The phosphorylation of nucleoside analogs requires three steps involving the action of deoxyribonucleoside kinase Keywords cidofovir; human UMP-CMP kinase; MABA-CDP; Mant-ATP; phosphona
Trang 1fluorescent derivatives ) a screening based on affinity
for the UMP-CMP binding site
Dimitri Topalis1,*, Hiroki Kumamoto2,*, Maria-Fernanda Amaya Velasco3,*, Laurence Dugue´4, Ahmed Haouz5, Julie Anne C Alexandre1, Sarah Gallois-Montbrun6,†, Pedro Maria Alzari3,
Sylvie Pochet4, Luigi Andre´ Agrofoglio2and Dominique Deville-Bonne1
1 Laboratoire d’Enzymologie Mole´culaire et Fonctionnelle, FRE 2852 CNRS-Paris 6, Institut Jacques Monod, Paris, France
2 Institut de Chimie Organique et Analytique, UMR CNRS 6005, FR 2708, Universite´ d’Orle´ans, UFR Sciences, Orle´ans, France
3 Unite´ de Biochimie Structurale, URA CNRS 2185, Institut Pasteur, Paris, France
4 Unite´ de Chimie Organique, URA CNRS 2128, Institut Pasteur, Paris, France
5 Plate-Forme 6- Cristalloge´ne`se et Diffraction des Rayons X, Institut Pasteur, Paris, France
6 Unite´ de Re´gulation Enzymatique des Activite´s Cellulaires, CNRS URA 2185, Institut Pasteur, Paris, France
Human UMP-CMP kinase (UCK) plays a key role in
the ribonucleoside and deoxyribonucleoside salvage
pathway and in the anabolic phosphorylation of
nucleo-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: ddeville@ccr.jussieu.fr
*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 human UMP-CMP kinase, a nucleoside salvage pathway kinase Like all NMP kinases, UMP-CMP kinase binds the phosphodonor, usually ATP, and the NMP at different binding sites The reaction results from an in-line phosphotransfer from the donor to the acceptor The probe for the donor site was displaced by the bisubstrate analogs of the Ap5X series (where
X¼ U, dT, A, G), indicating the broad specificity of the acceptor site Both CMP and dCMP were competitors for the acceptor site probe To find antimetabolites for antivirus and anticancer therapies, we have devel-oped a method of screening acyclic phosphonate analogs that is based on the affinity of the acceptor-binding site of the human UMP-CMP kinase Several uracil vinylphosphonate derivatives had affinities for human UMP-CMP kinase similar to those of dUMP and dUMP-CMP and better than that of cidofovir, an acyclic nucleoside phosphonate with a broad spectrum of antiviral activities The uracil derivatives were inhibitors rather than substrates of human UMP-CMP kinase Also, the 5-halogen-substituted analogs inhibited the human TMP kinase less efficiently The broad speci-ficity of the enzyme acceptor-binding site is in agreement with a large substrate-binding pocket, as shown by the 2.1 A˚ crystal structure
Abbreviations
Ap5A, P 1 -(5¢-adenosyl) P 5 -(5¢-adenosyl) pentaphosphate; Ap5dT, P 1 -(5¢-adenosyl) P 5 -[5¢-(2¢-deoxy-thymidyl)] pentaphosphate; Ap5G,
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.
