Radiolabeled 6-(2, 3-dichlorophenyl)-n4-methylpyrimidine-2, 4-diamine (th287): A potential radiotracer for measuring and imaging mth1

Article abstract of DOI:10.3390/ijms21228860

MTH1 (MutT homolog 1) or NUDT1 (Nudix Hydrolase 1), also known as oxidized purine nucleoside triphosphatase, has potential as a biomarker for monitoring cancer progression and quantifying target engagement for relevant therapies. In this study, we validate one MTH1 inhibitor TH287 as a PET MTH1 radiotracer. TH287 was radiolabeled with tritium and the binding of [³H]TH287 to MTH1 was evaluated in live glioblastoma cells (U251MG) through saturation and competitive binding assays, together with in vitro enzymatic assays. Furthermore, TH287 was radiolabeled with carbon-11 for in vivo microPET studies. Saturation binding assays show that [³H]TH287 has a dissociation constant (K_d) of 1.97 ± 0.18 nM, B_max of 2676 ± 122 fmol/mg protein for U251MG cells, and n_H of 0.98 ± 0.02. Competitive binding assays show that TH287 (K_i: 3.04 ± 0.14 nM) has a higher affinity for MTH1 in U251MG cells compared to another well studied MTH1 inhibitor: (S)-crizotinib (K_i: 153.90 ± 20.48 nM). In vitro enzymatic assays show that TH287 has an IC₅₀ of 2.2 nM in inhibiting MTH1 hydrolase activity and a K_i of 1.3 nM from kinetics assays, these results are consistent with our radioligand binding assays. Furthermore, MicroPET imaging shows that [¹¹C]TH287 gets into the brain with rapid clearance from the brain, kidney, and heart. The results presented here indicate that radiolabeled TH287 has favorable properties to be a useful tool for measuring MTH1 in vitro and for further evaluation for in vivo PET imaging MTH1 of brain tumors and other central nervous system disorders.

Full text of DOI:10.3390/ijms21228860

International Journal of
Molecular Sciences
Article
Radiolabeled 6-(2, 3-Dichlorophenyl)-N4
-methylpyrimidine-2, 4-diamine (TH287): A Potential
Radiotracer for Measuring and Imaging MTH1
Huaping Chen 1, Sadia Afrin 1, Yingqiu Guo 1, Wenhua Chu 1, Tammie L.S. Benzinger 1,
Buck E. Rogers 2, Joel R. Garbow 1, Joel S. Perlmutter 1,3, Dong Zhou 1,* and Jinbin Xu 1,
*
1
Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA;
chenhuaping@wustl.edu (H.C.); sadia@wustl.edu (S.A.); yingqiu.guo@duke.edu (Y.G.);
chuw@wustl.edu (W.C.); benzingert@wustl.edu (T.L.S.B.); garbow@wustl.edu (J.R.G.);
perlmutterjoel@wustl.edu (J.S.P.)
Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO 63110, USA;
b.rogers@wustl.edu
Department of Neurology, Washington University School of Medicine, St. Louis, MO 63110, USA
Correspondence: zhoud@wustl.edu (D.Z.); jinbinxu@wustl.edu (J.X.); Tel.: +314-362-9072 (D.Z.);
+314-747-0693 (J.X.)
2
3
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 7 October 2020; Accepted: 20 November 2020; Published: 23 November 2020
Abstract: MTH1 (MutT homolog 1) or NUDT1 (Nudix Hydrolase 1), also known as oxidized purine
nucleoside triphosphatase, has potential as a biomarker for monitoring cancer progression and
quantifying target engagement for relevant therapies. In this study, we validate one MTH1 inhibitor
TH287 as a PET MTH1 radiotracer. TH287 was radiolabeled with tritium and the binding of [3H]TH287
to MTH1 was evaluated in live glioblastoma cells (U251MG) through saturation and competitive
binding assays, together with in vitro enzymatic assays. Furthermore, TH287 was radiolabeled
with carbon-11 for in vivo microPET studies. Saturation binding assays show that [3H]TH287 has
a dissociation constant (Kd) of 1.97
and nH of 0.98 0.02. Competitive binding assays show that TH287 (Ki: 3.04
affinity for MTH1 in U251MG cells compared to another well studied MTH1 inhibitor: (S)-crizotinib
(Ki: 153.90 20.48 nM). In vitro enzymatic assays show that TH287 has an IC50 of 2.2 nM in inhibiting
±
0.18 nM, Bmax of 2676
±
122 fmol/mg protein for U251MG cells,
±
±
0.14 nM) has a higher
±
MTH1 hydrolase activity and a Ki of 1.3 nM from kinetics assays, these results are consistent with
our radioligand binding assays. Furthermore, MicroPET imaging shows that [11C]TH287 gets into
the brain with rapid clearance from the brain, kidney, and heart. The results presented here indicate
that radiolabeled TH287 has favorable properties to be a useful tool for measuring MTH1 in vitro
and for further evaluation for in vivo PET imaging MTH1 of brain tumors and other central nervous
system disorders.
Keywords: MTH1; radiotracer; TH287; binding assay; PET
1. Introduction
Improved cancer diagnosis and cancer-specific therapeutics are needed to reduce cancer mortality
rates. Positron emission tomography (PET) can measure disease-based changes in cell function at the
molecular level. PET combined with computed tomography (CT) or magnetic resonance imaging (MRI)
provides a powerful hybrid of anatomical and functional assessments of cancer and other diseases.
Dysfunctional redox regulation and increased reactive oxygen species (ROS) cause oxidative
damage to nuclear and mitochondria DNA that contributes to the pathogenesis and progression of
cancer and other diseases [1]. Thus, in vivo measures of oxidative stress or related DNA damage
Int. J. Mol. Sci. 2020, 21, 8860; doi:10.3390/ijms21228860
www.mdpi.com/journal/ijms

