Oncometabolites D- and L-2-hydroxyglutarate Inhibit the AlkB Family\nDNA Repair Enzymes under Physiological Conditions
Cancer-associated mutations often lead to perturbed cellular energy metabolism and accumulation of potentially harmful oncometabolites. One example is the chiral molecule 2-hydroxyglutarate (2HG); its two stereoisomers (D- and L-2HG) have been found with abnormally high concentrations in tumors featuring anomalous metabolic pathways. 2HG has been demonstrated to competitively inhibit several \xce\xb1-ketoglutarate (\xce\xb1KG)- and non-heme iron-dependent dioxygenases, including some of the AlkB family DNA repair enzymes, such as ALKBH2 and ALKBH3. However, previous studies have only provided the IC50 values of D-2HG on the enzymes and the results have not been correlated to physiologically relevant concentrations of 2HG and \xce\xb1KG in cancer cells. In this work, we carried out detailed kinetic analyses of DNA repair reactions catalyzed by ALKBH2, ALKBH3 and the bacterial AlkB in the presence of D- and L-2HG in both double and single stranded DNA contexts. We determined kinetic parameters of inhibition, including kcat, KM, and Ki. We also correlated the relative concentrations of 2HG and \xce\xb1KG previously measured in tumor cells with the inhibitory effect of 2HG on the AlkB family enzymes. Both D- and L-2HG significantly inhibited the human DNA repair enzymes ALKBH2 and ALKBH3 under pathologically relevant concentrations (73\xe2\x80\x9388% for D-2HG and 31\xe2\x80\x9358% for L-2HG inhibition). This work provides a new perspective that the elevation of either D- or L-2HG in cancer cells may contribute to an increased mutation rate by inhibiting the DNA repair carried out by the AlkB family enzymes and thus exacerbate the genesis and progression of tumors.
INTRODUCTION<!>Oligonucleotide Synthesis<!>Expression and Purification of the AlkB, ALKBH2 and ALKBH3 Proteins<!>Enzymatic Reaction<!>Kinetic Studies<!>RESULTS<!>Oligonucleotide Synthesis and Protein Purification<!>Enzymatic Assay for Measuring Kinetic Constants<!>Kinetic Analyses<!>2HG Inhibition of the DNA Repair Reactions Catalyzed by the AlkB Family\nEnzymes<!>\xce\xb1KG Recovery of 2HG\xe2\x80\x99s Inhibition on the Repair\nEnzymes<!>Biological Implications of 2HG Inhibition of DNA Repair Enzymes<!>Strand Preference of the Three Repair Enzymes<!>Other \xce\xb1KG/Fe(II)-Dependent Enzymes may be Inhibited by\nOncometabolites<!>
<p>Mutations in the isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) are
frequently found in >75% human low grade glioma, secondary
glioblastoma, cartilaginous tumor and >20% of acute myeloid
leukemia.1–5 Tumor-derived mutant forms of IDH
catalyze the NAD-dependent dehydrogenation of Î±-ketoglutarate (Î±KG)
to D-2-hydroxyglutarate (D-2HG), a function that supplants the physiological
activity of IDH, which entails reductive decarboxylation of isocitrate to
αKG (Figure 1b).6–13 L-2HG, the stereoisomer of D-2HG, has been identified as an
oncometabolite with elevated concentrations in renal cell carcinoma9,10 neurodegenerative disorders,14,15 and in
tissues under oxygen limitation or hypoxic conditions.11,12 The
elevation of L-2HG under such conditions is key from either loss of expression of
L-2HG dehydrogenase or promiscuous substrate utilization by lactate dehydrogenase A
and malate dehydrogenases 1 and 2.10,12 Both D-2HG
(R-2HG) and L-2HG (S-2HG) and several other molecules have been identified as
oncometabolites because their accumulations in different tumor cells are originated
from dysregulated energy metabolism pathways and metabolic imbalance.16–20 Because of their structural similarity to αKG, both
D- and L-2HG could compete with Î±KG and inhibit enzymatic processes that use
Î±KG as a substrate. One important class of enzymes that utilizes
αKG—and thus potentially susceptible to inhibition by 2HG—is
the non-heme iron- and Î±KG-dependent dioxygenases, a family of enzymes with
over 80 different members.21–24 2HG has
been demonstrated to inhibit several Î±KG-dependent enzymes, such as histone
demethylases, prolyl hydroxylases, the TET family 5-methlycytosine (5mC)
hydroxylases,7 and some of
the AlkB family DNA repair enzymes, such as ALKBH2 and ALKBH3.8,25 However, in the case of the AlkB proteins, previous in
vitro studies have only provided the IC50 values of 2HG on
the enzymes and the results have not been extrapolated to physiologically relevant
concentrations of 2HG and Î±KG in cancer cells. While Wang et al.
