recombinant mouse Tet1 and Tet2 have shown that both succinate and fumarate
inhibit at 400–600 μM range, and modest changes in global 5hmC levels are
observed in human neuroblastoma cells treated with fumarate and succinate esters
[160]. Thus, it appears TET activities are sensitive to concentration flux in some
TCA cycle intermediates/oncometabolites in vitro and in cells, as found in other
2OG (3) oxygenases [169–171].
In 2015, a fluorescent polarisation assay based on a fluorophore-linked
hydroxamic acid probe (33) was reported for Tet1 from NgTET, a model protein
with 39% similarity to human TETs [163]. The hydroxamic acid motif is thought to
chelate the active site Fe(II). Indeed, the probe binds to NgTet1 with K d ¼ 250 nM
and competes with 2OG (NgTET K d ¼ 250 μM). N-Oxalylglycine (NOG, 30), a
close isostere and an inactive analogue of 2OG (3) and a broad-spectrum inhibitor of
2OG oxygenases [172], was also found to bind to NgTet1 (K d ¼ 49 μM), demonstrating the utility of FP assay for the identification of 2OG competitive binders of
the TET proteins.
While the human TET2-5hmC-DNA complex had been co-crystallised with
NOG [114], it was not until recently that NOG was demonstrated to inhibit the
catalytic activity of human TET2CD (IC 50 ¼ 149 μM) using MALDI-TOF MS assay
[108]. R-2HG was confirmed to weakly inhibit TET2 in a similar range as previously
reported and S-2HG (28) at IC 50 > 10 mM using this assay.
5.2.2 Non-metal-Chelating Inhibitors of TETs
We have recently reported the development of macrocyclic peptide inhibitors for the
human TETs [158]. Using mRNA display-based RaPID technology, macrocyclic
peptide binders of human TET1CD delΔ1099–1936 were selected from a pool of
>10
12 peptides. Three sequences (TiP1 (36), TiP2 (37), Tip3M15L (38)) were
confirmed to bind to TET1 (K d < 100–220 nM) using surface plasmon resonance
(SPR) and inhibit the catalytic activity at approximately IC 50 ¼ 1 μM using 5hmC
antibody-based AlphaScreen™-based assay. Interestingly, TiP1 selectively
inhibited TET1 over TET2, demonstrating that some selectivity between different
TET proteins is possible. Unlike IOX1 (34), a 2OG competitive, metal-chelating
broad-spectrum 2OG oxygenase inhibitor control [172], the macrocyclic peptides do
not compete directly with 2OG, demonstrating a novel mode of action and a
promising approach to developing selective inhibitors.
6 Conclusions
DNA methylation is the most conserved epigenetic modification, and in mammals it
plays an important role in gene regulation. It is involved in normal biological
process, but it is also aberrant in several human diseases [35]. In particular it is
well studied in cancer, and, for example, certain FDA-approved cancer diagnostic
276
R. Belle et al.
inhibit at 400–600 μM range, and modest changes in global 5hmC levels are
observed in human neuroblastoma cells treated with fumarate and succinate esters
[160]. Thus, it appears TET activities are sensitive to concentration flux in some
TCA cycle intermediates/oncometabolites in vitro and in cells, as found in other
2OG (3) oxygenases [169–171].
In 2015, a fluorescent polarisation assay based on a fluorophore-linked
hydroxamic acid probe (33) was reported for Tet1 from NgTET, a model protein
with 39% similarity to human TETs [163]. The hydroxamic acid motif is thought to
chelate the active site Fe(II). Indeed, the probe binds to NgTet1 with K d ¼ 250 nM
and competes with 2OG (NgTET K d ¼ 250 μM). N-Oxalylglycine (NOG, 30), a
close isostere and an inactive analogue of 2OG (3) and a broad-spectrum inhibitor of
2OG oxygenases [172], was also found to bind to NgTet1 (K d ¼ 49 μM), demonstrating the utility of FP assay for the identification of 2OG competitive binders of
the TET proteins.
While the human TET2-5hmC-DNA complex had been co-crystallised with
NOG [114], it was not until recently that NOG was demonstrated to inhibit the
catalytic activity of human TET2CD (IC 50 ¼ 149 μM) using MALDI-TOF MS assay
[108]. R-2HG was confirmed to weakly inhibit TET2 in a similar range as previously
reported and S-2HG (28) at IC 50 > 10 mM using this assay.
5.2.2 Non-metal-Chelating Inhibitors of TETs
We have recently reported the development of macrocyclic peptide inhibitors for the
human TETs [158]. Using mRNA display-based RaPID technology, macrocyclic
peptide binders of human TET1CD delΔ1099–1936 were selected from a pool of
>10
12 peptides. Three sequences (TiP1 (36), TiP2 (37), Tip3M15L (38)) were
confirmed to bind to TET1 (K d < 100–220 nM) using surface plasmon resonance
(SPR) and inhibit the catalytic activity at approximately IC 50 ¼ 1 μM using 5hmC
antibody-based AlphaScreen™-based assay. Interestingly, TiP1 selectively
inhibited TET1 over TET2, demonstrating that some selectivity between different
TET proteins is possible. Unlike IOX1 (34), a 2OG competitive, metal-chelating
broad-spectrum 2OG oxygenase inhibitor control [172], the macrocyclic peptides do
not compete directly with 2OG, demonstrating a novel mode of action and a
promising approach to developing selective inhibitors.
6 Conclusions
DNA methylation is the most conserved epigenetic modification, and in mammals it
plays an important role in gene regulation. It is involved in normal biological
process, but it is also aberrant in several human diseases [35]. In particular it is
well studied in cancer, and, for example, certain FDA-approved cancer diagnostic
276
R. Belle et al.