The global spread of antimicrobial resistance (AMR) necessitates the development of new antibacterial agents effective against resistant bacteria. To combat drug-resistant strains like MRSA and VRE, novel antibacterial agents that circumvent existing resistance mechanisms are crucial. Phospho-N-acetylmuramoyl-pentapeptide-transferase (MraY), a bacterial transmembrane enzyme essential for peptidoglycan biosynthesis, is a promising target. Several nucleoside antibiotics (tunicamycins, muraymycins, mureidomycins, and capuramycin) inhibit MraY and demonstrate activity against drug-resistant bacteria. These share a uridine moiety but exhibit structural diversity and varying antibacterial spectra. While co-crystal structures provide a foundation for structure-based drug design, MraY's conformational dynamics and the need for membrane permeability pose challenges. Optimizing MraY inhibition and bacterial accumulation simultaneously is difficult, especially given the complex and polar nature of these antibiotics. Synthesizing numerous analogues for structural optimization is also challenging due to complex multi-step synthesis. This study addresses this bottleneck by developing a platform for simplified, comprehensive analogue synthesis to accelerate the structural optimization of MraY inhibitory natural products.

Existing literature highlights the potential of MraY as an antibacterial drug target and the challenges associated with optimizing natural product inhibitors. Studies on nucleoside antibiotics that inhibit MraY show promising antibacterial activity against drug-resistant bacteria. However, rational design of MraY inhibitors is complicated by the enzyme's conformational flexibility and the requirement for membrane permeability. The complex synthesis and purification processes associated with these natural products significantly hinder the development of analogue libraries for structure-activity relationship (SAR) studies. Existing in situ screening strategies, while allowing for rapid synthesis, are often limited by cytotoxic reagents or byproducts, thus limiting their applicability to cell-based assays.

This research employed a build-up library approach combined with *in situ* screening to efficiently optimize MraY inhibitors. The natural products were divided into core and accessory fragments, designed to bind to the target and modulate properties, respectively. The hydrazone formation reaction, chosen for its chemoselectivity and production of only water as a byproduct, was used to ligate the core and accessory fragments directly on the assay plate. This avoided lengthy multi-step synthesis, purification, and characterization processes. The library, comprising 7 cores (four classes) and 98 accessory fragments, resulted in 686 analogues. MraY inhibitory activity was assessed using a fluorescence-based assay, and antibacterial activity was evaluated against six ESKAPE pathogens using a microdilution broth method. Hit compounds from the MraY sub-library underwent resynthesis and further characterization (LC-MS, 1H NMR). To improve stability, stable amide and anilide analogues were designed and synthesized. These were tested for MraY inhibition, antibacterial activity against ESKAPE pathogens, including drug-resistant strains, and cytotoxicity against HepG2 cells. The bactericidal effect and resistance emergence of a selected analogue (2) were also evaluated. Finally, *in vivo* efficacy was assessed using a mouse thigh infection model. Cryo-EM structural studies were performed on selected analogues to elucidate their unique binding modes to MraY.

The study successfully developed a streamlined approach for generating and evaluating large libraries of MraY inhibitors. A build-up library of 686 analogues was created and screened, identifying several potent and broad-spectrum antibacterial compounds. The hydrazone formation reaction proved efficient and suitable for *in situ* synthesis and evaluation. Key SAR trends were identified, highlighting the importance of long lipophilic alkyl chains for both MraY inhibition and antibacterial activity, particularly against Gram-positive bacteria. In the muraymycin sub-library, analogues with basic amino acid residues (Lys and Arg) exhibited the most potent activity against both Gram-positive and Gram-negative bacteria. Stable amide and anilide analogues were synthesized, exhibiting similar or even improved activity compared to their hydrazone counterparts. Analogue 2 demonstrated bactericidal activity *in vitro*, minimal resistance emergence, and *in vivo* efficacy in a mouse thigh infection model. Cryo-EM structural analysis revealed distinct interaction patterns of analogues 2 and 3 with MraY, suggesting unique binding modes compared to previously known inhibitors. Analogues 1-8 displayed minimal cytotoxicity.

The developed build-up library strategy significantly simplifies and accelerates the process of optimizing natural product-based MraY inhibitors. The *in situ* screening method reduces the need for extensive purification and characterization of individual compounds, allowing for high-throughput screening and efficient identification of potent lead molecules. The identified SAR trends provide valuable insights into the structural requirements for both MraY inhibition and antibacterial activity. The findings highlight the potential of MraY inhibitors as novel antibacterial agents, particularly given their effectiveness against drug-resistant strains. The structural analysis provides a detailed understanding of the unique binding modes of the novel analogues, informing future drug design efforts. The minimal cytotoxicity observed suggests a favorable therapeutic index for this class of compounds. The *in vivo* efficacy of analogue 2 validates the approach and highlights the potential of MraY as a target for new antibacterial drugs. This study demonstrates the versatility of the strategy, which was successfully applied to tubulin-binding natural products as well, suggesting broader applicability.

This study successfully developed a novel strategy for the rapid optimization of natural product-based MraY inhibitors, utilizing a build-up library and *in situ* screening. The identified analogues demonstrate potent, broad-spectrum antibacterial activity, are effective against drug-resistant strains, and exhibit a favorable safety profile. The unique binding modes revealed by cryo-EM studies provide a basis for further optimization and development of this promising class of antibacterial agents. Future work could focus on further optimization of these lead compounds to enhance their potency, selectivity, and pharmacokinetic properties for clinical development.

While the study demonstrates promising results, several limitations need to be acknowledged. The *in vivo* studies were conducted using a single mouse model, and further studies in other animal models are needed to confirm the findings and assess long-term safety and efficacy. The number of clinical isolates tested was relatively limited, and testing against a broader panel of clinical isolates would enhance the generalizability of the results. The evaluation of tubulin-binding natural products was mentioned but details were deferred to a future publication. Therefore, comprehensive data on this application is lacking in the current paper.