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The investigations described in this thesis lay out strategies aimed at advancing antibiotic research and development. The examples presented revolve around two main approaches: understanding drug-target interactions and target identification.Applications of microcalorimetry provide insights into the binding mechanism of known antibiotics and their target within the bacterial membranes. These studies provided the thermodynamic characterization of cell-wall active compounds and their cell-wall precursor or phospholipid targets.Furthermore, by repurposing a small molecule library in a microbial susceptibility screen, the discovery of two new antibiotic leads is described. A suite of target identification methods, including whole genome sequencing and MS-based chemical proteomics, led to the characterization of their mode of action. Structure activity optimization of the leads led to the discovery of a new class of DNA gyrase inhibitors, acting on a so-far unexploited site of this validated bacterial target, as well as the identification of previously unmapped pathways in S. aureus, orchestrated by series of known and unknown enzymes.

Global healthcare is on the verge of an antibiotic availability crisis as bacteria have evolved resistance to nearly all known antibacterials. Identifying new antibiotics that operate via novel modes-of-action is therefore of high priority.This thesis contains two drug discovery projects, originating from a antibacterial screen of a compound library. In both projects chemical hits are first structurally optimized, after which their mode-of-action is determined.The first project entails optimizing a hit with potency against MRSA into a submicromolar active antibiotic. By using a chemical proteomics approach, the targets of this compound were elucidated, along with the targets that are most important in its antibacterial activity.The second project concerns Gram-negative bacteria, where a hit molecule is optimized into the conformationally restricted LEI-800. The target of LEI-800 is found to be DNA gyrase, a common antibiotic target. However, it is that LEI-800 inhibits DNA gyrase differently, and more potently, than the status quo.