-based fragment screening campaign at XChem (DLS) on the S. aureus GyrB27:A56 fusion truncateCORE construct was completed, soaking over 500 crystals with small drug-like fragments for structure determination. Following extensive refinement and model building, four fragment hits were observed in notable binding pockets; three within the thiophene pocket and one in the GyrA dimer interface pocket.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Schools > Biosciences
Subjects:	Q Science > Q Science (General)
Date of First Compliant Deposit:	21 May 2026
Last Modified:	21 May 2026 13:11
URI:	https://orca.cardiff.ac.uk/id/eprint/187157

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