An Unbiased Chemical Proteomics Method Identifies FabI as the Primary Target of 6‑OH-BDE-47

Determination of
the physical interactions of environmental chemicals with cellular
proteins is important for characterizing biological and toxic mechanism
of action. Yet despite the discovery of numerous bioactive natural
brominated compounds, such as hydroxylated polybrominated diphenyl
ethers (OH-PBDEs), their corresponding protein targets remain largely
unclear. Here, we reported a systematic and unbiased chemical proteomics
assay (Target Identification by Ligand Stabilization, TILS) for target
identification of bioactive molecules based on monitoring ligand-induced
thermal stabilization. We first validated the broad applicability
of this approach by identifying both known and unexpected proteins
bound by diverse compounds (anticancer drugs, antibiotics). We then
applied TILS to identify the bacterial target of 6-OH-BDE-47 as enoyl-acyl
carrier protein reductase (FabI), an essential and widely conserved
enzyme. Using affinity pull-down and in vitro enzymatic assays, we
confirmed the potent antibacterial activity of 6-OH-BDE-47 occurs
via direct binding and inhibition of FabI. Conversely, overexpression
of FabI rescued the growth inhibition of Escherichia coli by 6-OH-BDE-47, validating it as the primary in vivo target. This
study documents a chemical proteomics strategy for identifying the
physical and functional targets of small molecules, and its potential
high-throughput application to investigate the modes-of-action of
environmental compounds.