Trang 2[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
kinase 2, 21% identical to that of dTMP kinase, and
20% identical to that of GMP kinase The structure of
human UCK has been determined recently, but in the
absence of a ligand [9] However, the active site of the
homologous enzyme from Dictyostelium complexed with
the bisubstrate inhibitor P1-(5¢-adenosyl) P5-(5¢-uridyl)
pentaphosphate (Ap5U) is known [10]
The human UCK efficiently phosphorylates the
monophosphorylated forms of arabinocytidine,
gemcit-abine and 3¢thiancytidine, which are used to treat
leukemia, pancreatic cancer and AIDS [11] In
con-trast, the dCMP acyclic phosphonate mimic, cidofovir
[(S)-1-(3-hydroxyl-2-phosphonomethoxy-propyl)
cyto-sine], which is approved for treating cytomegalovirus
retinitis in patients with AIDS and has more recently
been approved for persons infected with monkeypox
virus, is poorly phosphorylated by human UCK [6]
The bioavailablity of cidofovir is below 5% but its
in vitro activity against herpes and orthopox viruses
can be increased by several orders of magnitude by
esterification with lipidic groups, which improve its
penetration into cells [12] Cidofovir, like the other
antiviral nucleoside phosphonates, is very stable and
has a long half-life in the body [7] Among other
licensed antiviral nucleoside phosphonates, the
anti-hepatitis B virus agent, adefovir dipivoxil
[9-(2-phos-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 Mant-ATP, in which the methylanthranylate group is bound to the 2¢-OH and 3¢-OH of the ribose [15] Mant-ATP binding to the enzyme resulted in a large increase in fluorescence intensity (220%) Titration of Mant-ATP with human UCK fitted the Langmuir binding equation with a stoichiometry of 1 and an equilibrium dissociation constant, KD, of 3.5 lm (results not shown) Mant-ATP binding was not modi-fied by CMP, in contrast to Escherichia coli CMP kinase [17] Mant-ATP was displaced by ATP with
a KATPD ¼ 10 lm in a competitive titration and in an indirect titration assay for several ATP concentrations (not shown) Other nucleotides such as CTP and ADP also displaced Mant-ATP The bisubstrate analog Ap5U displaced Mant-ATP (KAp5UD ¼ 0.15 lm) better than ATP (Fig 1) The other bisubstrate analogs in which U in Ap5U was replaced by A, G or even dT were also competitors, although they were less efficient than Ap5U (Fig 1) The KDvalue for P1-(5¢-adenosyl)
P5-[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)
Trang 3Dictyostelium 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 KMABACDPD ¼ (8.5 ± 1.0) lm
when fitted to the Langmuir binding equation The
fluorescence of the MABA-CDP ⁄ enzyme complex was
decreased by CDP and other nucleotides (Fig 3A,
inset) The fluorescence of human UCK was unaffected
by ATP, unlike that of UCK from Dictyostelium, indi-cating that MABA-CDP does not probe the human UCK ATP site [16] The concentrations of nucleotide needed to get half the signal (IC50) were determined by competition experiments (Fig 3B) The equilibrium dissociation constants KD are summarized in Table 1 CDP was better at displacing MABA-CDP than CMP, perhaps because the enzyme⁄ MABA-CDP conforma-tion favors the reverse reacconforma-tion (CDP + ADPfi CMP + ATP)
CDP, CMP, UMP, dCMP and dUMP all com-peted with MABA-CDP at the acceptor-binding site, showing that the same NMP site binds ribonucleo-sides and deoxyribonucleoribonucleo-sides under our experimen-tal conditions (5 mm magnesium ions) This does not
fit the hypothesis that acceptor sites use either ribo-nucleoside or deoxyribonucleoside monophosphate [18] Cytidine nucleotides had higher affinities than uridine nucleotides, as reported for the Dictyostelium enzyme The comparison between cytidine and uridine monophosphates in Table 1 is in agreement with the preference for ribonucleotides rather than deoxyribo-nucleotides The 5–6-fold difference in KD 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 KD, 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 KD 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-bindbind-ing 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 kcat(kcat¼ 0.06 s)1, Km¼ 1 mm) resulting
in a low catalytic efficiency, about 60 m–1Æs)1 These
Fig 1 Fluorescence competition assays with Mant-ATP bound to
the human UCK donor-binding site Mant-ATP (3 l M ) + human UCK
(10 l M ), resulting in 50% fluorophore bound, was titrated with
Ap5U (d), Ap5A (j), Ap5G (n), Ap5dT (s) and ATP (m) with IC50¼
1 l M , 4.4 l M , 11 l M , 25 l M and 50 l M , respectively (k excitation ¼
350 nm, k emission ¼ 436 nm, excitation slit ¼ 1 nm, k emission
slit ¼ 4 nm) The values for the equilibrium constants K D
calcula-ted as in Experimental procedures are: 0.15 l M for Ap5U, 0.7 l M
for Ap5A, 1.7 l M for Ap5G, 3.4 l M for Ap5dT, and 9.5 l M for ATP.
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).
