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Updated: Jul 4, 2025
Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase
Published on: October 15, 2018
Focused Screening Identifies Different Sensitivities of Human TET Oxygenases to the Oncometabolite 2-Hydroxyglutarate
Roman Belle1,2, Hilal Saraç1,2,3, Eidarus Salah1,4
1Chemistry Research Laboratory, Department of Chemistry, University of Oxford, 12 Mansfield Road, OX1 3TA Oxford, United Kingdom.
Abstract:
Ten-eleven translocation enzymes (TETs) are Fe(II)/2-oxoglutarate (2OG) oxygenases that catalyze the sequential oxidation of 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine in eukaryotic DNA. Despite their roles in epigenetic regulation, there is a lack of reported TET inhibitors. The extent to which 2OG oxygenase inhibitors, including clinically used inhibitors and oncometabolites, modulate DNA modifications via TETs has been unclear. Here, we report studies on human TET1-3 inhibition by a set of 2OG oxygenase-focused inhibitors, employing both enzyme-based and cellular assays. Most inhibitors manifested similar potencies for TET1-3 and caused increases in cellular 5hmC levels. (R)-2-Hydroxyglutarate, an oncometabolite elevated in isocitrate dehydrogenase mutant cancer cells, showed different degrees of inhibition, with TET1 being less potently inhibited than TET3 and TET2, potentially reflecting the proposed role of TET2 mutations in tumorigenesis. The results highlight the tractability of TETs as drug targets and provide starting points for selective inhibitor design.
Insights
Ten-eleven translocation (TET) enzymes, crucial for DNA demethylation, are explored as drug targets. Researchers identified inhibitors that increase 5-hydroxymethylcytosine (5hmC) levels, highlighting TETs
Area of Science:
- Epigenetics and Gene Regulation
- Enzymology
- Cancer Biology
Background:
- Ten-eleven translocation (TET) enzymes are key regulators of DNA demethylation, catalyzing the oxidation of 5-methylcytosine to various oxidized forms.
- Despite their critical roles in epigenetics and potential links to cancer, specific inhibitors for TET enzymes are scarce, and their modulation by existing 2-oxoglutarate (2OG) oxygenase inhibitors remains unclear.
- Understanding TET enzyme inhibition is crucial for developing targeted epigenetic therapies.
Purpose of the Study:
- To investigate the inhibition of human TET1, TET2, and TET3 by a panel of 2-oxoglutarate (2OG) oxygenase-focused inhibitors.
- To assess the impact of these inhibitors on cellular 5-hydroxymethylcytosine (5hmC) levels.
- To evaluate the potential of TET enzymes as drug targets and explore starting points for selective inhibitor design.
Main Methods:
- Utilized enzyme-based assays to determine the inhibitory potencies of various 2OG oxygenase inhibitors against human TET1-3.
- Employed cellular assays to measure the effects of inhibitors on intracellular 5hmC levels.
- Investigated the differential inhibition of TET1, TET2, and TET3 by the oncometabolite (R)-2-hydroxyglutarate.
Main Results:
- Most tested 2OG oxygenase inhibitors showed similar potency against TET1, TET2, and TET3.
- Inhibition of TET enzymes led to increased cellular 5hmC levels across the board.
- (R)-2-hydroxyglutarate exhibited differential inhibition, with TET1 being less sensitive than TET2 and TET3, potentially correlating with TET2's role in tumorigenesis.
Conclusions:
- TET enzymes are druggable targets, demonstrating tractability for therapeutic intervention.
- The study provides a foundation for designing selective TET inhibitors.
- Differential inhibition patterns, particularly for (R)-2-hydroxyglutarate, offer insights into TET enzyme function in cancer.