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repair through PET could provide a critical metric of disease progression and a target for drug
intervention. Furthermore, PET results on oxidative DNA damage can serve as a prognostic indicator
for radiotherapy in cancer patients [
biological systems to produce ROS and subsequent oxidative DNA damage, in addition, to directly
cause DNA breaks. Aerobic glycolysis is also a marker of tumor growth and aggressiveness [ ].
2,3] since ionizing radiation can directly interact with water in the
4
Therefore, a reliable biomarker targeting DNA damage repair could be valuable for monitoring cancer
progression and evaluating response to chemo- or radio-therapeutics, as well as pseudo-progression
and radiation necrosis, which are difficult to separate clinically using currently available MRI and PET
imaging approaches [5].
Cancer cells require high ROS concentrations due to their high proliferation rate. In mitochondria,
where ~90% of total oxygen consumption occurs, about 1–4% consumed oxygen molecules are
partially reduced by electrons to generate ROS including superoxide, hydrogen peroxide, and hydroxyl
radicals [
proteins, and nucleic acids in the living cells [
oxidized by hydroxyl radicals, a reaction that results in the formation of 8-oxo-2’-deoxyguanosine
6]. ROS causes cellular dysfunctions such as cell death and mutagenesis by damaging lipids,
7
]. Guanine is the most susceptible nucleobase to be
(8-oxo-dG) or 8-oxo-2’-deoxyguanosine-5’-triphosphate (8-oxo-dGTP) [8]. The concentration of
8-oxo-dG within a cell reflects the degree of oxidative stress [9].
Misincorporation of oxidized nucleoside triphosphates into DNA/RNA during replication
and transcription can cause mutations that may lead to carcinogenesis, senescence, apoptosis,
or neurodegeneration [10,11]. MTH1, or NUDT1, an oxidized purine nucleoside triphosphatase,
hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-dGTP, 8-oxo-dATP,
2-hydroxy-dATP, and 2-hydroxy rATP, to monophosphates. These molecules suppress the accumulation
of oxidative damage in nucleic acids and prevent the misincorporation of the oxidized nucleoside
triphosphates into DNA/RNA [11–16]. MTH1 is upregulated in a wide variety of cancers, including
brain tumors [17 18], lung cancer [19
,
,
20], breast cancer [21 22], liver cancer [23], renal cancer [24],
,
colorectal cancer [25], esophageal cancer [26], gastric cancer [27], and multiple myeloma [28].
Furthermore, as tumors progress to advanced stages, MTH1 expression upregulates. MTH1 levels also
correlate with the overall survival of HCC (Hepatocellular carcinoma) patients [23].
Thus, monitoring MTH1 status in patients through in vivo imaging would facilitate the design
of therapeutic strategies that target oxidative DNA damage involved diseases such as cancer.
TH287 (Figure 1) is among the known MTH1 inhibitors and is a potent first-in-class molecule
that inhibits MTH1 specifically in in vitro enzymatic assays [29]. However, whether TH287 has a high
affinity to MTH1 in live cells remains unknown.
Figure 1. Chemical structure of TH287.
In the present study, the affinity of TH287 to MTH1 in live glioblastoma U251MG cells and in vivo
was investigated using its radiolabeled form. TH287 was radiolabeled with tritium and the binding of
[3H]TH287 to MTH1 in glioblastoma cells (U251MG) was evaluated in vitro using saturation binding,
competitive binding, and enzymatic assays. Furthermore, TH287 was radiolabeled with carbon-11 to
study whether [11C]TH287 is a suitable radiotracer for PET. The uptake and distribution of [11C]TH287
in different organs of mice were investigated with microPET imaging. This is the first study that
explores the feasibility of developing a radiolabeled MTH1 inhibitor for use as an in vitro binding
assay or in vivo PET imaging radiotracer.

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2. Results
2.1. Saturation binding assays in U251MG cells
To validate TH287 as a potential radiotracer for microPET imaging, its affinity to MTH1 was
first characterized in live cells through saturation binding studies. The assays were carried out by
incubating increasing concentrations of [3H]TH287 with U251MG cells. The saturation curve, Scatchard
plot, and Hill plot are shown in Figure 2. [3H]TH287 has a Kd value of 1.97
of 2676.26 122.38 fmol/mg protein, and a nH value of 0.98
suggest TH287 is within the pharmaceutic limit to be adopted as a radiotracer for microPET imaging.
±
0.18 nM, a Bmax value
0.024 for U251MG cells. These values
±
±
A
B
[3H]TH287: Saturating binding
[3H]TH287: Scatchard analysis
2000
1500
1000
500
0
1400
1200
1000
800
600
400
200
0
Total Bound
Specific Bound
Nonspecific Bound
Kd=1.97 ± 0.18 nM
Bmax=2676.26 ± 122.38 fmol/mg protein
0
500 1000 1500 2000 2500 3000
0
2
4
6
8
10 12 14 16
[3H]TH287 (nM)
Specific Bound (fmol/mg protein)
C
[3H]TH287: Hill Plot
1
0.8
0.6
0.4
0.2
0
nH = 0.98 ± 0.024
-0.2
-0.4
-0.6
-9.5
-9
-8.5
-8
-7.5
Log L
Figure 2. Scatchard analysis of [3H]TH287 binding to the MTH1 in glioblastoma cells (U251MG).
): Representative saturation binding experiments that show the total bound, non-specific bound,
and specific bound. ( ): Representative Scatchard plots were used to determine Kd and Bmax values.
Kd = 1.97 0.8 nM, Bmax = 2676.26 122.38 fmol/mg protein. ( ): Representative Hill plot for
(
A
B
±
±
C
determining the Hill coefficient (nH values). nH = 0.98 ± 0.024. n = 3, samples in triplicate.
2.2. Knockdown MTH1 Expression through siRNA Abolishes [3H]TH287 Binding to U251MG Cells
To exclude potential off-target effects caused by [3H]TH287 in the saturation binding assays,
MTH1 was further knocked down through reverse transfection of MTH1 siRNA into U251MG cells.
MTH1 knockdown completely abolished [3H]TH287 binding as shown by radioligand binding readings
of MTH1 siRNA transfection groups similar to cells treated with 10 µM TH086, which is normally

