Nucleotide binding to human UMP-CMP kinase using
fluorescent derivatives ) a screening based on affinity
for the UMP-CMP binding site
Dimitri Topalis
1,
*, Hiroki Kumamoto
2,
*, Maria-Fernanda Amaya Velasco
3,
*, Laurence Dugue
´
4
,
Ahmed Haouz
5
, Julie Anne C. Alexandre
1
, Sarah Gallois-Montbrun
6,†
, Pedro Maria Alzari
3
,
Sylvie Pochet
4
, Luigi Andre
´
Agrofoglio
2
and Dominique Deville-Bonne
1
1 Laboratoire d’Enzymologie Mole

side analogs used as antiviral and anticancer agents. The
phosphorylation of nucleoside analogs requires three
steps involving the action of deoxyribonucleoside kinase
Keywords
cidofovir; human UMP-CMP kinase; MABA-
CDP; Mant-ATP; phosphonates
Correspondence
D. Deville-Bonne, Laboratoire d’Enzymologie
Mole
´
culaire et Fonctionnelle, FRE 2852
CNRS-Paris 6, Institut Jacques Monod,
4, place Jussieu, 75251 Paris Cedex 05,
France
Fax: +33 1 44 27 59 94
Tel: +33 1 44 27 59 93,
E-mail: [email protected]
*These authors contributed equally to this
work

Present address
Department of Infectious Diseases, Guy’s,
King’s and St Thomas’ Medical School,
King’s College London, GKT Guy’s Hospital,
London
(Received 12 February 2007, revised
25 May 2007, accepted 29 May 2007)
doi:10.1111/j.1742-4658.2007.05902.x
Methylanthraniloyl derivatives of ATP and CDP were used in vitro as
fluorescent probes for the donor-binding and acceptor-binding sites of

P
1
-(5¢-adenosyl) P
5
-(5¢-guanosyl) pentaphosphate; Ap5U, P
1
-(5¢-adenosyl) P
5
-(5¢-uridyl) pentaphosphate; cidofovir, (S)-1-(3-hydroxy-2-
phosphonylmethoxypropyl) cytosine; MABA-CDP, cytidine diphospho-b-(N¢-methylanthraniloylaminobutyl)-phosphoramidate; Mant,
N-methylanthraniloyl; UCK, UMP-CMP kinase; UVP, uracil vinylphosphonate.
3704 FEBS Journal 274 (2007) 3704–3714 ª 2007 The Authors Journal compilation ª 2007 FEBS
[1], NMP kinase [2] and finally NDP kinase [3] and ⁄ or
one of the enzymes capable of synthesizing ATP, such
as phosphoglycerate kinase [4,5], pyruvate kinase or
creatine kinase [6]. However, acyclic nucleoside phos-
phonates, a new class of antiviral analogs [7], do not
require the first phosphorylation step and depend on
cellular NMP kinases or, in some cases, on viral NMP
kinases. Human UCK, also known as pyrimidine nucle-
oside monophosphate kinase [6], is an NMP kinase.
These enzymes all have a highly conserved fold. The
family includes six isoforms of AMP kinase, one TMP
kinase and one GMP kinase. Like most NMP kinases,
human UCK is located in the cytosol, but isoforms 2
and 3 of AMP kinases are found in the mitochondria
and isoform 6 in the nucleus. Another dTMP kinase, as
yet unidentified, may be located in mitochondria [8].
The primary sequence of human UCK is 40% identical
to that of AMP kinase 1, 27% identical to that of AMP

phono-methoxylethyl) adenine dipivoxil] and the
anti-human immunodeficiency virus agent tenofovir
disoproxil fumarate (9-[2-(R)-(phosphonomethoxy)
propyl] adenine disoproxil fumarate) [7], once the pro-
tecting groups have been removed, are activated ineffi-
ciently by phosphorylation with cellular AMP kinases
[13,14]. The first phosphorylation of antiviral nucleo-
side phosphonates, catalyzed by NMP kinases, is prob-
ably a bottleneck in their activation. We have looked
for new acyclic phosphonate derivatives that interact
better with NMP kinases and are more readily phos-
phorylated to give active forms. We have studied the
specificity of binding at the acceptor site of human
UCK in order to identify potential ligands among new
acyclic nucleoside phosphonates considered to be cid-
ofovir analogs. We used N-methyl anthraniloyl (Mant)
nucleotides (Mant-ATP [15] and cytidine diphospho-
b-(N¢-methylanthraniloylaminobutyl)-phosphoramidate
(MABA-CDP ) [16]) as fluorescent probes to monitor
the binding of nucleotides to UCK. These assays were
used to determine the binding affinity of bisubstrates
and new phosphonate analogs and to validate a fluor-
escent approach for high-throughput screening of new
compounds.
Results and Discussion
Competitive fluorescence experiments to
determine the binding of natural substrates to
the donor and the acceptor sites of human UCK
The ATP-binding site (‘donor-binding site’) of human
UCK was probed with the fluorescent nucleotide