demonstrated that the accumulation of DNA damage in cells producing high levels of
D-2HG is consistent with inhibition of ALKBH2 and/or ALKBH3,25 the detailed mechanism of
inhibition of the AlkB enzymes by 2HG has not been reported. A careful study of the
inhibitory effect of both D- and L-2HG on AlkB repair enzymes is needed to quantify
the extent of inhibition of the direct reversal DNA repair pathways; perturbations
in these pathways would lead to unrepaired mutagenic DNA lesions, which would cause
mutations that can accelerate tumor progression or enable metastatic growth. Such a
study would also facilitate the identification of druggable targets related to the
AlkB enzymes because many alkylating chemotherapeutic agents generate DNA adducts
that are repaired by this family of repair enzymes.26 Inhibition of these enzymes would thus afford a
clinical benefit in anti-tumor regimens.</p><p>The Escherichia coli AlkB protein was discovered to be an
Î±KG/Fe(II)-dependent dioxygenase that oxidizes the alkyl groups in DNA
adducts formed by alkylation agents, ultimately restoring the undamaged DNA bases
(Figure 1a).27–29
Nine human homologs of AlkB have been identified as ALKBH1-8 and FTO.29,30 Among these homologs, ALKBH231–36
and ALKBH337–40 have been identified as major DNA
repair enzymes for repairing small alkyl DNA lesions. Since the initial discovery of
the catalytic mechanism of AlkB in 2002,27,28 a range of alkyl
adducts have been identified as substrates for AlkB, ALKBH2 and ALKBH3, both
in vitro and in vivo24 The adducts include all of the seven N-methyl
adducts occurring at the Watson-Crick (W-C) base-pairing face of the four
nucleobases.41 The seven
adducts include 3-methylcytosine (m3C),
N4-methylcytosine, 1-methyladenine (m1A),
N
6-methyladenine, 3-methylthymine, 1-methylguanine, and
N2-methylguanine. AlkB has also been reported to
repair other DNA adducts, such as 1, N6-ethenoadenine,
1, N6-ethanoadenine,
3,N4-ethenocytosine, 3-ethylcytosine, 1,
N2-ethenoguanine, 3, N
4-α-hydroxyethanocytosine,
3,N4-α-hydroxypropanocytosine,
N2-ethylguanine,
N2-tetrahydrofuran-2-yl-methylguanine,
N2-furan-2-yl-methylguanine, malondialdehydeguanine,
α-hydroxypropanoguanine, and γ-hydroxypropanoguanine.41–49 The repair efficiency and substrate scope of the AlkB
family enzymes have been recently reviewed in detail.24,30,50–54</p><p>In this work, we carried out kinetic analyses of DNA repair reactions
catalyzed by ALKBH2, ALKBH3 and AlkB in the presence of the inhibitors D- and L-2HG.