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considered as non-specific binding, suggesting that [3H]TH287 binds specifically to MTH1 in U251MG
cells instead of other targets (Figure 3).
2000
MTH1 siRNA
MTH1 siRNA + TH086
Control siRNA
Control siRNA + TH086
Lipofectamine
Lipofectamine +TH086
1500
1000
500
0
0
2
4
6
8
10
12
14
16
[3H]TH287 (nM)
Figure 3. Saturation binding assay for [3H]TH287 to the MTH1 in MTH1 knockdown glioblastoma
cells (U251MG). Representative saturation binding experiments which show the binding of [3H]TH287
in control groups and MTH1 knockdown groups. n = 3, samples in triplicate.
2.3. Competitive Profile of Standard and TH287 in U251MG Cells
To further validate the potential of TH287 as a radiotracer for MTH1, full dose range competition
binding assays were performed between a well-studied MTH1 inhibitor, (S)-crizotinib, and [3H]TH287.
Competitive binding assays show that TH287 has a high binding affinity, with a Ki of 3.04
±
0.14 nM
while (S)-crizotinib shows a Ki of 153.90 20.48 nM, indicating that its affinity is much lower than
±
TH287. No binding was detected for Raclopride—a nonselective compound (Figure 4). The values of
the pseudo-Hill coefficient, n’H, are 0.88 and 0.70 for TH287 and (S)-crizotinib respectively.
A
B
Competitive Bingding Assay
TH287: PseudoHill Plot
2
100
1.5
n'H = 0.88 ± 0.06
80
60
40
20
0
1
0.5
0
-0.5
-1
TH287
(S)-crizotinib
Raclopride
-1.5
-2
-3
-2
-1
0
1
2
10-1
100
101
102
103
104
105
Log I
Competitor Concentration (nM)
Figure 4. Cont.

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C
(S)-crizoitinib: PseudoHill Plot
2
1.5
1
n'H = 0.70 ± 0.02
0.5
0
-0.5
-1
-1.5
-2
-4
-3
-2
-1
0
1
2
Log I
Figure 4. Competitive binding for inhibition of the [3H]TH287 binding to MTH1 in glioblastoma
cells (U251MG) by TH287, (S)-crizotinib and a known MTH1 nonselective compound: Raclopride.
(
A
): Representative competitive binding data Ki for TH287: 3.04
±
0.14 nM, (S)-crizotinib: 153.90
±
20.48 nM, Raclopride: >10,000 nM. ( ) (TH287) and ( ) ((S)-crizotinib): Representative PseudoHill
B
C
plots for determining the PseudoHill coefficient (n’H values). TH287: n’H = 0.88
n’H = 0.70 ± 0.02. n = 3, samples in triplicate.
±
0.06; (S)-crizotinib:
2.4. Inhibition Profile of Standard and TH287 on Enzyme Activity of MTH1
To confirm the inhibition potency of TH287, a MTH1 enzymatic inhibition assay using a PPiLightTM
inorganic pyrophosphate bioluminescent assay (Lonza Walkersville Inc., MD, USA), was conducted
to compare inhibition efficacy of TH287, TH086, and (S)-crizotinib (Figure 5). The MTH1 inhibition
assay shows that TH287, TH086, and (S)-crizotinib have high inhibition potency with IC50s at 2.2,
6.5, and 13 nM respectively. While the non-selective compound, Raclopride shows no inhibition of
MTH1 activity. Furthermore, competitive inhibition assays were performed and the Ki of TH287 was
determined to be ~1.3 nM (Figure 6). When TH287 is absent in the enzyme reaction, the Km for
MTH1 is 305 µM. When 2.5 nM of TH287 is present in the reaction, the Kmapp of MTH1 is 892 µM.
This suggests that TH287 competes with the substrate to bind to MTH1.
120
100
IC50s (nM Mean ± SEM)
80
60
40
20
0
TH287 2.2±0.1
TH086 6.5±2.4
(S)-crizotinib 13±2.3
Raclopride >10,000
-3 -2 -1
0
1
2
3
4
5
-20
Log (Inhibitor nM)
Figure 5. MTH1 inhibition assay shows that TH287, TH086, and (S)-crizotinib have high inhibition
potency, with IC50s 2.2, 6.5, and 13 nM respectively. While a non-active compound Raclopride was
used as a control compound; n = 2, samples in triplicate; substrate dGTP concentration: 100 uM,
and recombinant MTH1 protein: 0.5 nM.