-(5¢-adenosyl)
P
5
-[5¢-(2¢-deoxy-thymidyl)] pentaphosphate (Ap5dT)
(3.5 lm) was lower than that for ATP (10 lm), sug-
gesting that the dTDP moiety in Ap5dT contributes to
the binding energy. This is the first evidence that any
base including thymidine can be accommodated in the
NMP acceptor site of human UCK (Fig. 1).
The acceptor-binding site of human UCK was also
probed with the fluorescent nucleotide MABA-CDP,
in which the Mant group is bound to the b-phos-
phate of CDP through a butyl linker (Fig. 2A).
D. Topalis et al. UMP-CMP kinase and acyclic phosphonate analogs
FEBS Journal 274 (2007) 3704–3714 ª 2007 The Authors Journal compilation ª 2007 FEBS 3705
Dictyostelium UCK has been reported to specifically
bind MABA-CDP at the CMP-binding site [16]. Add-
ing human UCK increased the fluorescence of MABA-
CDP, and the spectrum shifted slightly towards blue.
Excess of CMP or CDP returned the fluorescence of
MABA-CDP to its initial value, demonstrating the
specificity of MABA-CDP binding to the acceptor-
binding site (Fig. 3A, inset). Titration of MABA-CDP
with the enzyme (Fig. 3A) indicated an increase in
fluorescence of 160% at saturation and an equilibrium
dissociation constant K
MABAÀCDP
D
¼ (8.5 ± 1.0) lm
when fitted to the Langmuir binding equation. The

D
values was
presumably due to the interaction of the sugar 2¢-OH
group with the carbonyl of Lys61 [9,10]. Both AMP
and also dTMP displaced MABA-CDP from the accep-
tor-binding site with submillimolar K
D
, in agreement
with the results in Fig. 1 for Mant-ATP. The affinity of
the human enzyme acceptor-binding site for NMPs was
5–20 times higher than that of Dictyostelium UCK,
despite identical active site residues [10,16]. The K
D
val-
ues in Table 1 are in agreement with the kinetic param-
eters of the natural nucleoside monophosphates
dCMP, dUMP and AMP [19]. GMP and dTMP were
not substrates of the enzyme, indicating that their bind-
ing to the NMP-binding site is unproductive.
UCK has been identified in human liver as the
enzyme that catalyzes the first phosphorylation step
for cidofovir [6]. The binding of cidofovir was substan-
tially weaker than that of natural substrates. It was
also a poor substrate for recombinant human UCK,
with a low k
cat
(k
cat
¼ 0.06 s
)1

Fig. 2. (A) Formula of MABA-CDP used in
the fluorescent competitive titration assay
to determine dissociation constants of acy-
clic nucleotides. (B) Formula of C5-substi-
tuted vinyl phosphonates (Y ¼ H, Cl, Br,
phenyl, fluorophenyl, phenyl-S).
UMP-CMP kinase and acyclic phosphonate analogs D. Topalis et al.
3706 FEBS Journal 274 (2007) 3704–3714 ª 2007 The Authors Journal compilation ª 2007 FEBS
properties are comparable to those of the human liver
enzyme [6]. The MABA-CDP competition assay gave a
K
D
of 0.3 mm for cidofovir, similar to that of dUMP
(Fig. 3B and Table 1). The ratio between cidofovir and
dCMP binding affinities was no more than 5, a value
similar to the ratio between the CMP and dCMP equi-
librium constants.
Binding of acyclic phosphonate nucleosides
to the acceptor site of human UCK using
fluorescent MABA-CDP
Several uracil vinylphosphonates (UVPs) modified at
the 5-position were produced by parallel synthesis [20]
and evaluated for human UCK activity in order to
find acyclic phosphonate nucleoside analogs possessing
a better affinity for human UCK than cidofovir
(Scheme 1 and Fig. 2B). None of them was a substrate
for human UCK, but they were all inhibitors. Their
binding affinities for human UCK were studied using
both the MABA-CDP fluorescent competition and
activity assays. A preliminary plate-adapted assay indi-

