Bioluminescence resonance energy transfer‐based detection of acetyl‐CoA

CoA metabolites play a critical role in the epigenetic regulation of gene expression. Of particular significance is acetyl-CoA, a primary metabolite used by histone acetyltransferases to modify histone tails and therefore control chromatin state. In cancer cells, the dysregulation of acetyl-CoA can lead to the aberrant expression of genes controlling cell growth. One challenge in studying these acetyl-CoA dependent functions is the lack of optical methods to detect CoA metabolites. Towards that goal, we report a bioluminescence energy transfer (BRET)-based approach to detect acetyl-CoA. Our strategy is inspired by recent biophysical and chemoproteomic analyses of human acetyltransferases, which revealed that members of the N-terminal (NAA) acetyltransferase family preferentially bind acetyl-CoA over CoA. To explore whether we could develop an acetyl-CoA selective ligand-displacement assay, we first recombinantly expressed and purified a NAA-Nanoluc fusion protein. Next, we demonstrated its ability to generate a BRET signal when incubated with CoA-fluorophore conjugates. Finally, we evaluated whether NAA-Nanoluc BRET was altered by the addition of CoA metabolites, which revealed a direct relationship between the affinity of the CoA and optical signal. We report the first tunable indicator displacement assay for CoA metabolites and provide a new approach for studying the engagement of acetyltransferase enzymes by small molecule inhibitors.