Trang 4properties are comparable to those of the human liver
enzyme [6] The MABA-CDP competition assay gave a
KD 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-cated that all the molecules except the 5-fluorophenyl derivative (compound 6e) competed with MABA-CDP (1 mm) The IC50values were measured individually by fluorometric competition titration, and the dissociation constants (KD) were calculated (Table 2) The displace-ment was total for all compounds except for com-pound 6d (5-Phe-UVP) (50%), which was not suitable for the assay, due to its poor solubility The IC50 val-ues were also evaluated with the human UCK activity assay under standard conditions, i.e 50 lm CMP and 0.5 mm ATP (Table 2) Both assays usually gave IC50 values in the same range The values for compound 6a (UVP) are less accurate, probably due to the poor
Fig 3 Fluorescence assays with MABA-CDP bound to the human UCK acceptor site (A) Dissociation equilibrium constant of MABA-CDP ⁄ enzyme complex determined by the fluorescence assay The fluorescent signal of MABA-CDP (2 l M ) was monitored after stepwise addition
of human UCK (k excitation ¼ 325 nm, k emission ¼ 430 nm, excitation slit ¼ 2 nm, emission slit ¼ 2 nm) The signal was fitted to the Langmuir binding equation with a fixed maximum enhancement of fluorescence of 160% The KDwas 8.5 ± 1.5 l M Inset: Fluorescence emission spectra of MABA-CDP (10 l M ) in T buffer (kexcitation¼ 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 l M human UCK + 5 m M 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 l M ) 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 KDvalues 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 m M Mg 2+
ND, not detectable.
Ligand KD(l M ) ⁄ MABA-CDP Km(m M ) kcat(s)1)
k cat ⁄ K m
( M )1Æs)1)
Cidofovir 300 ± 100 1.0 ± 0.3 0.06 ± 0.02 60
a Data from Pasti et al [19].
Trang 5affinity 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 bindCMP-bind-ing 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) (Ki¼
16 ± 3 lm] 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-tion in the 5-posisubstitu-tion (Br, Cl) improved binding: UVP (compound 6a) inhibited the enzyme (Ki¼ 0.7 mm) less well (Table 2) No clear binding was detected when the 5-halogen was replaced by a larger group such as Phe,
Scheme 1 Synthesis of novel acyclic phosphononucleoside analogs Reagents: (a) (i) benzenethiol (PhSH), N-chlorosuccinimide (NCS), pyrid-ine, MeCN, reflux; (ii) crotyl bromide, K2CO3, dimethylformamide; (iii) K2CO3, MeOH; (b) Bu3SnH, AIBN, toluene, reflux; (c) N-bromosuccini-mide (NBS) (or NCS), tetrahydrofan (THF) (for 4b and 4c, respectively) or aryliodide (RI), PdCl 2 (PPh 3 ) 2 (0.11 mmol), CuI, dimethylformamide,
rt (for 4d–f); (d) diethyl vinyl phosphonate (4 eq.), Nolan’s catalyst ¼, CH 2 Cl2, reflux; (e) TMSBr (4 eq.), CH2Cl2.
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 KD 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 m M ATP, and 5 m M Mg 2+ ) The
inhibi-tion constants Kiwere 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
KD(l M )
Activity assay
IC50(l M )
Competitive inhibitors
Ki(l M )
6d (5-Phe-UVP) 150
amplitude 50%
120 amplitude 50%
–
Fig 4 Inhibition of human UCK activity by 5-chloro-UVP (com-pound 6c) Double reciprocal plots are shown of the initial velocity
as a function of dCMP concentrations at fixed concentration of 5-Cl-UVP (compound 6c) The inset is a replot of slopes of the same data The concentration of human UCK was 17 n M (0.43 lgÆmL)1) The results are from a typical experiment repeated twice with the same results (15%): m, 0 l M 5-Cl-UVP; j, 1 l M 5-Cl-UVP; d, 50 l M 5-Cl-UVP.
Trang 6showing 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 Ki
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–1Æs)1) Both 5-Br-UVP (compound 6b)
and 5-Cl-UVP (compound 6c) inhibited human dTMP
kinase with a higher Ki (about 10 times less) than
human UCK (not shown)
The lack of phosphorylation of UVP derivatives by
human UCK could result from an unproductive
posi-tioning of the phosphate moiety in the acceptor site
We carried out a crystallographic study of human
UCK in the presence of several ligands, in order to
further understand the structures determining
sub-strate-binding specificity
Structural analysis of human UCK
The structure of human UCK was determined using
single-wavelength anomalous diffraction of the
sele-nomethione-labeled protein The overall structure was
quite similar to those of other monophosphate kinases
reported previously, with the three classical regions, the
NMP binding region (residues 34–37), the LID domain
(residues 130–137) and the CORE domain (residues 3–
31, 80–127, 160–194) [9] (Fig 5A) Superimposing the structures revealed that the largest differences are in the LID and NMP-binding regions of the protein, which in the ligand-free form of human UCK are partially dis-ordered on the electron density map Although the enzyme was cocrystallized with several ligands [UMP, CMP, ADP, adenosine-5¢(b-c)-methylene-diphosphate (AMP-PCP), Ap5U, cidofovir] in the presence of
Mg2+, we always obtained a crystal form isomorphous
to that of the open form of the ligand-free enzyme [9], indicating that crystal packing precluded ligand bind-ing This was surprising, as ligands such as Ap5U have nanomolar KD 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 Mg2+ 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 (Rs¼ 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.
