Chemoproteomics Yields a Selective Molecular Host for Acetyl-CoA

Chemoproteomic profiling is a powerful approach to define
the selectivity
of small molecules and endogenous metabolites with the human proteome.
In addition to mechanistic studies, proteome specificity profiling
also has the potential to identify new scaffolds for biomolecular
sensing. Here, we report a chemoproteomics-inspired strategy for selective
sensing of acetyl-CoA. First, we use chemoproteomic capture experiments
to validate the N-terminal acetyltransferase NAA50 as a protein capable
of differentiating acetyl-CoA and CoA. A Nanoluc-NAA50 fusion protein
retains this specificity and can be used to generate a bioluminescence
resonance energy transfer (BRET) signal in the presence of a CoA-linked
fluorophore. This enables the development of a ligand displacement
assay in which CoA metabolites are detected via their ability to bind
the Nanoluc-NAA50 protein “host” and compete binding
of the CoA-linked fluorophore “guest”. We demonstrate
that the specificity of ligand displacement reflects the molecular
recognition of the NAA50 host, while the window of dynamic sensing
can be controlled by tuning the binding affinity of the CoA-linked
fluorophore guest. Finally, we show that the method’s specificity
for acetyl-CoA can be harnessed for gain-of-signal optical detection
of enzyme activity and quantification of acetyl-CoA from cellular
samples. Overall, our studies demonstrate the potential of harnessing
insights from chemoproteomics for molecular sensing and provide a
foundation for future applications in target engagement and selective
metabolite detection.