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80000
80,000
60000
60,000
40000
40,000
TH287 0 nM, Km = 305 µM
TH287 2.5 nM, Kmapp = 892 µM
20000
20,000
Ki = 1.3 nM
0
0
2000
4000
6000
dGTP (µM)
Figure 6. Representative competitive inhibition of MTH1 by TH287 (MTH1 0.5 nM, substrate dGTP
ranges from 0 to 5000 uM) determined Ki of TH287 to be 1.3 nM. Km: the Michaelis constant; Kmapp
apparent Km; Ki: the inhibitory constant. n = 3, samples in triplicate.
:
2.5. TH287 Exhibits no Killing Effect on Cells
To test the toxicity of TH287 to cancer cells, U251MG cells were treated with TH287 ranging from
0.3~30 µM for 72 h. The same doses of (S)-crizotinib were also included in the experiments to compare
with TH287. TH287 exhibits no cytotoxic effect on U251MG at the majority of the doses tested except
slight proliferation inhibition at 30 µM (88.93%). While 30 µM (S)-crizotinib killed all the cells within
72 h of treatment (0.14%) and showed 92.95% cell viability with a 10
with the vehicle control (Figure 7).
µM treatment for 72 h compared
Figure 7. The effect of (S)-crizotinib and TH287 treatment on the viability of U251MG cells by MTT
assay. U251MG cells were treated with increasing concentrations of (S)-crizotinib or TH287 (range:
0~30 µM) for 72 h, MTT assays were then performed. One Way ANOVA and Dunnett’s Multiple
Comparison test were adopted for significance analysis. In assessing the significance of comparisons a
p-value p < 0.05 was considered significant. *, p < 0.05. n = 3, samples in triplicate.
2.6. MicroPET Imaging
To validate the potential of TH287 as a radiotracer for PET imaging on tumor detection, [11C]TH287
was synthesized and microPET imaging was performed in normal mice. In the microPET imaging
of normal mice, summed (0–10 min) frames of the microPET images (Figure 8 left panel) show that
[11C]TH287 (~0.4 mCi) had high initial uptake and fast clearance of [11C]TH287 from the brain, heart,
and kidney (Figure 8 right panel).

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Figure 8. Representative summed (0–10 min) frames microPET images (left panel) from n = 4 male
severe combined immunodeficient mice (SCID) mice (~25 g, 3 months old) show that [11C]TH287 had
rapid brain uptake and fast clearance in a normal mouse; right panel shows [11C]TH287 time-activity
curves in brain, heart, and kidney.
3. Discussion
In this study, we explored the feasibility of developing TH287 as a PET radiotracer for cancer by
investigating the affinity of TH287 to MTH1 in live cells and its distribution and clearance in mice.
TH287 exhibits a high affinity for MTH1 in the nanomolar range (Ki: 3.04
MTH1 inhibitors besides TH287, including TH086 and (S)-crizotinib, were also tested in the study to
±
0.14 nM). Several existing
validate TH287 as a specific MTH1 probe. TH287 has a higher affinity to MTH1 compared to all these
two ligands. The cell viability studies showed that up to 30
µM TH287 exhibits minimal inhibition
on U251MG cells while 30 M (S)-crizotinib killed all the cells via an off-target other than MTH1.
µ
MicroPET imaging shows that TH287 penetrates the blood-brain barrier of mice with a fast clearance.
During this research, several potent MTH1 inhibitors have been synthesized and validated,
including Tetrahydronaphthyridine 5 (IC50 = 0.043 nM) [30], BAY-707 (IC50 = 2.3 nM) [31], and other
compounds [32]. Although these compounds exhibit a much higher affinity compared with existing
inhibitors, none of them show potent cancer-killing efficacy, casting doubts on the potential of MTH1 to
provide effective cancer treatment drugs. However, these ligands may still provide useful biomarkers
for quantifying the treatment efficacy of current or new therapies.
Huber et al. showed that overexpression of MTH1 could rescue SW480 cells from being killed
by treatment with a MTH1 inhibitor, SCH5134 but not with (S)-crizotinib [33]. This indicates that
(S)-crizotinib could have other targets beyond MTH1. Here the cell viability data shows that up to 30
µM of TH287 treatment for 72 h exhibits minimal growth inhibition on U251MG cells, while the same
dose of (S)-crizotinib kills all the cells. This suggests that (S)-crizotinib may also have different targets
compared with TH287, although both molecules do target MTH1, which is consistent with Huber’s
data. Kawamura et al. showed that TH287 and TH588 can inhibit in vitro beta-tubulin polymerization
at concentrations higher than 30 µM [34]. However, Helleday et al. later showed that TH588 exhibits
a different mode of action compared to the anti-microtubule agent. This casts doubts on the opinion
that tubulin is an off-target of TH287 and TH588—especially considering the high doses of TH287
and TH588 used in Kawamura’s study [35]. TH1579 (Karonudib), an analog of TH588, can cause
a toxic 8-oxodG lesion into DNA in an MTH1-dependent manner. While MTH1 inhibitors (compounds
15, 19, 24, and IACS-4759), which have no toxicity to tumor cells failed to incorporate 8-oxodG into
DNA [35], suggesting the cancer cell killing effect of TH287 and TH588 is not necessarily caused by
off-target effects. Further study by Helleday et al. showed that MTH1 can also bind to tubulin and
promote mitotic progression to avoid oxidative DNA damage in cancer cells [36]. Those cytotoxic
MTH1 inhibitors can inhibit both roles of MTH1 while newly developed non-cytotoxic inhibitors can
only inhibit its enzymatic activity [36].