Oligonucleotides containing the methylated bases m1A and m3C were selected as
substrates for the repair reactions because they are most efficiently repaired by
these three enzymes.24 For each
substrate/enzyme/inhibitor combination, we determined a complete panel of kinetic
parameters, (kcat, KM, kcat/KM,
Ki), and correlated the relative concentrations of 2HG and
Î±KG found in tumor cells with the inhibitory effect of 2HG on the AlkB
family enzymes. Because ALKBH2 preferentially repairs adducts in double stranded
(ds) DNA and ALKBH3 prefers to repair lesions in a single stranded (ss) DNA context,
we tested the three repair enzymes with both ds and ss DNA substrates. This is the
first report of 2HG inhibition of AlkB family DNA repair evaluated in both single-
and double- stranded DNA. We also developed an HPLC-based method to study DNA repair
in the ds-DNA. The results showed that both ALKBH2 and ALKBH3, the major mammalian
direct reversal repair enzymes for alkylated DNA damage, were significantly
inhibited by D- and L-2HG under pathophysiologically relevant conditions.</p><!><p>Sixteen-mer oligonucleotides were synthesized with the sequence
5'-GAAGACCTXGGCGTCC-3' containing the lesions m1A and m3C at the
X position.43,55 The complementary 23mer oligonucleotides were
synthesized with the sequence of 5′-CTGGGACGCCYAGGTCTTCACTG-3′,
where Y represents the position incorporating the regular bases T or G, and
named as 23mer-Tcp or 23mer-Gcp. The 23mer oligonucleotides complementary to
23mer-Tcp and 23mer-Gcp were also synthesized, with the sequence
5′-CAGTGAAGACCTZGGCGTCCCAG-3′, where Z was the regular base A or
C, named as 23mer-A or 23mer-C. All DNA syntheses employed solid-phase
phosphoramidite chemistry performed on a MerMade-4 Oligonucleotide
Synthesizer.55 The
oligonucleotides were purified by HPLC (Thermo Fisher Scientific) on a DNAPac
PA-100 Semi-Preparative column (Phenomenex). Solvent A was 100 mM 1:1
triethylamine-acetic acid (TEAA) in water and solvent B was 100%
acetonitrile. The concentration of DNA was determined by UV absorbance at 260nm
by NanoDrop. The oligonucleotides were characterized by HPLC-electrospray
ionization triple quadrupole time of flight mass spectrometry (AB Sciex) (Table S1).</p><!><p>ALKBH2, ALKBH3 and AlkB were expressed and purified as described and
shown previously.47,55 In the following section, AlkB
is used as an example to illustrate the purification protocols. Briefly,
His-tagged AlkB was obtained by transforming pET28a-AlkB into E.
coli Rosetta2(DE3)pLysS or BL21(DE3)pLysS cells and protein
expression was induced by the addition of 1mM
isopropyl-b-D-thiogalactopyranoside (IPTG) at 37 °C (37 °C for
ALKBH2 and 30 °C for ALKBH3). The expressed protein was purified by
affinity chromatography. Thrombin was used to digest His-tag containing AlkB
protein. The final purified protein was stored −80 °C in AlkB
storage buffer as previously described.55</p><!><p>To assay the AlkB family demethylase activity toward the two substrates
in ss- and ds-DNA, the enzymatic reactions were performed at 37 °C at
different time points for the kinetic study of the AlkB reaction in buffer [70.0
µM
Fe(NH4)2(SO4)2·6H2O,
0.93 mM Î±KG (0.1 mM for 2-HG inhibition assay), 1.86 mM ascorbic acid
and 46.5 mM HEPES (pH 8.0)]. The reactions were stopped by adding 10 mM EDTA
followed by heating at 95 °C for 5 min. Typically, the purified proteins
were incubated with oligonucleotides containing DNA adducts in the presence of
all cofactors in a 20 µL reaction volume. In order to separate substrate
and product, 16mer m1A and A or 16mer m3C and C, the HPLC condition started with
a 5 min gradient of 1.5 M ammonium acetate from 50% to 65%,
followed by 2 min 70% ammonium acetate. The column was DNApac PA-100
(4× 250 mm) (Thermo Scientific). The UV detection wavelength was at 260
nm. Each reaction was carried out in triplicate.</p><p>For the double-stranded DNA substrates, 1.5 equivalents of the 23mer
complementary oligonucleotides, 23mer-Tcp or 23mer-Gcp, were annealed with 16mer
oligos by heating the mixture at 80 °C for 10 min and then cooling down
to room temperature with the rate of 1 °C/10 s. The post-reaction
treatments were similar to those for the ss-DNA reactions, except 1.75
equivalents of 23mer-A and 23mer-C were added together with 10 mM EDTA followed
by heating up to 95 °C for 10 min and then cooled down to room
temperature with the same rate as used for annealing. The quantification method
was the same as described above. Each reaction was carried out in
triplicate.</p><!><p>To determine KM, kcat and Ki values for