Trang 7to a molecular mass of about 35 kDa for a globular
protein [19] Thus, the enzyme might form
homo-dimers in solution, and these may be stabilized in
the crystal form, so precluding substrate binding Such
inactive dimers presumably occur in high-salt
condi-tions and perhaps also in concentrated protein
solu-tions Their involvement in physiologic regulation is
therefore unlikely
Human UCK was modeled in a closed conformation,
using the available structures of homologous enzymes
(Fig 5A) and their complexes with ligands (Fig 6)
The model revealed a wide acceptor-binding site, which
accounts for the broad specificity Superimposing the
acceptor sites of CMP and cidofovir (Fig 6A) shows
that the acyclic part of cidofovir with a CH2-OH group
can be accommodated in a structurally permissive
region of the acceptor-binding site, with the OH group
interacting with R39 5-Cl-UVP also fits freely in the
active site (Fig 6B) The phosphonate group is
prob-ably too far from the three critical Arg residues (R39,
R96 and R140) that tightly maintain the phosphate
group of dCMP This prevents the transfer of the
c-phosphate from ATP The flexibility of the acyclic
part of the tested compounds may prevent these
inter-actions The size of the acyclic moiety could be
import-ant: Choo et al showed that analogs with an acyclic
moiety containing five carbons and a double bond have
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,
P1-(5¢-adenosyl) P5-(5¢-adenosyl) pentaphosphate (Ap5A),
P1-(5¢-adenosyl) P5-(5¢-guanosyl) pentaphosphate (Ap5G) and Ap5dT were purchased from Jena Biosciences (Jena,
R39
R134 R140 D142
R151
E36 R96
N100
T68 V63
K61
Mg2+
H2O
K61
V63
T68 N100
E36
R96
R134
R140
D142 R151
Mg2+
H2O 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).
Trang 8Germany) 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 N1-position
and the debenzoylation of the N3-position, with an overall
yield of 65% The resulting compound was sulfur-extrusive
stannylated to give the key intermediate compound 3
Compound 3 was converted to the 5-bromo (compound 4b)
and chloro (compound 4c) derivatives by simple treatment
with, respectively, N-bromosuccinimide and
N-chlorosuc-cinimide Several phenyl derivatives (compounds 4d–f) were
obtained from compound 3 via the Pd-catalyzed
Stille-coupling reaction [24] The acyclic cross-metathesis [20] of
compounds 4a–f with vinylphosphonate gave products
(compounds 5a–f) in the desired (E)-configuration Finally,
compounds 5a–f were incubated with trimethylsilyl bromide
in CH2Cl2for 2–3 days, to give the free phosphonates 6a–f
in good yields All compounds were purified by ion
exchange chromatography NMR, UV and mass analyses
confirmed their structures The detailed process will be
published elsewhere (Kunamoto H & Agrofoglio L,
unpub-lished results)
Protein expression and purification
His-tagged human UCK was expressed and purified to
homogeneity as previously described [19] The recombinant
enzyme used in biochemical studies was produced in E coli
BL21(DE3) (Novagen, Merck KGaA, Darmstadt,
Ger-many) transformed with the pDIA17 expression plasmid,
and purified in one step on an Ni–nitrilotriacetic acid
col-umn (Qiagen, Courtabeuf, France) using a linear gradient
of imidazole (10–250 mm) at pH 8 The purified protein
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 37C An aliquot of the culture (3 mL) was
centri-fuged (1 min at 6000 g at 4C), and the pellet was
resuspended in 100 mL of M9 minimum medium (plus
When the cells reached an D600 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 20C, and 1 mm isopropyl thio-b-d-galactoside was added to induce protein production Growth was continued for a further 12 h at
20C The cells were harvested by centrifugation (30 min
at 6000 g at 4C), 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
as determined by SDS⁄ PAGE)
Fluorescence measurements
All fluorescence measurements were performed at 20C in
T buffer (50 mm Tris⁄ HCl, pH 7.5, containing 5 mm MgCl2, 50 mm KCl, 5% glycerol and 1 mm dithiothreitol)
on a PTI spectrofluorometer Quantamaster (Birmingham,
NJ, USA) MABA-CDP was titrated with the enzyme by adding successive aliquots of the protein to MABA-CDP (2 lm) (kexcitation¼ 350 nm, kemission¼ 430 nm, 1 nm excitation slit and 2 nm emission slit) The fluorescent sig-nal was corrected for dilution The inner filter effect was found to be negligible Experimental ligand–protein binding curves were fitted to the Langmuir binding equation (Eqn 1) for determining the MABA-CDP (MC) dissociation constant, KMC