was 8.5 ± 1.5 lM. Inset: Fluorescence emission
spectra of MABA-CDP (10 l
M) in T buffer (k
excitation
¼ 325 nm, excitation slit ¼ 2 nm, emission slit ¼ 2 nm). (a) MABA-CDP alone. (b)
MABA-CDP + 50 l
M human UCK. (c) MABA-CDP + 50 lM human UCK + 5 mM CDP or CMP. (B) Fluorescence competition assays with
MABA-CDP bound to the human UCK acceptor-binding site. MABA-CDP (8 l
M) + human UCK (24 lM) titrated with CMP (d), dCMP (s),
UMP (j), AMP (·) TMP (h) and cidofovir (m).
Table 1. Equilibrium dissociation constants and kinetic parameters
of human UCK for natural NMP and cidofovir. The K
D
values were
obtained from fluorescence competition assays with MABA-CDP
bound to the human UCK acceptor-binding site. The conditions are
shown in Fig. 3B. The kinetic constants were measured under
standard conditions in the presence of 1 m
M ATP and 5 mM Mg
2+
.
ND, not detectable.
Ligand K
D
(lM) ⁄ MABA-CDP K
m
(mM) k
cat
(s
)1

3
dTMP 750 ± 150 ND ND ND
Cidofovir 300 ± 100 1.0 ± 0.3 0.06 ± 0.02 60
AMP 100 ± 30 > 5
a
ND 10
3a
GMP 500 ± 100 ND ND ND
a
Data from Pasti et al. [19].
D. Topalis et al. UMP-CMP kinase and acyclic phosphonate analogs
FEBS Journal 274 (2007) 3704–3714 ª 2007 The Authors Journal compilation ª 2007 FEBS 3707
affinity of the enzyme for this compound. The 6e
derivative (5-F-Phe-UVP) was only detected in the
enzymatic assay, indicating that the inhibition may
involve binding to a site different from the CMP-bind-
ing site. Cidofovir binding was detected only in the
MABA-CDP competition assay, a fact that was expec-
ted, as the enzymatic assay did not detect inhibition by
substrates.
Kinetic studies were also performed with 0.5 mm
ATP and dCMP as acceptor substrate. Several vinyl-
phosphonates were clearly competitive inhibitors of
dCMP [Fig. 4 for the 6c derivative (5-Cl-UVP) (K
i
¼
16±3lm]. The dissociation constant for 5-Br-vinyl-
phosphonate (compound 6b) was in the same range as
that for 5-Cl-UVP (compound 6c) and about 17 times
smaller than that for cidofovir. The halogen substitu-

2
Cl
2
.
Table 2. Binding affinities of human UCK for new uracil acyclic
phosphonates determined in the MABA-CDP fluorescent assay and
inhibitory constants in activity assays. The conditions for determin-
ing the K
D
values from fluorescence competition assays with
MABA-CDP bound to human UCK are reported in Fig. 3B. The IC
50
values with human UCK were measured at 37 °C (substrate con-
centrations: 50 m
M CMP, 0.5 mM ATP, and 5 mM Mg
2+
). The inhibi-
tion constants K
i
were measured as shown in Fig. 4 at 37 °C. The
substrate was here dCMP rather than CMP, as CMP is itself pre-
sent at high concentrations [19]. All the experiments were done at
least two times, and standard deviations were about 20%. ND, not
determined.
Ligand
Fluorescent
assay MABA-CDP
K
D
(lM)

twice with the same results (15%): m,0l
M 5-Cl-UVP; j,1lM
5-Cl-UVP; d,50lM 5-Cl-UVP.
UMP-CMP kinase and acyclic phosphonate analogs D. Topalis et al.
3708 FEBS Journal 274 (2007) 3704–3714 ª 2007 The Authors Journal compilation ª 2007 FEBS
showing that the Phe substitution was not accommoda-
ted in the binding site. However, the presence of an F
in compound 6e or a thiol (compound 6f) on the ben-
zene ring (5-F-Phe, 5-Phe-S) was beneficial, as the K
i
values were still smaller than that of cidofovir, even
though the affinity was lower than that of 5-Cl-UVP.
As 5-halogen-substituted uridylate derivatives are
often considered to be thymidine analogs, we assayed
the activity of human dTMP kinase with the UVPs.
There was no activity in the presence of 0.5 mm
ATP, even at a high concentration of the enzyme
(30 lm), except for 5-Cl-UVP (compound 6c)at
[dTMP] < 0.1 mm, resulting in a very low catalytic
efficiency (80 m
–1
Æs
)1
). Both 5-Br-UVP (compound 6b)
and 5-Cl-UVP (compound 6c) inhibited human dTMP
kinase with a higher K
i
(about 10 times less) than
human UCK (not shown).
The lack of phosphorylation of UVP derivatives by