the repair reactions, initial rates were obtained by keeping the DNA substrate
and enzyme concentration constant and varying Î±KG concentration with or
without various concentrations of D- or L-2HG (0, 1.0, 3.0, 5.0, 7.0, 9.0, 37.3
mM). All reactions were performed at 37 °C in triplicate and the data
were analyzed by GraphPad Prism 5 with the Michaelis-Menten kinetics model. The
inhibition curves were fit to the equation:
1/V0=1/Vmax+(KM/Vmax)×(1+[I]/Ki)×(1/[S]).</p><!><p>To test the inhibitory effect of D- and L-2HG on the AlkB family enzymes, we
first chemically synthesized oligonucleotides by site-specifically incorporating m1A
and m3C, the major substrates of the AlkB family enzymes.24,43 We also
expressed and purified recombinant human ALKBH2 and ALKBH3 proteins, and the
E. coli AlkB protein.55 Then, we performed kinetic experiments to determine the
kcat and KM of the three enzymes as they repair the two
adducts in both ds- and ss-DNA. After that, we measured the Ki of D- and
L-2HG on the repair reactions, and finally evaluated the inhibitory effect of the
oncometabolites in the concentration range reported to occur in certain human
cancers.</p><!><p>Two 16mer oligonucleotides containing m1A and m3C were chemically
synthesized with the sequence 5'-GAAGACCTXGGCGTCC-3' (X denotes
the alkylated base).43 After
HPLC purification, the identity of the oligonucleotides was confirmed by
comparing the theoretical m/z of the oligonucleotides with the
observed m/z from high resolution LC-MS (Table S1). The genes for
E. coli AlkB and its human homologs ALKBH2 and ALKBH3 were
cloned into pET28a+ expression vector; the incorporation of the correct
sequences was confirmed by sequencing the corresponding plasmids. The three
proteins were then expressed in E. coli hosts, isolated and
purified by affinity chromatography as described in Experimental
Section.55</p><!><p>For each enzymatic reaction, the adduct-containing oligonucleotide was
incubated with the necessary cofactors for the AlkB reaction: Fe(II),
Î±KG, and ascorbic acid (see Experimental Section) in either ss- or
ds-DNA. Below, m1A will be used as an example to explain the HPLC analyses. For
the ss-DNA reactions, the starting material 16mer m1A (1.5 min in Figure 2a) and product 16mer A (2.8 min in
Figure 2b) were well separated by anion
exchange HPLC, and the amount of each was quantified by reference to standard
curves. For ds-DNA repair reactions, we initially used a 16mer complementary
oligonucleotide. However, the dsDNA of starting material (16mer complementary
plus 16mer m1A) and the dsDNA of product (16mer complementary plus 16mer A)
could not be fully separated under various HPLC conditions, thus making the
quantification of reactions challenging. Therefore, we adopted a longer
complementary oligonucleotide (23mer Tcp, 5.6 min in Figure 2c), which provided a similar repair efficiency as
the 16mer complementary oligonucleotide. In the analysis of the 23mer reaction,
the dsDNA of starting material (23mer Tcp plus16mer m1A, 7.5min in Figure 2c) and the dsDNA of product (23mer
Tcp plus 16mer A, 7.7min in Figure 2c)
still could not be fully separated under the HPLC condition. Consequently, we
designed another 23mer oligonucleotide that was fully complementary to 23mer Tcp
(23mer A, 5.5min in Figure 2d). After the
dsDNA reaction with 23mer Tcp, 23mer A was added to the reaction mixture, and
the mixture was heated to 80 °C for 10 min and then slowly cooled down
to room temperature. The addition of 23mer A allowed the 23mer Tcp formed
perfect dsDNA with 23mer A (9.1 min in Figure
2d), thus releasing 16mer m1A and 16mer A from their previous
complementarity with 23mer Tcp. Under these conditions, the 16mer m1A and 16mer
A in the dsDNA repair reaction were well separated and quantified by the HPLC
analyses (Figure 2d). A similar analytical
strategy was successfully applied to m3C dsDNA repair reactions.</p><!><p>After setting up a reliable procedure to quantify the conversion of the
repair reactions, we carried out systematic kinetic analyses of the AlkB family
enzymes repairing m1A and m3C. Because the purpose of this work was to measure
the possible inhibition of D-and L-2HG on the repair reactions catalyzed by
Î±KG-dependent AlkB family enzymes, the kinetic parameters of Î±KG
in the repair reactions were first measured. In a typical kinetic analysis
(e.g., ALKBH2 repairing m1A), 5 µM of oligonucleotide substrate, and 0.2
µM ALKBH2 enzyme were mixed with different concentrations of αKG
(5.0–70.0 µM) and the extent of the repair reaction was
quantified at different time points (see Experimental section for details).
