Báo cáo khoa học: Nucleotide binding to human UMP-CMP kinase using fluorescent derivatives ) a screening based on affinity for the UMP-CMP binding site potx

Nucleotide binding to human UMP-CMP kinase using fluorescent derivatives ) a screening based on affinity for the UMP-CMP binding site ´ Dimitri Topalis1,*, Hiroki Kumamoto2,*, Maria-Fernanda Amaya Velasco3,*, Laurence Dugue4, Ahmed Haouz5, Julie Anne C Alexandre1, Sarah Gallois-Montbrun6,†, Pedro Maria Alzari3, ´ Sylvie Pochet4, Luigi Andre Agrofoglio2 and Dominique Deville-Bonne1 ´ Laboratoire d’Enzymologie Moleculaire et Fonctionnelle, FRE 2852 CNRS-Paris 6, Institut Jacques Monod, Paris, France ´ ´ ´ Institut de Chimie Organique et Analytique, UMR CNRS 6005, FR 2708, Universite d’Orleans, UFR Sciences, Orleans, France ´ Unite de Biochimie Structurale, URA CNRS 2185, Institut Pasteur, Paris, France ´ Unite de Chimie Organique, URA CNRS 2128, Institut Pasteur, Paris, France ´ ` Plate-Forme 6- Cristallogenese et Diffraction des Rayons X, Institut Pasteur, Paris, France ´ ´ ´ Unite de Regulation Enzymatique des Activites Cellulaires, CNRS URA 2185, Institut Pasteur, Paris, France Keywords cidofovir; human UMP-CMP kinase; MABACDP; Mant-ATP; phosphonates Correspondence D Deville-Bonne, Laboratoire d’Enzymologie ´ Moleculaire et Fonctionnelle, FRE 2852 CNRS-Paris 6, Institut Jacques Monod, 4, place Jussieu, 75251 Paris Cedex 05, France Fax: +33 44 27 59 94 Tel: +33 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 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 X ¼ 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 developed 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 UMPCMP kinase similar to those of dUMP and dCMP 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 specificity of the enzyme acceptor-binding site is in agreement with a large ˚ substrate-binding pocket, as shown by the 2.1 A crystal structure (Received 12 February 2007, revised 25 May 2007, accepted 29 May 2007) doi:10.1111/j.1742-4658.2007.05902.x 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 Abbreviations Ap5A, P1-(5¢-adenosyl) P5-(5¢-adenosyl) pentaphosphate; Ap5dT, P1-(5¢-adenosyl) P5-[5¢-(2¢-deoxy-thymidyl)] pentaphosphate; Ap5G, P1-(5¢-adenosyl) P5-(5¢-guanosyl) pentaphosphate; Ap5U, P1-(5¢-adenosyl) P5-(5¢-uridyl) pentaphosphate; cidofovir, (S)-1-(3-hydroxy-2phosphonylmethoxypropyl) 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 D Topalis et al [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 phosphonates, a new class of antiviral analogs [7], 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 nucleoside 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 and of AMP kinases are found in the mitochondria and isoform 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, gemcitabine and 3¢thiancytidine, which are used to treat leukemia, pancreatic cancer and AIDS [11] In contrast, the dCMP acyclic phosphonate mimic, cidofovir [(S)-1-(3-hydroxyl-2-phosphonomethoxy-propyl) cytosine], 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 antihepatitis B virus agent, adefovir dipivoxil [9-(2-phosphono-methoxylethyl) adenine dipivoxil] and the anti-human immunodeficiency virus agent tenofovir disoproxil fumarate (9-[2-(R)-(phosphonomethoxy) propyl] adenine disoproxil fumarate) [7], once the protecting groups have been removed, are activated inefficiently by phosphorylation with cellular AMP kinases [13,14] The first phosphorylation of antiviral nucleoside phosphonates, catalyzed by NMP kinases, is probably a bottleneck in their activation We have looked UMP-CMP kinase and acyclic phosphonate analogs for new acyclic phosphonate derivatives that interact better with NMP kinases and are more readily phosphorylated 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 cidofovir analogs We used N-methyl