Inhibition of CTP synthase from Escherichia coli by xanthines and uric acids

Article abstract of DOI:10.1016/j.bmcl.2009.11.017

CTP synthase (CTPS) catalyzes the conversion of UTP to CTP and is a recognized target for the development of anticancer, antiviral, and antiprotozoal agents. Xanthine and related compounds inhibit CTPS activity (IC₅₀ = 0.16-0.58 mM). The presence of an 8-oxo function (i.e., uric acids) enhances inhibition (IC₅₀ = 0.060-0.121 mM). An intact purine ring with anionic character favors inhibition. In general, methylation of the purine does not significantly affect inhibition.

Full text of DOI:10.1016/j.bmcl.2009.11.017

Bioorganic & Medicinal Chemistry Letters 20 (2010) 141–144
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibition of CTP synthase from Escherichia coli by xanthines and uric acids
a
a
a,b,*
Alexander C. Roy , Faylene A. Lunn , Stephen L. Bearne
a
Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5
Department of Chemistry, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4J3
b
a r t i c l e i n f o
a b s t r a c t
Article history:
CTP synthase (CTPS) catalyzes the conversion of UTP to CTP and is a recognized target for the develop-
ment of anticancer, antiviral, and antiprotozoal agents. Xanthine and related compounds inhibit CTPS
activity (IC50 = 0.16–0.58 mM). The presence of an 8-oxo function (i.e., uric acids) enhances inhibition
Received 3 October 2009
Revised 5 November 2009
Accepted 5 November 2009
Available online 12 November 2009
(IC50 = 0.060–0.121 mM). An intact purine ring with anionic character favors inhibition. In general, meth-
ylation of the purine does not significantly affect inhibition.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
CTP synthase
Methylated xanthines
Methylated uric acids
Inhibition kinetics
Structure–activity relationship
The glutamine amidotransferase cytidine 5-triphosphate synthase
EC 6.3.4.2; UTP–ammonia ligase (ADP-forming); CTPS] catalyzes
L
-glutamate
L
-glutamine + H O
2
[
the ATP-dependent formation of CTP from UTP using either
O
NH3 (or exogenous NH3)
NH
2
1
L-glutamine (Gln) or NH
3
as the nitrogen source (Scheme 1).
The enzyme is regulated in a complex fashion. GTP acts a
positive allosteric effector for Gln-dependent CTP formation;
HN
N
2
,3
O
N
R
O
N
R
however, at concentrations exceeding 0.15 mM, GTP inhibits
Gln-dependent CTP formation.4 Moreover, GTP is an inhibitor of
ATP
ADP + Pi
4
NH
3
-dependent CTP formation at all concentrations. The enzyme
UTP
R = ribose 5'-triphosphate
CTP
5–7
exhibits positive cooperativity for ATP and UTP, and these nucleo-
tides act synergistically to promote tetramerization of the enzyme
to its active form. The product CTP acts as a feedback inhibitor.
Scheme 1. Reactions catalyzed by CTP synthase.
7
5
8
–10
of these and other chemotherapy drugs;28–36 and CPEC treatment
has been associated with cardiotoxic effects. Hence, the develop-
ment of new, potent, and selective CTPS inhibitors is required.
Recently, we demonstrated that the inhibition of Escherichia coli
CTPS by GTP does not require the presence of the ribose 5 -triphos-
phate moiety. We demonstrated that 6-thioguanine inhibits CTPS
activity, suggesting that inhibition arises primarily through inter-
actions between CTPS and the purine base. To test this hypothesis
further, we examined the inhibition of CTPS by xanthine, uric acid,
and their methylated derivatives. Herein, we provide the first dem-
onstration that soluble purine derivatives are potent inhibitors of
E. coli CTPS.
The ability of xanthines, uric acids, and analogues (Fig. 1; Sigma
-Aldrich Canada, Ltd. Mississauga, ON) to inhibit both NH - and
Gln-dependent CTP formation, catalyzed by CTPS from E. coli,
The human enzyme
URA7 gene,
and the yeast enzyme, encoded by the
are also regulated by phosphorylation.
The central role of CTP in the biosynthesis of nucleic acids,
1
1,12
37
1
3
1
4,15
16
phospholipids,
and sialic acid makes CTPS an attractive tar-
get for the development of antineoplastic, antiviral, and anti-
13
17
0
1
8–20
22
protozoal
agents. However, very few studies have focused on
the development of nucleotide analogues as inhibitors of this en-
2
1–23
0
24
zyme.