Because the repair of one molecule m1A to A requires the conversion of one
molecule of Î±KG to succinate (Figure
1), the concentrations of the product 16mer A were used to calculate
the kcat and KM of Î±KG. To ensure that the kinetic
parameters reflect initial velocity, the DNA and enzyme concentrations were
optimized to make sure the conversion of the repair reactions was less than
20%. All reactions were carried out in triplicate.</p><p>For ALKBH2 repair of m1A in ds-DNA (Table 1, Table S2,
and Figure S1), the kcat of αKG was 2.5 ±
0.1 min−1 and the KM was 7.3 ± 0.9
µM, which are comparable to the literature reported kinetics parameters
of other αKG dependent enzymes.7,25,56–61 The kcat/KM value of
ds-repair reaction (0.34 min−1
·µM−1) shows that the repair was more
efficient than in ss-DNA (0.28
min−1·µM−1), which
agrees with the literature on the reported strand preference of
ALKBH2.59 The kinetic
data of ALKBH2 repair of m3C showed a similar trend (Table 1, Table S2, Table S4 and Figure S2). In contrast to ALKBH2's
preference for ds-DNA substrates, the kinetic parameters of ALKBH3 repair of
ds-DNA substrates could not be measured due to the low conversion ratio even
with very high enzyme loading, such as 5.0 µM of ALKBH3 to 5.0
µM substrate. Conversely, ALKBH3 could efficiently repair both DNA
adducts in ss-DNA (Table 1). These
results confirm the previously reported preference of ALKBH3's repair of
ss-DNA substrates.48,55 The kinetic factors of the
E. coli AlkB protein were also measured and the
kcat and KM values agreed well with the literature
reported kcat and KM of the reactions (Table 1, Table S2 and S3, and Figure S3
and S4).59,60
The kcat/KM values of AlkB repair confirm that the enzyme
prefers to repair m1A and m3C in ss-DNA as compared with ds-DNA.55</p><!><p>With reliable kcat and KM parameters of the three
enzymes, we set out to measure the Ki values of D-2HG and L-2HG
together with a positive control, N-oxalylglycine (N-OG), a commonly used
inhibitor of Î±KG dependent enzymes.7 Because D-2HG and L-2HG are chiral molecules,
polarimetry (P-2000 Digital Polarimeter, JASCO Inc.) was utilized to measure
their optical activity in a 1 decimeter cell. The optical rotation of D-2HG was
±9.5° (c = 1.0, 0.1M NaOH), which agreed well to the value
provided by the commercial source ([α]/D
±8.5±1.5°, c = 1.0 in NaOH, Sigma-Aldrich Co LLC).