D As the fluorescence enhancement is directly proportional to binding, the observed fluorescence signal
Fobs¼ (Fmax) F0)A + F0, where A is the molar fraction of bound MC (A¼ [MC.E] ⁄ [MC]t), Fo is the initial fluores-cence before adding the protein, and Fmax is the fluores-cence after saturation by the protein The concentration of the complex [MC.E] is given by Eqn (1):
½MC.E ¼ KDþ ½MCtþ ½Et
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
KDþ ½MCtþ ½Et 4½MCt½Et q
ð1Þ where KD is the dissociation constant, [MC]t is the total MABA-CDP concentration, and [E]t is the total protein concentration with one binding site per protein
Nucleotide and analog binding was investigated in com-petitive experiments The 1 mL cell contained MABA-CDP (8 lm) and human UCK (24 lm) corresponding to, respect-ively, 1KDand 3KD, and resulting in the half-saturation of the enzyme at the start of the experiment [25] The fluores-cence decreased after each addition of unlabeled ligand Total displacement was checked by adding excess CDP A microplate assay was used for initial screening under the same conditions, and fluorescence values were determined (FluorstarGalaxy fluorometer; BMG Labtech, Champigny sur Marne, France) with 340 nm excitation and 446 nm emission filters Those compounds (1 mm) that displaced MABA-CDP were further studied in competition titrations The IC50 value at half-displacement was related to the
Trang 9dissociation constants KD for the competitor and KMC
D for MABA-CDP using Eqn (2) [26,27]:
KD¼ IC50þ KMC
D B=½AP þ BðP A þ B KMC
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
350 nm; kemission¼ 436 nm, excitation slit 1 ¼ nm, emission
slit¼ 4 nm)
Enzymatic activity measurements
The catalytic activity of the NMP kinases was determined
in a spectrophotometer by measuring ADP formation [28]
1 mm phosphoenolpyruvate and the auxiliary enzymes
pyruvate kinase (4 U) and lactate dehydrogenase (4 U)
The enzyme was diluted in a stabilizing solution (50 mm
Tris⁄ HCl, 5 mm MgCl2, 5 mm KCl, 1 mm dithiothreitol
and 10% glycerol) The reaction at 37C was started by
adding the enzyme followed by a phosphate acceptor at
the desired concentration The absence of inhibition of the
coupled system was carefully checked by measuring the
reaction with 10 lm ADP with and without the tested
analog Concentrations of UVP derivatives below 0.5 mm
produced no inhibition The reaction of
monophosphoryl-ated cidofovir with pyruvate kinase may have caused a
slight overestimation of the rates It was considered to be
negligible during the less than 10 min reaction time, as the
amount of monophosphorylated cidofovir produced was
quite low [6]
Crystallographic studies
Human UCK was crystallized using the hanging drop vapor
diffusion method by mixing 1.5 lL of protein solution
(8 mgÆmL)1) in 50 mm Tris⁄ HCl (pH 7.5), 10 mm
dithio-threitol, 20 mm NaCl, 5 mm MgCl2 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 P6522, with
cell dimensions a¼ 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
of model building with the program o [33] and refinement with the programs refmac5 [34] and arp⁄ warp [35] The refined model converged to an Rfactor⁄ Rfreeof 0.218⁄ 0.249 at 2.1 A˚ resolution, and was very similar to that previously reported for the ligand-free enzyme [8] (Protein Data Bank code 1TEV; rmsd of 0.4 A˚ for 188 residues) All cocrystalli-zation assays with ligands produced crystals isomorphous to those of the ligand-free protein, and no bound ligand could
be identified from difference Fourier calculations in any of seven different crystal structures analyzed
Structural models
Docking of 3-Cl-UVP and cidofovir was performed using arguslabsoftware [36] The closed (ligand-bound) form of human UCK was modeled from the atomic coordinates of the Dictyostelium UCK in complex with ADP and CMP (Protein Data Bank code 2UKD) Docking precision was set at ‘high’, and the ‘flexible ligand docking’ mode was used for each docking run The complexes were visualized with the program pymol [37]
Acknowledgements
We thank William Shepard (ESRF, Grenoble, France) for help with crystallographic data collection, Johan 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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