values. Segura-Pena et al. found that
human UCK could be crystallized only at low pH
(4–6), which may have weakened the substrate binding
and the Mg
2+
ion coordination [9]. However, we
obtained crystals of human UCK at higher pH (7.5), so
pH may not be the only explanation for the lack of
bound ligands in the crystal.
The ligand-free enzyme crystallized as a dimer
(Fig. 5B) in which intermolecular contacts between the
LID and the NMP-binding regions prevent substrate
binding. The reversible dissociation of such dimers
might be involved in regulatory mechanisms [18]. Sev-
eral human kinases, such as dTMP kinase, deoxyguan-
osine kinase and deoxycytidine kinase, are known to
exist as stable dimers, as does deoxynucleoside kinase
from Drosophila [1,21]. Gel filtration experiments with
the recombinant human UCK showed that the protein
was eluted with an estimated molecular mass of 32–35
kDa, a value significantly higher than the molecular
mass (22 222 kDa) of the protein [18]. Human UCK is
a monomer in low-salt solution (R
s
¼ 2.2 nm), but the
Stokes radius is 2.8 nm in 0.2 m KCl, corresponding
Fig. 5. (A) Superpositions of the polypeptide backbone of human UCK (green) and pig adenylate kinase (orange; Protein Data Bank code 3ADK),
Dictyostelium discoideum UCK (blue; 2UKD), human adenylate kinases 1 (cyan; 1Z83) and 2 (light gray; 2C9Y), yeast uridylate kinase (yellow;
1UKY) and yeast adenylate kinase (pink; 1DVR). (B) Crystallographic dimer of human UCK, with each monomer shown in a different color.
D. Topalis et al. UMP-CMP kinase and acyclic phosphonate analogs

antiviral activities when used as prodrugs [22], indica-
ting indirectly that human NMP kinases can phos-
phorylate them in the cell.
Conclusion
The binding studies on human UCK highlight the broad
specificity of the acceptor site. As the structure of the
human enzyme active site in complex with natural or
exogenous ligands is still unknown, the structure of the
Dictyostelium enzyme was analyzed. The presence of
several water molecules in the acceptor-binding site of
this enzyme explains its ability to accommodate several
chemical modifications of the acceptor [10]. The fluores-
cence competition assay data correlate well with the
inhibition constants determined using the activity assay,
and could thus be useful for screening new analogs. The
fluorescence competition assay does not replace the
assays for antiviral activity and cytotoxicity, but may
contribute to the knowledge of the interaction of deriva-
tives with cellular targets. The binding of dTMP and
5-halogenated acyclic derivatives to human UCK indi-
cates that human UCK and human dTMP kinase may
have unexpected common ligands that could contribute
to the toxicity of therapeutic analogs.
Experimental procedures
Materials
Mant-ATP, d4TMP and the bisubstrate analogs Ap5U,
P
1
-(5¢-adenosyl) P
5

R140
AB
D142
R151
Mg
2+
H
2
O
R39
Fig. 6. Model of the acceptor-binding site in the closed form of human UCK. (A) Superposition of the acceptor site with bound CMP (blue)
and cidofovir (red). (B) Superposition of the acceptor site with bound CMP (blue) and UVP (green).
UMP-CMP kinase and acyclic phosphonate analogs D. Topalis et al.
3710 FEBS Journal 274 (2007) 3704–3714 ª 2007 The Authors Journal compilation ª 2007 FEBS
Germany). Cidofovir was a gift from J Neyts (Rega Insti-
tute, Leuven, Belgium). The fluorescent CDP analog (Pb)-
MABA-CDP (Fig. 2) was synthesized by the procedure
of Rudolph et al. [16], slightly modified as described for
MABA-dTDP in Topalis et al. [23].
Synthesis of UVPs
The synthesis of the novel unsaturated acyclic phosphono-
nucleosides (compounds 6a–f) is outlined in Scheme 1. The
5-phenylthio derivative (compound 2) was prepared from
compound 1 by introduction of a phenylthio group at the
5-position, followed by a crotylation of the N
1
-position
and the debenzoylation of the N
3
-position, with an overall

