Small Molecule Inhibitors of TET Dioxygenases: Bobcat339 Activity Is Mediated by Contaminating Copper(II)

Ten eleven translocation (TET) dioxygenases 1-3 are non-heme Fe(II) and alpha-ketoglutarate dependent enzymes that catalyze oxidation of 5-methylcytosine (SmC) in DNA to hydroxymethyl-C, formyl-C, and carboxy-C. This typically leads to gene activation and epigenetic remodeling. Most known inhibitors of TET are alpha-ketoglutarate mimics that may interfere with other alpha-ketoglutarate dependent enzymes. Recently, a novel cytosine-based inhibitor of TET, Bobcat339, was reported to have mid-mu M inhibitory activity against TET1 and TET2. The molecule is now sold as a TET inhibitor by several vendors. We independently prepared Bobcat339 in our laboratory and observed that it had minimal inhibitory activity against human TET1 and TET2 via a quantitative LC-ESI-MS/MS assay. Furthermore, the inhibitory activity of commercial Bobcat339 preparations was directly correlated with Cu(II) content. We therefore conclude that Bobcat339 alone is not capable of inhibiting TET enzymes at the reported concentrations, and that its activity is enhanced by contaminating Cu(II).

Automated summary

Ten eleven translocation (TET) dioxygenases are enzymes that play important roles in gene activation and epigenetic remodeling. Inhibitors of TET, such as alpha-ketoglutarate mimics, have been developed, but they may interfere with other enzymes. A recent study reported a new cytosine-based inhibitor called Bobcat339, but further research found that its inhibitory activity against TET enzymes is minimal and mainly comes from contaminating copper ions (Cu(II)).