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Additionally, Helleday et al. found that knocking down MTH1 through siRNA transfection does
not inhibit the proliferation of a certain type of cancer cells, while TH588 and TH1579 are more potent
in killing cancer cells than siRNA mediated knocking down of MTH1 [29,35]. The discrepancy in
data of these two approaches could be due to the development of a compensatory mechanism during
the gradual exhaustion of MTH1 protein in the siRNA knocking down approach. While the MTH1
inhibitors in this study allow no time for alternative mechanisms to develop, which argues against the
opinion that TH588 and related MTH1 inhibitors have off-targets beyond MTH1.
To further validate the potential of TH287 as a radiotracer for MTH1, full dose range competition
binding assays were performed between a well-studied MTH1 inhibitor, (S)-crizotinib, and [3H]TH287.
Competitive binding assays show that TH287 has a high binding affinity, with a Ki of 3.04
±
0.14 nM
while (S)-crizotinib shows a Ki of 153.90 20.48 nM, indicating that its affinity is much lower than
±
TH287. No binding was detected for Raclopride—a nonselective compound (Figure 4). The values of
the pseudo-Hill coefficient, n’H, are 0.88 and 0.70 for TH287 and (S)-crizotinib respectively.
Our competitive assay in U251MG cells shows that the Ki of TH287 is found to be close to the
IC50 value from pure MTH1 enzymatic assay, and the n’H is close to unity, which obeys a single-site
binding model. However, the Ki of (S)-crizotinib is much lower than the IC50 value from the MTH1
enzymatic assay, and the n’H is way off the unity. This suggests that (S)-crizotinib binds to off-targets
other than MTH1.
It has been reported that TH287 can be rapidly metabolized in human and mouse liver microsomes
through the N-dealkylation of the aminomethyl substituent. The major metabolite is TH586 (IC50:
77 nM), which has a much higher IC50 than TH287 (0.8
labeled part is removed and not included in TH586, so TH586 should have minimal impact on applying
11C-TH287 as a radiotracer for PET imaging.
±
0.1 nM). It is worth noting that the 11C
Although the possibility of killing cancer cells through targeting MTH1 is still controversial [30–32],
a promising yet unexplored application of MTH1 inhibitors is to monitor disease progression that
involves oxidative stress since elevated oxidative stress in cells occurs in a large number of chronic
diseases including different types of cancers.
Another potential application of this radioligand is to monitor the progression of neurodegenerative
diseases such as Alzheimer’s disease (AD) or Parkinson’s disease (PD). In AD, loss of aerobic glycolysis
is associated with the presence of neurofibrillary tangles in tau PET studies [37]. Evidence of oxidative
stress in diseased brains has been widely reported although direct correlations between upregulated
expression of MTH1 and the oxidative status of corresponding brain tissue awaits to be established.
Elevated 8-OHG, 8-OHA, and 5,6-diamino-5-formamidopyrimidine have been detected in both nuclear
and mitochondrial DNA (mtDNA) extracted from vulnerable brain regions in amnestic mild cognitive
impairment, the earliest clinical manifestation of AD, indicating that oxidative stress could be an
early event in AD [38]. Thus, monitoring oxidative stress in a noninvasive way though indirectly,
through examining MTH1 level in patients’ brains by PET scan, could provide useful information
for early intervention.
The work in this study would be essential for further characterizing and developing MTH1
probes for in vitro quantitative autoradiography and in vivo PET to monitor oxidative damage repair.
The development of these probes would be highly significant for determining the expression level of
MTH1 in both in vitro and in vivo and thus provide a potential marker for therapeutics that target
DNA damage and repair.
In conclusion, we have thoroughly evaluated radiolabeled TH287 as a radioligand for MTH1 and
compared it with the other unlabeled MTH1 probes: TH086 and (S)-crizotinib. The results of our study
indicate that TH287, which possesses a nanomolar affinity for MTH1 is a superior ligand to (S)-crizotinib
for measuring MTH1 density and function using in vitro binding assays. TH287 exhibits enrichment
in brain tissue and penetrates the blood-brain barrier with a fast clearance rate. The knowledge
gained from this study thus lays a solid foundation for further development of radiotracers for in vitro

Int. J. Mol. Sci. 2020, 21, 8860
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quantitative autoradiography and in vivo PET and will have broad applications to a variety of cancers
and other noteworthy diseases (CNS disorders, diabetes, cardiovascular injuries, etc.).
4. Materials and Methods
4.1. Materials
Chemical reagents and the standard compounds (Raclopride and (S)-crizotinib) were purchased
from Sigma-Aldrich (St. Louis, MO, USA) and Tocris (Ellisville, MO, USA). N, N-Dimethylformamide
(DMF), dimethyl sulfoxide (DMSO), or ethanol was used to dissolve the various compounds.
Different concentrations were then achieved by diluting stock solutions with assay buffer for the
enzymatic assay or RPMI 1640 medium for cell-binding assays. [3H]TH287 was customer synthesized
by ViTrax (Placentia, CA, USA) via direct catalytic tritium gas exchange, a radiochemical purity of
>99% was determined by the high performance liquid chromatography (HPLC). Product identity
was confirmed by Mass spectrometry and HPLC co-elution with Authentic Standard (Retention
time: 8.39 min; Column: Zorbax SB-Phenyl, 4.6
×
150mm, 3.55 µm; Gradient elution: mobile phase
A (0.05% TFA/water) and mobile phase B (acetonitrile). From 10% B to 90% B over 10 min at flow rate
1 mL/min.).Specific Activity was determined to be 31.2 Ci/mmol by Mass Spec. The location of the
tritium label has not been determined, however, can be assumed to be located on the aromatic ring.
4.2. Synthesis of Precursor and Radiosynthesis of [11C]TH287
4.2.1. Synthesis of Precursor
The synthesis of the C-11 labeling precursor of TH287 (
5) is shown in Scheme 1. At first, compound
2
was synthesized by coupling dichlorophenyl boronic acid and 4,6-dichloropyrimidin-2-amine (
1) with
Pd(PPh3)4 as the catalyst. The amine group of
compound . The chloro group of compound
2
3
was then protected with a Boc group to give
was substituted with an azide group to afford
3
compound 4. the azide group of 4 was reduced to an amine group with PPh3 to give the precursor 5.
Reagents: (a) Pd(PPh3)4, Na2CO3 (2,3-dichlorophenyl)boronic acid, dioxane/H2O; (b) Boc2O; (c) NaN3; (d)
Ph3P.
Scheme 1. Synthesis of TH287 standards and precursors for radiolabeling with 11C.