Similarly, the optical rotation of L-2HG was −8.0°, which was
consistent with the reported −8.5±1.5° value. These
values confirm the chirality and purity of the two enantiomers.</p><p>For the inhibition of the ALKBH2 repair reaction on m1A in ds- and
ss-DNA, the Ki values for D-2HG are 280 ± 61 µM and
405 ± 61 µM, respectively (Table
2, Figure 3a and 3b, Table S5). These data
indicate that D-2HG has a stronger binding affinity for the complex of ALKBH2
with ds-DNA than ss-DNA. For L-2HG reactions, the Ki values are
similar but smaller (stronger inhibition) than with the corresponding D-2HG
reactions (Table 2, Figure 3d and 3e, Table S5). The Ki values of N-OG show much
stronger inhibition (with about 10 times more potency, Table 2) of all repair reactions with the Ki
values ranging from 6 to 40 µM. For the inhibition of ALKBH3, a similar
trend was observed for each individual reaction for the Ki values:
D-2HG > L-2HG > N-OG (Table
2). For the inhibition of AlkB-catalyzed reactions, there is no clear
trend in the inhibitory potency between the D- and L-2HG; N-OG, however, is a
stronger inhibitor than either of 2HG isomers. We also measured the
IC50 of D- and L-2HG on the three enzymes (Table S7); in general,
the IC50 values correlate well with the Ki values.</p><p>To make our experiments more relevant with regard to the anticipated
cellular concentrations of metabolites/oncometabolites observed in human tumors,
we also evaluated the extent inhibition of the ALKBH2 and ALKBH3 repair
reactions by varying the ratios of D- or L- 2HG to Î±KG. For D-2HG
inhibition, we tested a ratio of concentrations for D-2HG:Î±KG = 373:1,
which was observed in glioma patients with IDH mutations (detailed information
see the Discussion section).6
The concentration of αKG was fixed at 100 µM to make sure that
the kinetic analyses reflected steady state catalysis (Figure S5). We found that
the repair efficiencies of ALKBH2 and ALKBH3 were 73–88%
inhibited under such conditions, (Figure
3c, Table 3). For L-2HG
inhibition, we tested a ratio of L-2HG:Î±KG= 28:1, which was reported in
patients with kidney cancers (see Discussion section).10 We found 48–58% of ALKBH2 and
31–40% of ALKBF3's activity was inhibited under this
condition. These results suggest that the strong inhibition on DNA repair
observed in the in vitro experiments may also occur in tumor
cells of cancer patients.</p><!><p>Because 2-HG and Î±KG are structurally similar, researchers
hypothesized that 2HG is able to replace Î±KG in the active site of
Î±KG dependent enzymes and competitively inhibit their enzymatic
activities.7 Crystal
structures of histone demethylases show that D-2HG binds to the same site as
Î±KG in the catalytic center.7 We tested the competition between 2HG and Î±KG in the
DNA repair reactions. Using ALKBH2 repair of m1A as an example, the repair
ratios without adding 2HG were controlled to be around 60% under
different Î±KG concentrations (0.1, 0.5 and 1.0 mM, Figure 4 and Table S6). For the inhibition reactions, D-2HG was added at
a fixed concentration (10 mM) in the reaction mixture, which contained ALKBH2
and necessary cofactors. Then, different concentrations of Î±KG were
added and mixed. After that the reaction was initiated by adding the
oligonucleotide substrates. When 0.1 mM Î±KG was present, the conversion
decreased to 22%. When 0.5 mM and 1.0 mM Î±KG were added, the
repair ratio increased to 35% and 38%, respectively (Figure 4 and Table S6). This observed
trend of reactivity recovery is consistent with the notion that D-2HG acts as a
competitive inhibitor in the Î±KG-dependent DNA repair
reactions.7 Similar
recovery patterns were observed for all other D- and L-2HG inhibition reactions
on all three enzymes (Figure 4 and Table S6).</p><!><p>In the current study, we have shown that both D- and L-enantiomers of
the oncometabolite 2HG can significantly inhibit the human DNA repair enzymes
ALKBH2 and ALKBH3 under physiologically relevant concentrations. The
concentrations of D-2HG and Î±KG on average in glioma cells are 15.5