anthraniloyl (Mant) nucleotides (Mant-ATP [15] and cytidine diphosphob-(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 fluorescent 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 and an equilibrium dissociation constant, KD, of 3.5 lm (results not shown) Mant-ATP binding was not modified by CMP, in contrast to Escherichia coli CMP kinase [17] Mant-ATP was displaced by ATP with a K ATP ¼ 10 lm in a competitive titration and in an D 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 (K Ap5U ¼ 0.15 lm) better D 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 KD value for P1-(5¢-adenosyl) P5-[5¢-(2¢-deoxy-thymidyl)] pentaphosphate (Ap5dT) (3.5 lm) was lower than that for ATP (10 lm), suggesting 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-phosphate of CDP through a butyl linker (Fig 2A) FEBS Journal 274 (2007) 3704–3714 ª 2007 The Authors Journal compilation ª 2007 FEBS 3705 UMP-CMP kinase and acyclic phosphonate analogs D Topalis et al Fig Fluorescence competition assays with Mant-ATP bound to the human UCK donor-binding site Mant-ATP (3 lM) + human UCK (10 lM), resulting in 50% fluorophore bound, was titrated with Ap5U (d), Ap5A (j), Ap5G (n), Ap5dT (s) and ATP (m) with IC50 ¼ lM, 4.4 lM, 11 lM, 25 lM and 50 lM, respectively (kexcitation ¼ 350 nm, kemission ¼ 436 nm, excitation slit ¼ nm, kemission slit ¼ nm) The values for the equilibrium constants KD calculated as in Experimental procedures are: 0.15 lM for Ap5U, 0.7 lM for Ap5A, 1.7 lM for Ap5G, 3.4 lM for Ap5dT, and 9.5 lM for ATP Dictyostelium UCK has been reported to specifically bind MABA-CDP at the CMP-binding site [16] Adding human UCK increased the fluorescence of MABACDP, 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 acceptorbinding 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 MABẦCDP ¼ (8.5 ± 1.0) lm D 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, indicating 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 CDP was better at displacing MABA-CDP than CMP, perhaps because the enzyme ⁄ MABA-CDP conformation favors the reverse reaction (CDP + ADP fi CMP + ATP) CDP, CMP, UMP, dCMP and dUMP all competed with MABA-CDP at the acceptor-binding site, showing that the same NMP site binds ribonucleosides and deoxyribonucleosides under our experimental conditions (5 mm magnesium ions) This does not fit the hypothesis that acceptor sites use either ribonucleoside 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 is in agreement with the preference for ribonucleotides rather than deoxyribonucleotides 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 acceptor-binding site with submillimolar KD, in agreement with the results in Fig 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 values in Table are in agreement with the kinetic parameters of the natural nucleoside monophosphates dCMP, dUMP and AMP [19] GMP and dTMP were not substrates of the enzyme, indicating that their binding 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 substantially weaker than that of natural substrates It was also a poor substrate for recombinant human UCK, with a low kcat (kcat ¼ 0.06 s)1, Km ¼ mm) resulting in a low catalytic efficiency, about 60 m–1Ỉs)1 These Fig (A) Formula of MABA-CDP used in the fluorescent competitive titration assay to determine dissociation constants of acyclic nucleotides (B) Formula of C5-substituted vinyl phosphonates (Y ¼ H, Cl, Br, phenyl, fluorophenyl, phenyl-S) 3706 FEBS Journal 274 (2007) 3704–3714 ª 2007 The Authors Journal compilation ª 2007 FEBS D Topalis et al UMP-CMP kinase and acyclic phosphonate analogs Fig 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 lM) was monitored after stepwise addition of human UCK (kexcitation ¼ 325 nm, kemission ¼ 430 nm, excitation slit ¼ nm, emission slit ¼ nm) The signal was fitted to the