3-Deazauridine 5 -triphosphate (IC50 ꢀ18
l
l
m) and
0
m)25 are
cyclopentenyl cytosine (CPEC) 5 -triphosphate (IC50 ꢀ6
substrate and product analogues, respectively, and are the most
0
extensively studied inhibitors of CTPS. In addition, the 5 -triphos-
phate of 2,2-difluorodeoxycytidine (gemcitabine) is also believed
to exert its chemotherapeutic effects, in part, through the inhibi-
2
6,27
tion of CTPS.
Unfortunately, mutations in CTPS lead to the loss
3
of feedback inhibition by CTP and resistance to the cytotoxic effects
was examined. Recombinant CTPS bearing an N-terminal hexahis-
tidine tag was overexpressed in E. coli BL21(DE3) cells and purified
using Ni -affinity chromatography as described previously. The
*
Corresponding author. Tel.: +1 902 494 1974; fax: +1 902 494 1355.
E-mail address: sbearne@dal.ca (S.L. Bearne).
2
+
22
0
960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.017

1
42
A. C. Roy et al. / Bioorg. Med. Chem. Lett. 20 (2010) 141–144
R3
O
6
Xanthine (1) and its derivatives, differing in the location and de-
gree of methylation (2–5), inhibited CTPS-catalyzed NH - and Gln-
dependent CTP formation with IC50 values ranging between 0.22–
.58 mM and 0.16–0.44 mM, respectively (Table 1). Fitting the ki-
netic data to Eq. 1 (where and are the initial velocities in
the presence and absence of inhibitor, respectively, [I] is the con-
7
R1
H
N
O
3
N
5
4
N
NH2
1
2
8
O
3
0
N
9
N
H
N
O
N
H
O
v
i
v
o
R2
2
1
3, allantoin
0
n
centration of inhibitor, K = IC , and n is the Hill number) revealed
1
3
NH2
50
1
2
3
4
5
, R = R = R = H
that caffeine inhibited CTPS activity in a sigmoid fashion (Fig. 2).
1
2
3
H
N
, R = R = R = CH
3
0
N
Eqs. 2, 3, and 4 describe the dependence of K on substrate concen-
1
2
3
, R = R = CH , R = H
3
tration ([S]) for a competitive, uncompetitive, and noncompetitive
1
2
3
, R = CH , R = H, R = CH
, R = H, R = R = CH
N
N
38,39
0
3
3
O
multisite inhibitor, respectively.
independent of [substrate], indicating that caffeine behaves as a
noncompetitive, multisite inhibitor with respect to NH , and also
Table 2 shows that K was
1
2
3
H
3
1
4, adenine
3
R3
O
O
with respect to ATP (Fig. 2). Unfortunately, a detailed kinetic mech-
anism accounting for the inhibition of GTP-dependent activation
R1
N
N
NH
2
2
O
and inhibition is not available, making determination of the exact
mode of inhibition with respect to GTP difficult. Consequently, we
have relied on IC50 values as an empirical estimate of inhibitor po-
tency. For all compounds examined, the kinetic data were fit to Eq.
1 and n >1 was observed in all cases indicating cooperativity in
inhibitor binding.
N
R2
N
O
N
O
R4
R2
R1
1
2
3
4
1
2
6
7
8
9
1
1
1
, R = R = R = R = H
15, R = β-D-ribose, R = H
1
2
3
4
1
2
, R = CH , R = R = R = H
16, R = R = H
3
1
2
3
4
1
2
, R = R = CH , R = R = H
17, R = H, R = CH CO H
3
2 2
0
1
2
3
4
v
v
i
K
, R = CH , R = H, R = CH , R = H
3
3
¼
ð1Þ
0
n
1
2
3
4
o
K þ ½Iꢁ
K þ ð
K þ ð
0, R = H, R = R = CH , R = H
3
1
2
3
4
0
n
n
i
1, R = R = R = CH , R = H
3
K ¼
a
a
K =K
Þ½Sꢁ ðcompetitiveÞ
ð2Þ
ð3Þ
ð4Þ
i
S
1
2
3
4
2, R = R = R = R = CH
3
0
n
n
K ¼
a
a
K K
S
Þ=½Sꢁ ðuncompetitiveÞ
ðnoncompetitiveÞ
i
i
Figure 1. Structures of xanthine (1), uric acid (6), their methylated derivatives (2–5
and 7–12, respectively), and other analogues (13–17).