µmol/g, and 0.0415 µmol/g, respectively, which correspond to a
concentration ratio between D-2HG and Î±KG of 373 to 1,6,7 Under this ratio condition, the repair activities of
ALKBH2 and ALKBH3 were 73–88% inhibited (Table 3). The concentrations of L-2HG and αKG on
average in kidney cancer cells are 1.15 µmol/g and 0.0484
µmol/g, respectively, which corresponds to a concentration ratio between
L-2HG and αKG of 28 to l.10 Under this ratio condition, ALKBH2 and ALKBH3's
repair activities are 31–58% inhibited (Table 3). Although the relative concentration of L-2HG (1.15
µmol/g) is more than 10 times lower than D-2HG (15.5 µmol/g),
the ALKBH2 and ALKBH3 enzymes are still soundly inhibited by L-2HG partially due
to the higher binding affinity of L-2HG (i.e., lower Ki) than D-2HG
(Table 2). The extent of inhibition
in both cases was measured when the concentration of Î±KG was fixed at
100 µM, to ensure steady state catalysis. However, at lower
concentrations of αKG, (i.e., 50 or 20 µM), the efficiency of
adduct repair decreased even further. The cellular concentrations of Î±KG
are typically around 40 to 50 µM (0.0415 and 0.0484 µmol/g or
mM) in cancer patients,6,10 which are near to the
50–100 µM range used in our experiments. Our data also show
that, consistent with competitive inhibition of 2HG, the inhibition activity in
the repair reaction reflects primarily the ratio between 2HG and Î±KG.
ALKBH2 and ALKBH3 are enzymes that repair alkyl DNA damage; hence, inhibition of
DNA repair leads to alkylation product accumulation, less cellular survival, and
increased mutations, which affect the resistance/sensitivity balance to
alkylating chemotherapeutics. The elevation of both D- and L-2HG in cancer cells
may contribute to the increased mutation rate and exacerbate tumorigenesis and
progression.</p><!><p>According to the literature, ALKBH2 prefers to repair ml A and m3C in
ds-DNA, whereas ALKBH3 and AlkB prefer to repair those adducts in
ss-DNA.24,30,55 We tested the repair activity in both ss-DNA and ds-DNA
substrates in this study. The experimental results reported in this paper
provide a strong kinetic basis for the previous observations. For ALKBH2, the
kcat/KM values of ds-repair are higher than the repair
in ss-DNA (Table 1). By contrast, the
kcat/KM values of AlkB repair are higher for ss-DNA
substrates than for ds-DNA substrates (Table
1). For ALKBH3, we were only able to measure the kinetic parameters
for ss-repair, as the ds-repair reactions were too inefficient to evaluate.
These results agree with and add quantitative detail to previous observations
that ALKBH3 strongly prefers to repair adducts in ss-DNA.</p><!><p>There are about 80 proteins in the Î±KG/Fe(II)-dependent enzyme
family, including jmjc, prolyl hydroxylase, TET, and the AlkB family
enzymes.21–23 Studies have demonstrated that
D- and L-2HG inhibit jmjc and TET family proteins.7,62 In
addition to 2HG, intermediates in the TCA cycle such as succinate and fumarate
have also been found to exhibit higher-than-normal concentrations in different
cancer cells (Figure 1b).18 Given their structural
similarities to Î±KG and 2HG, these metabolites could also perturb
Î±KG-dependent enzymatic activities in the cell, especially DNA repair
processes that are related to the AlkB family enzymes. Systematic studies are
needed to explore these possibilities and correlate these biochemical results
with clinical observations. These studies are also pivotal for the design and
development of therapeutic agents that target the abnormal metabolic pathways of
cancer.</p><!><p>
          ASSOCIATED CONTENT
</p><p>
          Supporting Information
</p><p>The Supporting
Information is available free of charge on the ACS Publications
website at DOI: XXX.</p><p>Tables of molecular weights and m/z values, initial rate for kinetic
studies, and IC50 values of inhibition reactions; figures for
steady-state kinetics, repair percentage of reactions, and inhibition curves of
different repair enzymes (PDF)</p><p>The authors declare no competing financial interest.</p>