Langmuir binding equation with a fixed maximum enhancement of fluorescence of 160% The KD was 8.5 ± 1.5 lM Inset: Fluorescence emission spectra of MABA-CDP (10 lM) in T buffer (kexcitation ¼ 325 nm, excitation slit ¼ nm, emission slit ¼ nm) (a) MABA-CDP alone (b) MABA-CDP + 50 lM human UCK (c) MABA-CDP + 50 lM human UCK + mM CDP or CMP (B) Fluorescence competition assays with MABA-CDP bound to the human UCK acceptor-binding site MABA-CDP (8 lM) + human UCK (24 lM) titrated with CMP (d), dCMP (s), UMP (j), AMP (·) TMP (h) and cidofovir (m) Table Equilibrium dissociation constants and kinetic parameters of human UCK for natural NMP and cidofovir The KD 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 mM ATP and mM Mg2+ ND, not detectable Ligand KD (lM) ⁄ MABA-CDP Km (mM) kcat (s)1) kcat ⁄ Km (M)1Ỉs)1) CDP CMP dCMP UMP dUMP dTMP Cidofovir AMP GMP 10 60 90 400 750 300 100 500 – 0.07a 1.1a 0.13a 1.3 ± 0.3 ND 1.0 ± 0.3 > 5a ND 130a 75a 130a 8±1 ND 0.06 ± 0.02 ND ND 106a · 104a · 105a · 103 ND 60 103a ND a ± ± ± ± ± ± ± ± ± 10 10 50 150 100 30 100 Data from Pasti et al [19] properties 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 equilibrium 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 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 indicated that all the molecules except the 5-fluorophenyl derivative (compound 6e) competed with MABA-CDP (1 mm) The IC50 values were measured individually by fluorometric competition titration, and the dissociation constants (KD) were calculated (Table 2) The displacement was total for all compounds except for compound 6d (5-Phe-UVP) (50%), which was not suitable for the assay, due to its poor solubility The IC50 values 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 FEBS Journal 274 (2007) 3704–3714 ª 2007 The Authors Journal compilation ª 2007 FEBS 3707 UMP-CMP kinase and acyclic phosphonate analogs D Topalis et al Scheme Synthesis of novel acyclic phosphononucleoside analogs Reagents: (a) (i) benzenethiol (PhSH), N-chlorosuccinimide (NCS), pyridine, MeCN, reflux; (ii) crotyl bromide, K2CO3, dimethylformamide; (iii) K2CO3, MeOH; (b) Bu3SnH, AIBN, toluene, reflux; (c) N-bromosuccinimide (NBS) (or NCS), tetrahydrofan (THF) (for 4b and 4c, respectively) or aryliodide (RI), PdCl2(PPh3)2 (0.11 mmol), CuI, dimethylformamide, rt (for 4d–f); (d) diethyl vinyl phosphonate (4 eq.), Nolan’s catalyst ¼, CH2Cl2, reflux; (e) TMSBr (4 eq.), CH2Cl2 Table 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 determining the KD values from fluorescence competition assays with MABA-CDP bound to human UCK are reported in Fig 3B The IC50 values with human UCK were measured at 37 °C (substrate concentrations: 50 mM CMP, 0.5 mM ATP, and mM Mg2+) The inhibition constants Ki were measured as shown in Fig at 37 °C The substrate was here dCMP rather than CMP, as CMP is itself present at high concentrations [19] All the experiments were done at least two times, and standard deviations were about 20% ND, not determined Ligand Cidofovir 6a (UVP) 6b (5-Br-UVP) 6c (5-Cl-UVP) 6d (5-Phe-UVP) Fluorescent assay MABA-CDP Activity assay KD (lM) IC50 (lM) 300 400 50 20 150 amplitude 50% 6e (5-F-Phe-UVP) > 5000 6f (5-Phe-S-UVP) 100 Competitive inhibitors Ki (lM) ND ND 1200 700 35 17 30 16 120 – amplitude 50% 230 > mM 50 28 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-binding site Cidofovir binding was detected only in the MABA-CDP competition assay, a fact that was expected, 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 vinyl3708 Fig Inhibition of human UCK activity by 5-chloro-UVP (compound 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 nM (0.43 lgỈmL)1) The results are from a typical experiment repeated twice with the same results (15%): m, lM 5-Cl-UVP; j, lM 5-Cl-UVP; d, 50 lM 5-Cl-UVP phosphonates were clearly competitive