0
n
K ¼
a
Ki
While xanthine and its methylated derivatives afforded only
modest inhibition of CTPS activity, the presence of an 8-oxo group,
as in uric acid (6) and its derivatives with varying degrees and posi-
tions of methylation (7–12), enhanced the inhibition ꢀ4–7-fold
with IC values ranging between 0.072–0.121 mM and 0.060–
affinity tag was removed using thrombin-catalyzed cleavage and
the CTPS-catalyzed conversion of UTP to CTP activity was followed
using
a continuous spectrophotometric assay as described
5
0
2
2
previously.
3
0.113 mM for the inhibition of NH - and Gln-dependent CTP
Table 1
IC50 Values for the inhibition of E. coli CTPS-catalyzed NH
3
- and Gln-dependent CTP formation by xanthine, uric acid, and their derivativesa
NH
Clb
Inhibitor
4
Glnc
IC50 (mM)
n
IC50 (mM)
n
Xanthine (1), pH 8.0
Xanthine (1), pH 8.5
Caffeine (2)
Theophylline (3), pH 8.0
Theophylline (3), pH 8.5
Paraxanthine (4), pH 8.0
Paraxanthine (4), pH 8.5
Theobromine (5)
0.37 ± 0.02
0.22 ± 0.01
0.51 ± 0.02
0.55 ± 0.02
0.40 ± 0.01
0.48 ± 0.03
0.36 ± 0.04
0.58 ± 0.04
0.087 ± 0.006
0.113 ± 0.004
0.096 ± 0.004
0.072 ± 0.001
0.099 ± 0.001
0.119 ± 0.008
0.109 ± 0.001
0.090 ± 0.009
0.07 ± 0.01
0.121 ± 0.002
No inhibition
12.9 ± 1.0
2.9 ± 0.6
2.6 ± 0.3
0.23 ± 0.01
0.16 ± 0.02
0.39 ± 0.02
0.43 ± 0.01
0.30 ± 0.07
0.44 ± 0.03
0.20 ± 0.04
0.42 ± 0.01
0.060 ± 0.003
0.06 ± 0.01
0.101 ± 0.001
0.088 ± 0.004
0.064 ± 0.007
0.113 ± 0.008
0.061 ± 0.002
0.063 ± 0.004
0.067 ± 0.003
0.079 ± 0.006
No inhibition
15.8 ± 0.5
1.9 ± 0.2
1.9 ± 0.2
1.9 ± 0.1
d
2.57 ± 0.03
1.55 ± 0.08
2.22 ± 0.08
2.11 ± 0.08
2.88 ± 0.05
1.9 ± 0.6
2.5 ± 0.2
2.8 ± 0.4
2.5 ± 0.1
2.64 ± 0.05
2.8 ± 0.2
3.0 ± 0.2
2.6 ± 0.3
3.4 ± 0.4
2.8 ± 0.4
3.0 ± 0.3
–
3.01 ± 0.07
2.0 ± 0.6
1.9 ± 0.1
2.0 ± 0.1
1.80 ± 0.06
1.98 ± 0.07
2.0 ± 0.6
1.84 ± 0.07
2.0 ± 0.1
1.9 ± 0.3
1.7 ± 0.1
1.8 ± 0.1
2.4 ± 0.4
2.4 ± 0.3
2.7 ± 0.2
–
d
d
Uric acid (6), pH 8.0
Uric acid (6), pH 8.5
d
1
1
1
1
1
3
1
1
-Methyluric acid (7)
,3-Dimethyluric acid (8), pH 8.0
,3-Dimethyluric acid (8), pH 8.5d
,7-Dimethyluric acid (9), pH 8.0
,7-Dimethyluric acid (9), pH 8.5d
,7-Dimethyluric acid (10)
,3,7-Trimethyluric acid (11)
,3,7,9-Tetramethyluric acid (12)
e
Allantoin (13)
Adenine (14)
Uridine (15)
Uracil (16)
Uracil-4-acetic acid (17)
1.6 ± 0.1
2.5 ± 0.4
3.1 ± 0.8
2.4 ± 0.2
2.1 ± 0.1
2.3 ± 0.2
3.0 ± 0.5
2.3 ± 0.3
4.6 ± 0.2
4.7 ± 0.9
3.17 ± 0.03
3.1 ± 0.1
4.2 ± 0.2
2.6 ± 0.1
a
b
c
Errors are the standard deviations obtained from triplicate determinations of IC50 values.
Concentration of inhibitor required to yield 50% of the activity (i.e., = 0.5) observed when CTPS (26
Concentration of inhibitor required to yield 50% of the activity observed when CTPS (26 g /mL) is assayed with Gln (10 mM) and GTP (0.15 mM).
