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Abstract: Carbapenem-resistant Enterobacterales (CRE), including Escherichia coli and Klebsiella pneumoniae are increasingly common pathogens. Resistance in CRE is often associated with production of K. pneumoniae (KPC) carbapenemases. We report a structurally modified meropenem derivative, JDB-1-200 (i.e., 8-epi-meropenem, in which the stereochemistry of the C8 hydroxyl is inverted), displaying enhanced activity relative to meropenem against KPC-producing CRE. JDB-1-200 alone displayed superior activity (MICs of 0.5 to 1 mg/L, compared with >8 mg/L for meropenem) against several clinically relevant Enterobacterales, including KPC-producing K. pneumoniae and Citrobacter freundii. Crystallographic analysis of JDB-1-200 complexes with the E166Q deacylation-deficient KPC-2 mutant, compared with an analogous meropenem (8R-hydroxyl) complex, indicates that the improvement arises from the JDB 8S-hydroxyl group acting as a hydrogen-bond donor to the hydrolytic water, slowing acyl-enzyme hydrolysis, with similar interactions observed in a CTX-M-15:JDB-1-200 complex. Kinetic inhibition data for KPC-2 and CTX-M-15 indicate that JDB-1-200 could be used alone or alongside a β-lactamase inhibitor for effective CRE treatment.

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Abstract: Tuberculosis remains one of the leading major global health challenges, driven by the emergence of multidrug-resistant and extensively drug-resistant bacterial strains. Resistant strains complicate treatment, which often requires prolonged use of toxic second- and third-line drugs. Protein phosphorylation plays critical roles in Mycobacterium tuberculosis, with serine/threonine kinases PknA, PknB, and PknG being essential for survival, virulence, and persistence. In this study, we screened an in-house kinase inhibitor library to identify compounds targeting these kinases. Four structurally diverse hits from the screening inhibiting all three kinases in vitro were selected for further analysis. Hits were evaluated for their ability to inhibit M. tuberculosis growth and characterized structurally using X-ray crystallography, molecular docking, and isothermal titration calorimetry. Our findings provide a structural framework for the development of multitargeting kinase inhibitors, offering a potential strategy to combat drug-resistant M. tuberculosis.

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Abstract: The widespread emergence of bacterial resistance to β-lactam antibiotics, driven primarily by serine β-lactamases (SBLs), continues to threaten the clinical utility of this cornerstone class of antibacterial agents. Although β-lactamase inhibitors such as sulbactam and tazobactam have historically restored the activity of partner antibiotics, their effectiveness has been eroded by the proliferation of inhibitor-resistant enzymes, particularly class C (AmpC) and class D (OXA) β-lactamases. Consequently, there remains a pressing need for new inhibitor scaffolds that combine potent enzyme inhibition with improved chemical stability and tunable physicochemical properties. This thesis explores sulfoximine substitution on the penam scaffold as a strategy to expand the chemical and biological properties of β-lactamase inhibitors. Sulfoximines offer distinct advantages over classical sulfones, including stereochemical control, dual hydrogen-bond donor/acceptor capability, and enhanced opportunities for structural diversification. Leveraging recent advances in nitrene-transfer chemistry, robust and stereocontrolled synthetic routes to penam-sulfoximines were developed from sulbactam-derived sulfoxides. Both (S)- and (R)-configured penam sulfoximines were prepared, including synthetically challenging NH-sulfoximines that closely mimic the parent sulbactam framework. Single-crystal X-ray diffraction confirmed stereochemical integrity and structural assignments. The scope and limitations of sulfoximine N-functionalization were systematically investigated, enabling access to amide, urea, and N-aryl derivatives while revealing intrinsic stability constraints of the penam core under basic or reductive conditions. These studies establish a versatile synthetic platform for the generation of structurally diverse penam-sulfoximines suitable for biological evaluation. Biochemical and microbiological studies demonstrated that penam-sulfoximines are potent inhibitors of clinically relevant SBLs, including AmpC and OXA-type enzymes. A free NH penam-sulfoximine displayed particularly strong enzyme inhibition and measurable antibacterial activity against Acinetobacter baumannii, supported by enzyme inhibition assays, protein-binding studies, antimicrobial susceptibility testing, and structural characterization of enzyme–inhibitor complexes. However, many substituted sulfoximines exhibited reduced stability under physiological conditions, highlighting a trade-off between potency and chemical robustness. Overall, this work establishes penam-sulfoximines as a chemically accessible and biologically active class of β-lactamase inhibitors, delineates key structure–stability–activity relationships, and provides a foundation for the future design of sulfoximine-based agents targeting resistant Gram-negative pathogens.

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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.