inhibitors of dCMP [Fig for the 6c derivative (5-Cl-UVP) (Ki ¼ 16 ± lm] The dissociation constant for 5-Br-vinylphosphonate (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 substitution in the 5-position (Br, Cl) improved binding: UVP (compound 6a) inhibited the enzyme (Ki ¼ 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, FEBS Journal 274 (2007) 3704–3714 ª 2007 The Authors Journal compilation ª 2007 FEBS D Topalis et al showing that the Phe substitution was not accommodated in the binding site However, the presence of an F in compound 6e or a thiol (compound 6f) on the benzene 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–1Ỉs)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 positioning 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 substrate-binding specificity Structural analysis of human UCK The structure of human UCK was determined using single-wavelength anomalous diffraction of the selenomethione-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– UMP-CMP kinase and acyclic phosphonate analogs 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 disordered 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 binding 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] Several human kinases, such as dTMP kinase, deoxyguanosine 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 (Rs ¼ 2.2 nm), but the Stokes radius is 2.8 nm in 0.2 m KCl, corresponding Fig (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 (cyan; 1Z83) and (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 FEBS Journal 274 (2007) 3704–3714 ª 2007 The Authors Journal compilation ª 2007 FEBS 3709 UMP-CMP kinase and acyclic phosphonate analogs D Topalis et al A B R151 R140 R140 D142 R134 R151 D142 K61 R134 K61 R39 Mg2+ R39 Mg2+ V63 E36 R96 E36 R96 V63 H2O H2O N100 N100 T68 T68 Fig 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) to a molecular mass of about 35 kDa for a globular protein [19] Thus, the enzyme might form homodimers in solution, and these may be stabilized in the crystal form, so precluding substrate binding Such inactive dimers presumably occur in high-salt conditions and perhaps also in concentrated protein solutions 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 probably 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 interactions The size of the acyclic moiety could be important: 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], indicating indirectly that human NMP kinases can phosphorylate them in the cell 3710 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 fluorescence 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 derivatives with cellular targets The binding of dTMP and 5-halogenated acyclic derivatives to human UCK indicates 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, FEBS Journal 274 (2007) 3704–3714 ª 2007 The Authors Journal compilation ª 2007 FEBS D Topalis et al Germany) Cidofovir was a gift from J Neyts (Rega Institute, 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 phosphononucleosides (compounds 6a–f) is outlined in Scheme The 5-phenylthio derivative (compound 2) was prepared from compound 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 Compound was converted to the 5-bromo (compound 4b) and chloro (compound 4c) derivatives by simple treatment with, respectively, N-bromosuccinimide and N-chlorosuccinimide Several phenyl derivatives (compounds 4d–f) were obtained from compound via the Pd-catalyzed Stillecoupling 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 CH2Cl2 for 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, unpublished 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, Germany) transformed with the pDIA17 expression plasmid, and purified in one step on an Ni–nitrilotriacetic acid column (Qiagen, Courtabeuf, France) using a linear