Assays at pH 8.5 were conducted in TAPS buffer (70 mM, pH 8.5) rather than Hepes buffer (70 mM, pH 8.0).
Approximately 20% reduction in was observed at [allantoin] = 37.57 mM.
v
i
/
v
o
4
lg/mL) is assayed with 150 mM NH Cl.
l
d
e
i o
v /v

A. C. Roy et al. / Bioorg. Med. Chem. Lett. 20 (2010) 141–144
143
formation, respectively (Table 1). This observation is consistent
with our previous report that 8-oxoguanosine inhibited NH - and
Gln-dependent CTP formation with IC50 values that were reduced
3
2
2
2
.2- and 4.0-fold, respectively, relative to guanosine.
Xanthine (1, pKa3 = 7.7, pKa7/9 = 10.6), paraxanthine (4,
pKa3 = 8.8), and theophylline (3, pKa7/9 = 8.7) are partially ionized
4
0
under the assay conditions. To determine the extent to which
ionization affects inhibition, IC50 values were also determined at
pH 8.5 for these compounds and their corresponding uric acids
(
6, 9, and 8, respectively). Raising the pH from 8.0 to 8.5 resulted
in slightly increased inhibition of NH -dependent CTP formation
by the xanthines; however, the inhibition was slightly decreased
6 and 8) or remained roughly unchanged (9) for the correspond-
3
(
ing uric acids. On the other hand, raising the pH from 8.0 to 8.5 for
Gln-dependent CTP formation caused enhanced inhibition for
xanthines 1, 3, and 4, as well as for the methylated uric acids 8
and 9. Only the IC50 value for uric acid was unchanged. The origin
of this pH-effect on the inhibition of Gln-dependent CTP forma-
tion is unclear although it likely arises due to changes in the ion-
ization state of the enzyme affecting Gln- or GTP-binding since
the IC50 value of 8 was reduced 1.4-fold when the pH was in-
creased yet there is no significant change in the ionization state
of 8 within this pH range (pKa9 = 5.75, pKa3 = 10.3).40 Overall, it
appears that an increased negative charge on the purine ring
yields better inhibition as is evidenced by the lower IC50 values
observed for the xanthines at pH 8.5 and the observation that
the uric acids, which are fully ionized at the 7- or 9-position
(
pKa7/9 ꢂ 5.8–5.9) under our assay conditions, are the most potent
inhibitors. To test this conclusion, we examined inhibition by ura-
cil-4-acetic acid (17). Indeed, the addition of the negative carbox-
ylate resulted in a 1.5-fold reduction in the IC50 value relative to
that of uracil (16) or uridine (15). However, our observation that
Figure 2. Inhibition of CTPS-catalyzed NH
to NH (A–C) and ATP (D–F) as substrates. Error bars are the standard deviations.
Panel A: The dependence of the initial velocities ( ) on [NH ] in the presence of
varying [caffeine] (0 mM, d; 0.1 mM, ; 0.2 mM, j; 0.3 mM, ; 0.4 mM, .;
.5 mM, s; 0.6 mM, 4; 0.7 mM, h; and 0.8 mM, }) is shown. The standard assay
was conducted in Hepes buffer (70 mM, pH 8.0) containing EGTA (0.5 mM), MgCl
10 mM), CTPS (20 g/mL), NH Cl (10, 25, 75, 100, and 150 mM), ATP (1 mM), and
3
-dependent CTP formation with respect
3
v
i
3
1
7 was a relatively weak inhibitor and that allantoin (13) was
N
not an inhibitor indicated that an intact purine ring is required
for potent inhibition.
0
2
(
l
4
It is possible that inhibition of CTPS by the xanthines and uric
acids could arise due to an effect on the quaternary structure of
the enzyme. Tetramerization of CTPS to its active form can be in-
duced either through the synergistic action of UTP and ATP bind-
ing, or in the presence of elevated concentrations of ATP, UTP, or
UTP (1 mM) in a total volume of 1 mL. Initial velocities were fit to eqn. 1 by non-
linear regression analysis using KaleidaGraph v. 4.0 (Synergy Software, Reading, PA).
Panel B: The effect of caffeine on the relative initial velocities for NH
CTP formation ( ) is shown for various [NH ] (8.16 mM, d; 5.44 mM, s;
.72 mM, are relative to
; 1.36 mM, 4; and 0.544 mM, .). Values of
obtained with [NH ] = 8.16 mM and the curves shown are for data fit to eqn. 1.
