are separated and analysed by LC in tandem with MS (e.g. triple-quadrupole MS,
quadrupole-orbitrap MS) and quantitated against labelled nucleoside internal standards. The large dynamic range (from low femtomole range) allows diverse application of this technique, from gDNA to oligonucleotides. LC-MS/MS analysis has
been used to determine the substrate preference for TETs for 5mC oxidation over
5hmC/5fC-DNA [107, 118]. For example, steady-state kinetics analysis of TET2CD
(delΔ1099–1936) using LC-MS/MS revealed the affinities of modified cytosine to
reduce with increasing oxidation (K M ¼ 0.48, 0.9 and 1.3 μM for 5mC, 5hmC and
5fC, respectively) [118].
While LC-MS/MS methodology provides quantitative accuracy and robustness,
the multistep processing of DNA has hampered its use for high-throughput applications. A matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF)
MS method has provided a middle ground. This assay allows direct measurements
of intact DNA without the need for downstream processing steps (e.g. enzymatic and
chemical modifications), thus minimising the reagent requirement, processing error
and time. Michaelis-Menten kinetic parameters for 2OG (3) were determined to be
K M ¼ 15.7 μM (hTET2CD (delΔ1099–1936)) and K M ¼ 24.2 μM (hTET3CD (delΔ
689–1596)) using this method. The relatively low 2OG K M suggests that potency is
crucial for 2OG competitive inhibitors.
Assays using antibodies against modified cytosines, such as enzyme-linked
immunosorbent assay (ELISA) and AlphaScreen, have also helped improve the
sensitivity, the volumes and throughput of the assays [158]. Assays using
radiolabelling (e.g. 2-oxo[114 C]glutarate, [γ32 P]ATP) are also utilised for inhibitor
screening.
5.2 Inhibitors of TET Enzyme Activity
As for the other 2OG oxygenases, the majority of inhibitors target the catalytic Fe
(II) and often mimic or compete with the 2OG binding (see [162] for review of 2OG
oxygenase inhibitors (Fig. 8)).
5.2.1 2OG Analogues as TET Inhibitors
The first TET inhibitor to be identified was R-2-hydroxyglutarate (R-2HG, 28), an
‘oncometabolite’ associated with the gain-of-function mutations in isocitrate dehydrogenases (IDH). IDH is a tricarboxylic acid (TCA) cycle enzyme that catalyses the
oxidative decarboxylation of isocitrate to 2OG (3). IDH with mutations in the active
site (Arg100, Arg132 in IDH1, Arg140, Arg172 in IDH2 found in glioma/leukaemia) can further catalyse the reduction of 2OG (3) to R-2HG (28), leading to cellular
accumulation of R-2HG (28) (up to 30–50 mM, compared to <0.1 mM in normal
cells) [164–167]. R-2HG inhibits human (and mouse) TETs with IC 50 at 4–5 mM
range [159, 160]. Given the inhibitory effect of TETs by R-2HG and significantly
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R. Belle et al.
quadrupole-orbitrap MS) and quantitated against labelled nucleoside internal standards. The large dynamic range (from low femtomole range) allows diverse application of this technique, from gDNA to oligonucleotides. LC-MS/MS analysis has
been used to determine the substrate preference for TETs for 5mC oxidation over
5hmC/5fC-DNA [107, 118]. For example, steady-state kinetics analysis of TET2CD
(delΔ1099–1936) using LC-MS/MS revealed the affinities of modified cytosine to
reduce with increasing oxidation (K M ¼ 0.48, 0.9 and 1.3 μM for 5mC, 5hmC and
5fC, respectively) [118].
While LC-MS/MS methodology provides quantitative accuracy and robustness,
the multistep processing of DNA has hampered its use for high-throughput applications. A matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF)
MS method has provided a middle ground. This assay allows direct measurements
of intact DNA without the need for downstream processing steps (e.g. enzymatic and
chemical modifications), thus minimising the reagent requirement, processing error
and time. Michaelis-Menten kinetic parameters for 2OG (3) were determined to be
K M ¼ 15.7 μM (hTET2CD (delΔ1099–1936)) and K M ¼ 24.2 μM (hTET3CD (delΔ
689–1596)) using this method. The relatively low 2OG K M suggests that potency is
crucial for 2OG competitive inhibitors.
Assays using antibodies against modified cytosines, such as enzyme-linked
immunosorbent assay (ELISA) and AlphaScreen, have also helped improve the
sensitivity, the volumes and throughput of the assays [158]. Assays using
radiolabelling (e.g. 2-oxo[114 C]glutarate, [γ32 P]ATP) are also utilised for inhibitor
screening.
5.2 Inhibitors of TET Enzyme Activity
As for the other 2OG oxygenases, the majority of inhibitors target the catalytic Fe
(II) and often mimic or compete with the 2OG binding (see [162] for review of 2OG
oxygenase inhibitors (Fig. 8)).
5.2.1 2OG Analogues as TET Inhibitors
The first TET inhibitor to be identified was R-2-hydroxyglutarate (R-2HG, 28), an
‘oncometabolite’ associated with the gain-of-function mutations in isocitrate dehydrogenases (IDH). IDH is a tricarboxylic acid (TCA) cycle enzyme that catalyses the
oxidative decarboxylation of isocitrate to 2OG (3). IDH with mutations in the active
site (Arg100, Arg132 in IDH1, Arg140, Arg172 in IDH2 found in glioma/leukaemia) can further catalyse the reduction of 2OG (3) to R-2HG (28), leading to cellular
accumulation of R-2HG (28) (up to 30–50 mM, compared to <0.1 mM in normal
cells) [164–167]. R-2HG inhibits human (and mouse) TETs with IC 50 at 4–5 mM
range [159, 160]. Given the inhibitory effect of TETs by R-2HG and significantly
274
R. Belle et al.