For more than 60 years antibiotics have successfully been used to treat bacterial infections. However, current therapeutics are losing efficacy due to the rise of antibiotic resistance. Consequently, new antibiotic classes that are not subject to existing resistance mechanisms are urgently required. We are discovering new antibiotic classes that can be used to treat bacterial infections caused by the Gram-positive pathogen, Staphylococcus aureus.  One novel approach is to identify small molecules that target biotin utilisation. Biotin protein ligase (BPL) is an essential metabolic enzyme that is responsible for the post-translational attachment of biotin onto biotin-dependent enzymes, representing a novel antibiotic drug target (1). As humans also possess a BPL homologue, it is important to discover inhibitors that are selective for the bacterial enzyme over the human equivalent. Our team has discovered a new class of antibiotic, the biotin triazoles, that are chemical analogues of the reaction intermediate (bio-5'AMP) naturally employed by BPLs (1). Twenty three analogues based upon the biotin triazole pharmacophore have been synthesised and tested yielding a structure activity relationship (SAR) series. For this series, the adenosine moiety present in bio-5’AMP is replaced with mono- and di- substituted benzyl functional groups. This approach provides rapid access to a large number of new drug-like chemical structures for in vitro assays and anti S. aureus activities.  Enzyme assays have shown that the biotin triazoles inhibit S. aureus BPL but not the human equivalent, thereby providing a therapeutic window for selective inhibition. Importantly, these next generation inhibitors exhibit bacteriostatic activity against a methicillin sensitive strain of S. aureus, but are devoid of cytotoxic activity against HepG2 cells. The data from this SAR series has identified new chemical structures with further potential in inhibiting S. aureus BPL. Our goal is to chemically optimise these inhibitors for treating Staphylococcus aureus infections.