was equilibrated by dialysis against 20 mm Tris ⁄ HCl
(pH 7.5) buffer containing 20 mm NaCl, 1 mm dithiothrei-
tol and 50% glycerol.
The selenomethionine-labeled protein was obtained from
Bli5 E. coli cells transformed with the pET28a-huck plas-
mid and grown overnight in LB medium supplemented
with 30 lgÆmL
)1
kanamycin and 70 lgÆmL
)1
chloramphen-
icol at 37 °C. An aliquot of the culture (3 mL) was centri-
fuged (1 min at 6000 g at 4 °C), and the pellet was
resuspended in 100 mL of M9 minimum medium (plus
kanamycin and chloramphenicol) and grown at 37 °C.
When the cells reached an D
600
of 0.6, 50 mg each of
lysine, threonine and phenylalanine and 25 mg each of
leucine, isoleucine, valine and selenomethionine was added
to the culture, and incubation was continued for 40 min.
The temperature was then lowered to 20 °C, and 1 mm
isopropyl thio-b -d-galactoside was added to induce protein
production. Growth was continued for a further 12 h at
20 °C. The cells were harvested by centrifugation (30 min
at 6000 g at 4 °C), and suspended in lysis buffer contain-
ing 1 mm dithiothreitol and EDTA-free protease inhibitors
(Roche, Meylan, France). The protein was purified as pre-
viously described for the unlabeled protein [19], and an
almost pure protein was obtained (> 95% homogeneity

0
)A + F
0
, where A is the molar fraction of
bound MC (A ¼ [MC.E] ⁄ [MC]
t
), F
o
is the initial fluores-
cence before adding the protein, and F
max
is the fluores-
cence after saturation by the protein. The concentration of
the complex [MC.E] is given by Eqn (1):
½MC.E¼K
D
þ½MC
t
þ½E
t
À
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
K
D
þ½MC
t
þ½E
t
À 4½MC
t

The IC
50
value at half-displacement was related to the
D. Topalis et al. UMP-CMP kinase and acyclic phosphonate analogs
FEBS Journal 274 (2007) 3704–3714 ª 2007 The Authors Journal compilation ª 2007 FEBS 3711
dissociation constants K
D
for the competitor and K
MC
D
for
MABA-CDP using Eqn (2) [26,27]:
K
D
¼ IC
50
þ K
MC
D
B=½AP þ BðP À A þ B À K
MC
D
Þ ð2Þ
where B is the initial concentration of MABA-CDP bound
to the enzyme, A is its total concentration, and P is the
total concentration of human UCK. Data were analyzed
using kaleidagraph (Abelbeck Software, ALSYD, Mey-
lan, France). Similar measurements were done for the inter-
action of Mant-ATP with human UCK (k
excitation

Crystallographic studies
Human UCK was crystallized using the hanging drop vapor
diffusion method by mixing 1.5 lL of protein solution
(8 mgÆmL
)1
)in50mm Tris ⁄ HCl (pH 7.5), 10 mm dithio-
threitol, 20 mm NaCl, 5 mm MgCl
2
and 5–10 mm of the
different ligands (ADP, UMP, CMP, AMPPCP, Ap5U, or
cidofovir) with 1.5 lL of the reservoir solution [2.5 m
ammonium sulfate, 5% (v ⁄ v) glycerol, 25 mm sodium
citrate]. The crystals belonged to space group P6
5
22, with
cell dimensions a ¼ b ¼ 62.1 A
˚
, c ¼ 222.5 A
˚
. The Se-methi-
onine-labeled protein was produced as previously described
[29], and protein was synthesized and purified as above
(unlabeled enzyme). Diffraction data were collected at
100 K on single frozen crystals at the ESRF (beam lines
ID14.2 and ID29). Data were processed using programs
from the CCP4 software package [30]. The crystal structure
was determined using single-wavelength anomalous diffrac-
tion methods from a single crystal of SeMet-labeled protein,
using the programs shake’n’bake [31] and sharp [32]. Crys-
tallographic refinement was carried out by alternate cycles

Neyts (Rega Institute, Leuven, Belgium) for the gift of
cidofovir, Miche
`
le Reboud (FRE 2852 CNRS-Univer-
site
´
Paris 6) and Michel Ve
´
ron (Institut Pasteur) for
helpful discussions, and Ezequiel Panepucci (Institut
Pasteur) for help in modeling human UCK in the close
conformation. The English text was checked by Owen
Parkes. These studies were supported by a grant from
Sanofi-Aventis France (Sanofi-Aventis Group) and
Bayer Pharma as part of a multi-organism call for
proposals. We also thank the Agence Nationale de
Recherches (France) for grant ANR-05-BLAN-0368
(L. A. Agrofoglio and D. Deville-Bonne), and the
Agence Nationale de Recherche sur le SIDA (France)
to P. Alzari and D. Deville-Bonne. Part of this work
was presented during the XVIIth Round Table for
Nucleosides, Nucleotides and Nucleic Acids in Bern, in
September 2006.
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