gradient of imidazole (10–250 mm) at pH The purified protein was equilibrated by dialysis against 20 mm Tris ⁄ HCl (pH 7.5) buffer containing 20 mm NaCl, mm dithiothreitol and 50% glycerol The selenomethionine-labeled protein was obtained from Bli5 E coli cells transformed with the pET28a-huck plasmid and grown overnight in LB medium supplemented with 30 lgỈmL)1 kanamycin and 70 lgỈmL)1 chloramphenicol at 37 °C An aliquot of the culture (3 mL) was centrifuged (1 at 6000 g at °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 D600 of 0.6, 50 mg each of lysine, threonine and phenylalanine and 25 mg each of UMP-CMP kinase and acyclic phosphonate analogs leucine, isoleucine, valine and selenomethionine was added to the culture, and incubation was continued for 40 The temperature was then lowered to 20 °C, and 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 at 6000 g at °C), and suspended in lysis buffer containing mm dithiothreitol and EDTA-free protease inhibitors (Roche, Meylan, France) The protein was purified as previously 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 20 °C in T buffer (50 mm Tris ⁄ HCl, pH 7.5, containing mm MgCl2, 50 mm KCl, 5% glycerol and 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 MABACDP (2 lm) (kexcitation ¼ 350 nm, kemission ¼ 430 nm, nm excitation slit and nm emission slit) The fluorescent signal 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 MC constant, KD As the fluorescence enhancement is directly proportional to binding, the observed fluorescence signal Fobs ¼ (Fmax ) F0)A + F0, where A is the molar fraction of bound MC (A ¼ [MC.E] ⁄ [MC]t), Fo is the initial fluorescence before adding the protein, and Fmax is the fluorescence after saturation by the protein The concentration of the complex [MC.E] is given by Eqn (1): ½MC.EŠ ẳ KD ỵ ẵMCt ỵ ẵEt q KD ỵ ẵMCt ỵ ẵEt 4ẵMCt ẵEt 1ị 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 competitive experiments The mL cell contained MABA-CDP (8 lm) and human UCK (24 lm) corresponding to, respectively, 1KD and 3KD, and resulting in the half-saturation of the enzyme at the start of the experiment [25] The fluorescence 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 FEBS Journal 274 (2007) 3704–3714 ª 2007 The Authors Journal compilation ª 2007 FEBS 3711 UMP-CMP kinase and acyclic phosphonate analogs D Topalis et al dissociation constants KD for the competitor and K MC for D MABA-CDP using Eqn (2) [26,27]: MC MC KD ẳ IC50 ỵ KD B=ẵAP ỵ BP A ỵ B KD ị 2ị 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, Meylan, France) Similar measurements were done for the interaction of Mant-ATP with human UCK (kexcitation ¼ 350 nm; kemission ¼ 436 nm, excitation slit ¼ nm, emission slit ¼ nm) Enzymatic activity measurements Structural models The catalytic activity of the NMP kinases was determined in a spectrophotometer by measuring ADP formation [28] The assay mix contained 50 mm Tris ⁄ HCl (pH 7.4), 50 mm KCl, mm MgCl2, mm ATP, 0.2 mm NADH, 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, mm MgCl2, mm KCl, mm dithiothreitol and 10% glycerol) The reaction at 37 °C 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 monophosphorylated cidofovir with pyruvate kinase may have caused a slight overestimation of the rates It was considered to be negligible during the less than 10 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 mgỈmL)1) in 50 mm Tris ⁄ HCl (pH 7.5), 10 mm dithiothreitol, 20 mm NaCl, 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 a ¼ b ¼ 62.1 A, c ¼ 222.5 A The Se-methionine-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- 3712 tion methods from a single crystal of SeMet-labeled protein, using the programs shake’n’bake [31] and sharp [32] Crystallographic 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 ⁄ Rfree of 