Panel C: The dependence of K on [NH
dashed line) and uncompetitive (dotted line) inhibition were calculated using
3
-dependent
v
i
/
v
o
3
2
N
v
i
/
v
o
i o
v /v
7
,41,42
22
3
CTP.
However, using gel filtration-HPLC as described previ-
0
3
] is shown. Theoretical curves for competitive
ously, we have shown that neither caffeine nor 3,7-dimethyluric
acid at concentrations of 1 mM (i.e., 2ꢃ and 10ꢃ their IC50 values,
respectively) appear to impair the ability of CTPS to form tetra-
mers under the assay conditions (i.e., in the presence of 1 mM
each of ATP and UTP). Since the Hill numbers (n) for inhibition
by most of the compounds range between 2.0 and 4.0, there is
cooperativity in inhibitor binding. Such cooperativity likely arises
from the interaction between subunits within the intact CTPS
tetramer.
(
3
0
K
S
= 1.83 mM (assuming that K
tively. Panel D: The dependence of
0 mM, d; 0.25 mM, s; 0.35 mM,
Standard assay conditions were employed with [NH
effect of caffeine on (relative to for [ATP] = 1 mM) for NH
formation is shown for various [ATP] (1 mM, d; 0.75 mM, ; 0.5 mM, j; 0.25 mM,
0.167 mM, .; and 0.10 mM, s). Curves are fits to eqn. 1. Panel F: The
S
ꢂ K
m
), K = 0.080 mM, and eqns. 2 and 3, respec-
on [ATP] in the presence of varying [caffeine]
; 0.5 mM, 4; and 0.75 mM, .) is shown.
Cl] = 150 mM. Panel E: The
-dependent CTP
v
i
(
N
4
v
i
/
v
o
v
i
/
v
o
3
N
;
0
dependence of K on [ATP] is shown. Theoretical curves for competitive (dashed
line) and uncompetitive (dotted line) inhibition were calculated using
0
K
S
= 0.066 mM (assuming that K
S
ꢂ K
m
), K = 0.053 mM, and Eqs. 2 and 3, respec-
At present, the location of the inhibitor binding site is not
known. However, the fact that adenosine (IC50 = 11 mM for the
NH -dependent reaction ), adenine, uridine, and uracil (Table 1)
3
tively. In both panels C and F, the linear regression lines (solid lines) show no
significant dependence on [substrate], consistent with noncompetitive, multisite
inhibition.
2
2
are much weaker inhibitors than the xanthines and uric acids sug-
gests that the purine-based inhibitors are not binding at the ATP-
or UTP-binding sites. This conclusion is also supported by our
observation that caffeine behaves as a noncompetitive, multisite
inhibitor of CTPS with respect to ATP (Fig. 2). Moreover, the pur-
ine-based inhibitors do not appear to disrupt the ATP/UTP-depen-
dent formation of CTPS tetramers.
Thus, inhibition of E. coli CTPS can be achieved solely through
interactions between the enzyme and a xanthine heterocycle,
suggesting that the purine ring may serve as a useful scaffold
for the development of CTPS inhibitors. Studies to characterize
the architecture of the inhibitor binding site are currently
underway.
Table 2
Kinetic parameters for the multisite inhibition of CTPS-catalyzed NH
3
-dependent CTP
formation by caffeinea
0
[
NH
3
], mM
K
IC50, mM
n
8
5
2
1
0
.16
0.073 ± 0.007
0.067 ± 0.002
0.082 ± 0.002
0.090 ± 0.040
0.086 ± 0.015
0.080 ± 0.017
0.44 ± 0.01
0.45 ± 0.01
0.42 ± 0.01
0.45 ± 0.04
0.44 ± 0.01
0.44 ± 0.02
3.2 ± 0.1
3.4 ± 0.1
2.9 ± 0.1
3.1 ± 0.1
3.0 ± 0.2
3.1 ± 0.4
.44
.72
.36
.54
Average
a
Values are means of three experiments; errors are standard deviations.

1
44
A. C. Roy et al. / Bioorg. Med. Chem. Lett. 20 (2010) 141–144
1
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Acknowledgements
This work was supported by a Natural Sciences and Engineering
Research Council (NSERC) of Canada Discovery Grant and a Cana-
dian Institutes for Health Research Operating Grant to S.B. and
F.L. was supported through a graduate traineeship from the Dal-
housie Cancer Research Training Program. A.R. was supported
through a Norah Stephen Oncology Scholarship and an NSERC
USRA. We thank Kelli Flemming for technical assistance (supported
through an NSERC USRA).
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