Characterising the mutations involved in resistance to biotin protein ligase inhibitors, a novel class of antibacterial agent to combat Staphylococcus aureus

A Hayes1, J Satiaputra1, AS Paparella1, B Bianco-Rodriguez2, S Pedersen1, AD Abell2, GW Booker1 and SW Polyak1

  1. School of Biological Sciences, University of Adelaide
  2. School of Physical Sciences, University of Adelaide

Staphylococcus aureus is an important clinical pathogen for which current antibiotics are becoming ineffective. The development of new antibiotics is essential to combat these antibiotic resistant infections. One potential target for new antibiotics is the inhibition of the essential metabolic enzyme, biotin protein ligase (BPL). In S. aureus, BPL is a bifunctional protein that is both an enzyme and transcriptional repressor of biotin biosynthesis and transport genes. These alternative functions are regulated by the higher order structure of the protein, with the monomer being enzymatic and dimer able to bind DNA. Our approach to target BPL is to design, synthesise and characterise chemical analogues of the reaction intermediate employed by BPL. We have identified a potent antibacterial and pre-clinical candidate, BPL199 (MIC 0.25–0.5 µg/ml), with low cytotoxicity observed in vitro and in vivo. An important consideration during antibiotic development is drug resistance. In this study, bacterial isolates resistant to BPL199 were evolved in vitro (n=7) and then characterised using whole genome sequencing. One strain harboured a missense mutation in BPL, D200E, which resides in the dimerisation interface of BPL implying DNA-binding activity may be affected. We demonstrated that the transcriptional repressor activity of D200E was indeed compromised, using a variety of genetic and biochemical techniques including an Escherichia coli based reporter system, qPCR and protein cross-linking studies. We propose a novel resistance mechanism whereby the mutation increases the intracellular concentration of biotin that competes with the inhibitor to bind the BPL target.