Int. J. Mol. Sci. 2020, 21, 8860
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All chemicals were obtained from standard commercial sources and used without further
purification. All reactions were carried out by standard air-free and moisture-free techniques under an
inert nitrogen atmosphere with dry solvents unless otherwise stated. Flash column chromatography
was conducted using Scientific Adsorbents, Inc. silica gel, 60A, “40 Micron Flash” (32–63
µ
m). Melting
1
points were determined by using the MEL-TEMP 3.0 apparatus and are uncorrected. Routine H
spectra were recorded at 400 MHz on Agilent Technologies spectrometers. All chemical shifts were
reported as a part per million (ppm) downfield from tetramethylsilane (TMS). All coupling constants
(J) are given in Hertz (Hz). Splitting patterns are typically described as follows: s, singlet; d, doublet;
t, triplet; m, multiplet.
4-Chloro-6-(2,3-dichlorophenyl)pyrimidin-2-amine
(2)
A
solution
of
(2,3-dichlorophenyl)boronic acid (640 mg, 3.35 mmol) and 4,6-dichloropyrimidin-2-amine
(500 mg, 3.0 mmol) in dioxane (30 mL) and water (10 mL) was added Pd(PPh3)4 (116 mg, 0.1 mmol)
and Na2CO3 (646 mg, 6.10 mmol). The mixture was heated at 90 ◦C for 4 h. At room temperature, the
reaction mixture was diluted with ethyl acetate (100 mL), washed with water (50 mL
solution (75 mL), and dried over Na2SO4. After evaporation of the solvents, the crude product was
purified by column chromatography with CH2Cl2/EtOAc (5:1) to afford 306 mg (37%) of as a white
solid, mp 210.6–211.7 ◦C. 1H NMR (400 MHz, CDCl3)
7.56 (dd, J = 8.0 Hz, J = 1.2 Hz, 1H), 7.40 (dd,
×
2) and NaCl
2
δ
J = 7.4 Hz, J = 1.2 Hz, 1H), 7.31 (t, J = 8.0 Hz, 1H), 6.93 (s, 1H), 5.31 (s, 2H).
tert-Butyl (4-chloro-6-(2,3-dichlorophenyl)pyrimidin-2-yl)carbamate (3) A solution of
2 (275
mg, 1.0 mmol) and di-tert-butyl dicarbonate (273 mg, 1.25 mmol) in THF (20 mL) was added
N,N-dimethylpyridin-4-amine (122 mg, 1 mmol). The mixture was stirred at room temperature
overnight. After evaporation of the THF, the crude product was purified by column chromatography
1
with CH2Cl2 to afford 296 mg (79%) of
3
as a colorless oil. H NMR (400 MHz, CDCl3)
δ
7.90 (s, 1H),
(250 mg,
7.61 (d, J = 8.0 Hz, 1H), 7.56 (d, J = 7.6 Hz, 1H), 7.37 (t, J = 8.0 Hz, 1H), 1.46 (s, 9H).
tert-Butyl (4-azido-6-(2,3-dichlorophenyl)pyrimidin-2-yl)carbamate (4) A solution of
0.67 mmol) and NaN3 (130 mg, 2.0 mmol) in DMF (10 mL) was heated at 90 ◦C for 24 h. At room
temperature, the reaction mixture was diluted with ethyl acetate (75 mL), washed with water (50
3
mL
the crude product was purified by column chromatography with CH2Cl2 to afford 216 mg (85%) of
as a colorless oil. 1H NMR (400 MHz, CDCl3)
7.54 (d, J = 8.4 Hz, 1H), 7.50 (d, J = 3.2 Hz, 1H),
7.30 (t, emphJ = 7.6 Hz, 1H), 6.78 (s, 1H), 1.55 (s, 9H).
tert-Butyl (4-amino-6-(2,3-dichlorophenyl)pyrimidin-2-yl)carbamate (5) A solution of
×
2), and NaCl solution (50 mL), and dried over Na2SO4. After evaporation of the ethyl acetate,
4
δ
4
(150 mg,
0.39 mmol) and Ph3P (205 mg, 0.78 mmol) in THF (10 mL) and water (1 mL) was heated to reflux for 4 h
to complete the reaction. At room temperature, the reaction mixture was added ethyl acetate (50 mL),
and then was washed with water (30 mL
After evaporation of the ethyl acetate, the crude product was purified by column chromatography with
CH2Cl2/EtOAc (1:1) to afford 116 mg (83%) of
×
2) and NaCl solution (30 mL) and dried over Na2SO4.
1
5
as a colorless oil. H NMR (400 MHz, CDCl3)
δ
7.65
(s, 1H), 7.50 (d, J = 8.4 Hz, 1H), 7.41 (d, J = 6.8 Hz, 1H), 7.26 (t, J = 8.0 Hz, 1H), 6.36 (s, 1H), 5.39 (s, 2H),
1.51 (s, 9H).
4.2.2. Radiosynthesis of [11C]TH287
The C-11 radiolabeling of the precursor
5
to prepare [11C]TH287 is shown in Scheme 2.
As previously described [39], [11C]CH3I was produced from [11C]CO2 using a GE PETtrace MeI
Microlab. [11C]CO2 was produced at the Cyclotron Facility of Washington University School of
Medicine using a JSW BC-16/8 cyclotron by irradiating a gas target of 0.2% O2 in N2 for 15–30 min
with a 40 µ
A beam of 16 MeV protons. [11C]CO2 was converted to [11C]CH3I by the GE PETtrace
MeI Microlab using a nick◦el catalyst [Shimalite-Ni (reduced), Shimadzu, Japan P.N.221-27719]◦in
the presence of H2 at 360 C and followed by a reaction with iodine in the gas phase at 690 C.
[11C]CH3I was delivered in the gas phase with helium approximately 12 min following the end of
the bombardment.