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 cocrystallization 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 Docking of 3-Cl-UVP and cidofovir was performed using arguslab software [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, Michele Reboud (FRE 2852 CNRS-Univer´ ´ site Paris 6) and Michel Veron (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 References Eriksson S, Munch-Petersen B, Johansson K & Eklund H (2002) Structure and function of cellular deoxyribonucleoside kinases Cell Mol Life Sci 59, 1327–1346 FEBS Journal 274 (2007) 3704–3714 ª 2007 The Authors Journal compilation ª 2007 FEBS D Topalis et al Van Rompay AR, Johansson M & Karlsson A (2000) Substrate specificity and phosphorylation of nucleosides and nucleoside analogs by mammalian nucleoside monophosphate kinases Pharmacol Ther 87, 189–198 Schneider B, Xu YW, Sellam O, Sarfati R, Janin J, Veron M & Deville-Bonne D (1998) Pre-steady state of reaction of nucleoside diphosphate kinase with anti-HIV nucleotides J Biol Chem 273, 11491–11497 Krishnan P, Fu Q, Lam W, Liou JC, Dutschman GE & Cheng Y-C (2002) Phosphorylation of pyrimidine deoxynucleoside analog diphosphates: selective phosphorylation of 1-nucleoside analog diphosphates by 3-phosphoglycerate kinase J Biol Chem 277, 5453–5459 Gallois-Montbrun S, Faraj A, Seclaman E, Sommadossi J-P, Deville-Bonne D & Veron M (2004) Broad specificity of human phosphoglycerate kinase for antiviral nucleoside analogs Biochem Pharmacol 68, 1749–1756 Cihlar T & Chen MS (1996) Identification of enzymes catalyzing two-step phosphorylation of cidofovir and the effect of cytomegalovirus infection on their activities in host cells Mol Pharmacol 50, 1502–1510 De Clercq E & Holy A (2005) Acyclic nucleoside phosphonates: a key class of antiviral drugs Nat Rev Drug Disc 4, 928–940 Ferraro P, Nicolosi L, Bernardi P, Reichard P & Bianchi V (2006) Mitochondrial deoxynucleotide pool sizes in mouse liver and evidence for a transport mechanism for thymidine monophosphate Proc Natl Acad Sci USA 103, 18586–18591 Segura-Pena D, Sekulic N, Ort S, Konrad M & Lavie A (2004) Substrate-induced conformational changes in human UMP ⁄ CMP kinase J Biol Chem 32, 33882– 33889 10 Scheffzek K, Kliche W, Wiesmuller L & Reinstein J (1996) Crystal structure of the complex of UMP ⁄ CMP kinase from Dictyostelium discoideum and the bisubstrate inhibitor P1-(5¢-adenosyl) P5-(5¢-uridyl) penta˚ phosphate (UP5A) and Mg2+ at 2.2 A: implications for water-mediated specificity Biochemistry 35, 9716– 9727 11 Van Rompay AR, Johansson M & Karlsson A (1999) Phosphorylation of deoxycytidine analog monophosphates by UMP-CMP kinase: molecular characterisation of the human enzyme Mol Pharmacol 56, 562–569 12 Kern E, Hartline C, Harden E, Keith K, Rodriguez N, Beadle J & Hostetler K (2002) Enhanced inhibition of orthopoxvirus replication in vitro by alkoxyalkyl esters of codifovir and cyclic cidofovir Antimicrob Agents Chemother 46, 991–995 13 Robbins BL, Greenhaw J, Connelly MC & Fridland A (1995) Metabolic pathways for activation of the antiviral Antimicrob Agents Chemother 39, 2304–2308 14 Votruba I, Bernaerts R, Sakuma T, De Clercq E, Merta A, Rosenberg I & Holy A (1987) Intracellular phosphorylation of broad-spectrum anti-DNA virus agent UMP-CMP kinase and acyclic phosphonate analogs 15 16 17 18 19 20 21 22 23 24 25 26 27 (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl) adenine and inhibition of viral DNA synthesis Mol Pharmacol 32, 524–529 Hiratsuka T (1984) Affinity labeling of the myosin ATPase with ribose-modified fluorescent nucleotides and vanadate J Biochem (Tokyo) 96, 147–154 Rudolph MG, Veit TJH & Reinstein J (1999) The novel fluorescent CDP-analogue (Pb)MABA-CDP is a specific probe for the NMP binding site of UMP-CMP kinase Prot Sci 8, 2697–2704 Bucurenci N, Sakamoto H, Briozzo