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Abstract: Antimicrobial resistance is driving the search for new antibiotics and a greater understanding of their mechanism of action. Doxycycline is amongst the most-prescribed antimicrobials. It demonstrates a particularly low minimum inhibitory concentration against the zoonotic pathogen Coxiella burnetii. Doxycycline canonically targets the bacterial ribosome by blocking tRNA binding at the decoding centre (A site) of the small subunit. Using cryo-electron microscopy, we analysed doxycycline binding to C. burnetii and Escherichia coli ribosomes. Both structures reveal doxycycline binding at the exit tunnel in the large subunit. In C. burnetii three doxycycline molecules stack to block the tunnel. In E. coli one doxycycline molecule triggers a major change in the conformation of the ribosome. This rearrangement of the peptidyl transferase centre blocks tRNA binding and nascent chain accommodation, abolishing interactions that are fundamental to ribosome function. We identify a distinct ribosomal protein in the C. burnetii large subunit and characterise an additional member of the prokaryotic ribosome hibernation-promoting factor family. These insights into ribosome function and antibiotic action may aid the development of new ribosome inhibitor antibiotics.

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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.

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Abstract: The tRNA m1G37 methyltransferase (TrmD) is considered essential in various bacteria, including Staphylococcus aureus, a pathogen responsible for a wide range of diseases. Here, we have performed a high-throughput nanomole-scale synthesis campaign (nanoSAR) by late-stage copper(I)-catalyzed alkyne–azide cycloaddition (CuAAC)-functionalizing a library of structurally diverse azides (N = 320) to a pyrrolopyrimidone alkyne. We have identified selective S. aureus TrmD inhibitors with inhibitory activity in the nanomolar to low micromolar range using a direct-to-biology assay read-out. A carbamate-masked guanidine intermediate of the lead structure selectively inhibited S. aureus growth at low micromolar concentrations in cell-based assays, while Gram-negative bacteria and an off-target panel of methyltransferases were not affected. Subsequent cocrystallization resulted in a crystal structure of S. aureus TrmD bound to an inhibitor, providing detailed insights into its binding mode and enabling future structure-guided optimization.

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Abstract: Streptomyces bacteria are renowned for their multicellular lifestyle and as Nature’s medicine makers, producing the majority of the clinical antibiotics. A landmark event during early development is the lytic dismantling of the substrate mycelium. Degradation of the hyphal cell-wall leads to the accumulation of N-acetylglucosamine (GlcNAc) in the colonies, which is a metabolic checkpoint during the onset of development and antibiotic production. Here, we show that GlcNAc sensing requires a toxicity pathway dependent on the enzyme GlcNAc-6P dehydratase (NagS). Dehydration of GlcNAc-6P by NagS to 6P-chromogen I is an unprecedented reaction in central metabolism that is highly conserved in – and limited to – the Streptomycetaceae. 6P-chromogen I is metabolized into a structural analogue of ribose by a promiscuous activity of GlcNAc-6P deacetylase NagA. Toxicity is relieved by supplementing the growth media with ribose. Structure-function analysis of NagS not only highlighted key residues in the active site of the enzyme in interaction with its substrate GlcNAc-6P, but also revealed 6-phosphogluconate as its catalytic inhibitor. Our work uncovers a conserved metabolic toxicity pathway in Streptomyces that revolves around a novel enzyme that plays a key role in nutrient signaling.

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Abstract: As the global fight against antimicrobial resistance in bacteria becomes increasingly pressing, new tool compounds are needed to study and evaluate novel therapeutic targets. Here, cysteine-directed fragment-based drug discovery is coupled with high throughput chemistry direct-to-biology screening to target the catalytic cysteine of a family of bacterial effector proteins, the novel E3 ligases (NELs) from Salmonella and Shigella. These effector E3 ligases are attractive as potential drug targets because they are delivered into host cells during infection, have no human homologues and disrupt host immune response to infection. We successfully identify hit compounds against the SspH subfamily of NELs from Salmonella and show that these proteins are inhibited by compound treatment, representing an exciting starting point for development into specific and potent tool compounds.