Int. J. Mol. Sci. 2020, 21, 8860
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Scheme 2. Synthesis of 11C labeled MTH1 inhibitors [11C]TH287.
(1.1 mg) was treated with TFA (300 µ
L) at 80 ◦C for 10 min, and then
The boc-protected precursor
5
TFA was removed under a flow of nitrogen at 80 ◦C. The residual TFA was removed by the addition of
acetonitrile (1 mL) and heating at 80 ◦C under a flow of nitrogen. This process was repeated two more
times to obtain a white residue, which was transferred to the labeling vessel by DMF (2
×
100 µL).
[11C]MeI was produced at the Washington University in Saint Louis Cyclotron facility. Before the
trapping of [11C]MeI, NaH (1 mg) in DMF (100
µL) was added to the labeling vessel and the vessel
was kept at 0 ◦C to trap [11C]MeI. After trapping of [11C]MeI, the reaction was heated at 90 ◦C for 6
min and then diluted with 1:1 HPLC mobile phase/water for HPLC purification (Phenomenex Luna
250
×
100 mm, 34% MeCN/66%water/0.1%TFA, flow rate: 4 mL/min, UV: 270 nm). [11C]TH287 was
collected at 15 min in 50 mL of water and extracted by a Waters C18 cartridge. The cartridge was rinsed
with 10 mL of water and [11C]TH287 was eluted with ethanol and saline to achieve a final dose of 10%
ethanol/saline. The radiochemical yield (decay corrected) was 5.9
±
3.2% (n = 5), and the specific molar
activity at the end of the bombardment was 2494 1603 mCi/ mol (n = 5) and radiochemical purity
±
µ
was >99%. At this preliminary stage of our research, we have yet to optimize the labeling. We believe
the low yield is due to the slow methylation reaction on the aromatic amine, being limited by the short
half-life of carbon-11. The use of more reactive [C-11]methyl triflate may improve the yield under
mild conditions.
4.3. Cell Culture
U251MG cells (RRID: CVCL_0021) (originally obtained from Dr. Bigner, Duke University, Durham)
were grown in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal
bovine serum (Invitrogen, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Invitrogen, Carlsb◦ad,
CA, USA). Cells were maintained in a humidified environment of 5% CO2 and 95% air at 37 C.
Short tandem repeat (STR) profiling was carried annually from Cell Line Authentication Service from
the University of Arizona Genetics Core to ensure quality and integrity.
4.4. Knockdown MTH1 through siRNA Transfection
Reverse transfection of MTH1 siRNA was used to knockdown MTH1 expression and cells
transfected with control siRNA served as the experimental control. A Lipofectamine RNAiMAX group
only was also used as a control group. Briefly, 0.2
mixed with 0.6 L of 10 M siRNA in 20 L of Opti-MEM was added to each well of the 96-well plates.
Then, 100
µL Lipofectamine RNAiMAX (Invitrogen, CA, USA)
µ
µ
µ
µ
L of the cell suspension containing 8000 cells was added to each well. The culture medium
was changed after overnight incubation at 37 ◦C. 48 h after the transfection, saturation binding assays
were performed on all experimental groups of cells.

Int. J. Mol. Sci. 2020, 21, 8860
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4.5. MTH1 Binding Assay
4.5.1. Saturation Binding and Scatchard Analysis
U251MG cells (~16, 000 cells per well) were seeded 24 h before the assay. The cells were incubated
with [3H]TH287 in a total volume of 150 L of media at 37 ◦C in 96 well stripwell tissue culture plates
(Fisher Scientific, Pittsburgh, PA, USA) for 30 min. The concentrations of the radioligand ranged from
0.5–15 nM. After incubation, each well was washed with 200 L of ice-cold PBS and transferred to
µ
µ
a vial with 2 mL of scintillation fluid. A liquid scintillation system AccuFLEX LSC-800 (Hitachi Aloka
Medical, Ltd. Tokyo, Japan) was used to quantify the bound radioactivity. Nonspecific binding was
determined from cells treated with 10 µM TH086. Experiments were repeated three times.
The equilibrium dissociation constant (Kd) and the maximum number of binding sites (Bmax) were
determined by a linear regression analysis of the transformed data using the Scatchard method [40].
Data from saturation radioligand binding studies were transformed to determine the Hill
coefficient, nH, defined as:
Bs
log
= log Kd + nH log L
(1)
Bmax − Bs
Bs
max−Bs
where L is the concentration of radioligand. nH, Hill slope, was determined from Hill plot of log B
versus log L.
4.5.2. Competitive Binding
U251MG cells (~16,000 cells per well) were seeded the day before the assay and incubated in a total
volume of 150
L of media with [3H]TH287 at 37 ◦C in 96 well plates for 30 min. The final concentration
of the radioligand in each assay was 4 nM. Inhibitors (TH287, (S)-crizotinib) concentrations ranging
from 0.1 nM to 10 M were added to acquire the inhibition curves. After the reaction was completed,
the cells were washed once with ice-cold PBS and the bound radioactivity was counted and analyzed
as described above. Nonspecific binding was determined from cells treated with 10 M TH086.
Raclopride, a well-reported selective antagonist for dopamine D2 receptors [41 42], was also used to
µ
µ
µ
,
validate the assay. Raclopride shares a similar molecular weight with TH287, but structurally different
from TH287 and it has no reported binding affinity to MTH1.
Data from the competitive binding experiments were modeled using nonlinear regression
analysis to determine the concentration of inhibitor that inhibits 50% of the radioligand specific
binding (IC50 value). The competition curves were fitted to a single-site binding model using the
following equation:
"
#
(B0 ∗ I)
Bs = B0 −
340
(2)
(IC50 + I)
where, Bs is the amount of the radioligand bound specifically to the cells (i.e., Bs = Bt – Bns, where Bt is
the total bound radioactivity and Bns is the nonspecific binding of the radiotracer), B0 is the amount
of the radioligand bound in the absence of the competitive inhibitor, I is the concentration of the
competitive inhibitor and the IC50 is the concentration of the competitive inhibitor that blocks 50% of
the total specific binding, or 50% sites occupied by the competitor. The values for Bns and B0 were
constrained using experimentally derived values. Competitive inhibition constants (Ki values) were
calculated from the IC50 values using the Cheng and Prussoff equation [43]:
IC50
Ki =
(3)
Lt
1 +
Kd
where the Kd values of [3H]TH287 (1.97
the concentration of radioligand used in each assay.
±
0.18 nM) were derived from U251MG experiments and Lt is