P, Palibroda N, Serina L, Sarfati RS, Labesse G, Danchin A, Barzu O & Gilles A-M (1996) CMP kinase from Escherichia coli is structurally related to other nucleoside monophosphate kinases J Biol Chem 271, 2856–2862 Hsu CH, Liou JC, Dutschman GE & Cheng YC (2005) Phosphorylation of cytidine, deoxycytidine and their analog monophosphates by human UMP-CMP kinase is differentially regulated by ATP and magnesium Mol Pharmacol 67, 806–814 Pasti C, Gallois-Montbrun S, Munier-Lehmann H, Veron M, Gilles A-M & Deville-Bonne D (2003) Reaction of human UMP-CMP kinase with natural and analog substrates Eur J Biochem 270, 1784–1790 Amblard F, Nolan S & Agrofoglio L (2005) Metathesis strategy in nucleoside chemistry Tetrahedron 61, 7067– 7080 Sabini E, Ort S, Monnerjahn C, Konrad M & Lavie A (2003) Structure of human dCK suggests strategies to improve anticancer and antiviral therapy Nat Struct Biol 10, 513–519 Choo H, Beadle J, Kern E, Prichard M, Keith KA, Hartline C, Trahan J, Aldern K, Korba B & Hostetler K (2006) Antiviral activity of novel 5-phosphono-pent-2-en-1-yl nucleosides and their alkoxyalkyl phosphonoesters Antimicrob Agents Chemother 51, 611–615 Topalis D, Collinet B, Gasse C, Dugue L, Balzarini J, Pochet S & Deville-Bonne D (2005) Substrate specificity of vaccinia virus thymidylate kinase FEBS J 272, 6254– 6265 Agrofoglio LA, Gillaizeau I & Saito Y (2003) Palladium-assisted routes to nucleosides J Med Chem 103, 1875–1916 Dandliker WB, Hsu M-L, Levin J & Rao BR (1981) Equilibrium and kinetic inhibition assays based upon fluorescence polarisation Methods Enzymol 74, 3–28 Kenakin T (1993) Pharmacologic analysis of drug ⁄ receptor interaction pp 385–410, 2nd edn Raven Press, New York, NY Chen Y, Gallois-Montbrun S, Schneider B, Veron M, Morera S, Deville-Bonne D & Janin J (2003) Nucleotide binding to nucleoside diphosphate kinases: X-ray structure of human NDPK-A in complex with ADP FEBS Journal 274 (2007) 3704–3714 ª 2007 The Authors Journal compilation ª 2007 FEBS 3713 UMP-CMP kinase and acyclic phosphonate analogs 28 29 30 31 32 D Topalis et al and comparison to protein kinases J Mol Biol 332, 915–926 Blondin C, Serina L, Wiesmuller L, Gilles A-M & Barzu O (1994) Improved spectrophotometric assay of nucleoside monophosphate kinase activity using the pyruvate kinase ⁄ lactate dehydrogenase coupling system Anal Biochem 220, 219–221 Windfield PT (2000) Production of recombinant proteins Current Protocols Protein Sci 1, 5.3.9–53.14 CCP4 (1994) The CCP4 suite: programs for protein crystallography Acta Crystallogr D50, 760–763 Weeks CM & Miller R (1999) Optimizing Shake-andBake for proteins Acta Crystallogr D Biol Crystallogr 55, 492–500 Bricogne G, Vonrhein C, Flensburg C, Schiltz M & Paciorek W (2003) Generation, representation and flow of phase information in structure determination: recent 3714 33 34 35 36 37 developments in and around SHARP 2.0 Acta Crystallogr D 59, 2023–2030 Jones TA, Zou JY, Cowan SW & Kjeldgaard M (1991) Improved methods for building protein models in electron density maps and the location of errors in these models Acta Crystallogr A 47, 110–119 Murshudov GN, Vagin AA, Lebedev A, Wilson KS & Dodson EJ (1999) Efficient anisotropic refinement of macromolecular structures using FFT Acta Crystallogr D 55, 247–255 Perrakis A, Morris R & Lamzin VS (1999) Automated protein model building combined with iterative structure refinement Nat Struct Biol 6, 458–463 Thompson M (2004) ArgusLab Planaria Software LLC, Seattle, WA DeLano WL (2002) The PyMOL Molecular Graphics System DeLano Scientific, Palo Alto, CA FEBS Journal 274 (2007) 3704–3714 ª 2007 The Authors Journal compilation ª 2007 FEBS ... 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,... 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... 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,