Int. J. Mol. Sci. 2020, 21, 8860
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Data from competitive radioligand binding studies were transformed to determine the pseudo-Hill
coefficient, n’H, defined as:
Bs
B0 − Bs
log
= n0H log I − n0H log IC50
(4)
Bs
0
n’H, which is the negative of the Hill slope, was readily determined from the plot of log B −B
s
versus log I.
4.6. Enzyme Activity Assays
The enzymatic hydrolysis of dGTP by purified human recombinant MTH1 to form dGMP and
pyrophosphate was applied for screening purposes using a PPiLightTM inorganic pyrophosphate
bioluminescent assay—according to published procedures [29]. After shaking and color development,
the plates were analyzed in a Victor 3 Microplate reader (PerkinElmer, Waltham, MA, USA)
for luminescence. The inhibition assays were conducted using a series of dilutions of standard
compounds and new MTH1 inhibitors. The IC50 values were determined by fitting a dose-response
curve to the data points using nonlinear regression.
4.7. MTT Assays
MTT assays were used to test cell toxicity of compounds. 2
96-well plates and treated with different concentrations (0.3, 1, 3, 10, 30
at 37 ◦C for 72 h. After incubation with MTT for 4 h at 37 ◦C, the culture medium was aspirated and
200 L DMSO was added to each well to dissolve the formazan crystals, then the absorbance was
×
103 U251MG cells were plated into
M) of TH287 or (S)-crizotinib
µ
µ
measured at 540 nm using a Victor 3 Microplate reader (PerkinElmer, Waltham, MA, USA).
4.8. MicroPET Imaging of Normal Mice with [11C]TH287
The Washington University Animal Studies Committee approved all procedures used for the
mouse experiments described in the present study under ACS# 20,170,165 (approved on 9/1/2017).
Overall care of the animals was consistent with The Guide for the Care and Use of Laboratory Animals
from the National Research Council and the USDA Animal Care Resource Guide. Four male athymic
nude mice (~25 mg, 8 weeks old), purchased from Charles River Laboratories (Wilmington, MA, USA),
were included for the preliminary microPET imaging evaluation of MTH1 in vivo. The mice were
anesthetized with isoflurane and injected with ~400 µ
Ci of [11C]TH287 via the tail vein. The imaging
sessions were 1 h dynamic scans using the MicroPET® Focus 220 and Inveon scanners (Siemens Medical
Solutions USA Molecular Imaging, Knoxville, TN, USA). Acquired list mode data were histogrammed
into a 3D set of sinograms and binned to the following time frames: 5
×
60 s, 5
×
2 min, and 9
×
5 min.
Sinogram data was then processed using a filter back-projection algorithm with attenuation and
scatter corrections. Regions of interest (ROI) were manually drawn on the brain, heart, and kidney
of the mice using 0–10 min summarized images as references. The software Acquisition Sinogram
Image PROcessing using IDL’s Virtual MachineTM (ASIPro VMTM) (Siemens Medical Solutions USA
Molecular Imaging, Knoxville, TN, USA) was used to draw ROI and obtain the radioactivity uptake
(nCi/c.c.) curve over the time course of the scan, and the uptake data were further normalized by the
injected radioactivity dose to get %I.D./cc.
4.9. Data and Statistical Analysis
The data and statistical analysis comply with the recommendations on experimental design
and analysis in pharmacology [44]. Statistical analyses and figure plotting were performed using
KaleidaGraph (Synergy Software, Reading, PA, USA). One-Way ANOVA and Dunnett’s Multiple
Comparison test were adopted for significance analysis. In assessing the significance of comparisons
a p-value of <0.05 was considered statistically significant. All data were reported as mean
deviation if not otherwise noted.
±
standard

Int. J. Mol. Sci. 2020, 21, 8860
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Author Contributions: Study concept, design, and supervision: J.X. and D.Z. Qualitative analysis of data: H.C.,
S.A., Y.G., and J.X. Data analysis and interpretation: H.C. and J.X. Organic synthesis and radiosynthesis: W.C.
and D.Z. Drafting of the manuscript: J.X., H.C., and D.Z. Preparation of figures/tables: H.C., Y.G., and J.X. Critical
revision of the manuscript for important intellectual content: T.S.L.B., J.R.G., and J.S.P. Obtained funding: B.E.R.,
D.Z., and J.X. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded in part by NIH R01 NS092865, R21 EB028302, R01 AG054567, Department
of Defense, Emerson collective cancer research fund, Siteman cancer center, the American Parkinson Disease
Association (APDA), the Greater St. Louis Chapter of the APDA, and Washington University Molecular
Imaging Center.
Acknowledgments: The authors thank William Knight for editorial assistance.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
Abbreviations
MTH1
HCC
MutT homolog 1
Hepatocellular carcinoma
NUDT1 Nudix Hydrolase 1
PET
CT
Positron emission tomography
Computed tomography
MRI
ROS
IC50
Magnetic resonance imaging
Reactive oxygen species
The half-maximal inhibitory concentration
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