F-ara-AMP is a substrate of cytoplasmic 5′-nucleotidase II (cN-II): HPLC and NMR studies of enzymatic dephosphorylation

Article abstract of DOI:10.1080/15257770500458027

Intracellular accumulation of triphosphorylated derivatives is essential for the cytotoxic activity of nucleoside analogues. Different mechanisms opposing this accumulation have been described. We have investigated the dephosphorylation of monophosphorylated fludarabine (F-ara-AMP) by the purified cytoplasmic 5′-nucleotidase cN-II using HPLC and NMR. These studies clearly showed that cN-II was able to convert F-ara-AMP into its non phosphorylated form, F-ara-A, with a K m in the millimolar range and V max = 35 nmol/min/mg, with both methods. Cytoplasmic 5′-nucleotidase cN-II can degrade this clinically useful cytotoxic nucleoside analogue and its overexpression is thus likely to be involved in resistance to this compound. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770500458027

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F-ara-AMP is a substrate of cytoplasmic
5′-nucleotidase II (cN-II): HPLC and NMR
studies of enzymatic dephosphorylation
Lars P. Jordheim a , Emeline Cros a , Carlos M. Galmarini a , Charles
Dumontet a , Anne-Sophie Bretonnet b , Isabelle Krimm b , Jean-Marc
Lancelin b & Marie-Claude Gagnieu c
a Laboratoire de Cytologie Analytique, Faculté de Médecine
Rockefeller, INSERM U590, Université Claude Bernard Lyon I, Lyon,
France
b Laboratoire de RMN Biomoléculaire, Université Claude
Bernard—Lyon I, ESCPE Lyon, Villeurbanne, France
c UF de Pharmacologie Spécialisée, Fédération de Biochimie, Hôpital
Edouard Herriot, Lyon Cedex, France
Available online: 23 Aug 2006
To cite this article: Lars P. Jordheim, Emeline Cros, Carlos M. Galmarini, Charles Dumontet, Anne-
Sophie Bretonnet, Isabelle Krimm, Jean-Marc Lancelin & Marie-Claude Gagnieu (2006): F-ara-
AMP is a substrate of cytoplasmic 5′-nucleotidase II (cN-II): HPLC and NMR studies of enzymatic
dephosphorylation, Nucleosides, Nucleotides and Nucleic Acids, 25:3, 289-297
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Nucleosides, Nucleotides, and Nucleic Acids, 25:289–297, 2006
Copyright ꢀ Taylor & Francis Group, LLC
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ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770500458027
F-ara-AMP IS A SUBSTRATE OF CYTOPLASMIC
5ꢀ-NUCLEOTIDASE II (cN-II): HPLC AND NMR STUDIES
OF ENZYMATIC DEPHOSPHORYLATION
Lars P. Jordheim, Emeline Cros, Carlos M. Galmarini,
and Charles Dumontet
INSERM U590, Laboratoire de Cytologie Analytique,
2
Faculte´ de Me´decine Rockefeller, Universite´ Claude Bernard Lyon I, Lyon, France
Anne-Sophie Bretonnet, Isabelle Krimm, and Jean-Marc Lancelin
2
Laboratoire de RMN Biomole´culaire, Universite´ Claude Bernard—Lyon I, ESCPE Lyon,
Villeurbanne, France
Marie-Claude Gagnieu
UF de Pharmacologie Spe´cialise´e, Fe´de´ration de Biochimie,
2
Hoˆpital Edouard Herriot, Lyon Cedex, France
Intracellular accumulation of triphosphorylated derivatives is essential for the cytotoxic activity
2
of nucleoside analogues. Different mechanisms opposing this accumulation have been described.
We have investigated the dephosphorylation of monophosphorylated fludarabine (F-ara-AMP) by the
purified cytoplasmic 5 ꢁ-nucleotidase cN-II using HPLC and NMR. These studies clearly showed that
cN-II was able to convert F-ara-AMP into its non phosphorylated form, F-ara-A, with a Km in the
millimolar range and Vmax = 35 nmol/min/mg, with both methods. Cytoplasmic 5 ꢁ-nucleotidase
cN-II can degrade this clinically useful cytotoxic nucleoside analogue and its overexpression is thus
likely to be involved in resistance to this compound.
Keywords 5ꢁ-Nucleotidase; Fludarabine; cN-II; HPLC; NMR; Resistance
INTRODUCTION
Fludarabine (9-β-D-arabinosyl-2-fluoroadenine monophosphate; F-ara-
AMP) is an active agent for the treatment of chronic lymphocytic leukemia
(CLL). Although fludarabine monotherapy produces responses in 63–79%
Received 5 April 2005; accepted 1 August 2005.
L.P.J. and A.S.B. contributed equally to this work.
This study was supported by Mja˚lands Stiftelse for Kreftforskning and Stiftelsen Thomas Fearnley,
Heddy og Nils Astrup. L.P.J. is a recipient of a doctoral grant from the Association de Recherche sur le
Cancer. A.S.B. is a recipient of a doctoral grant from the Re´gion Rhoˆne-Alpes, France.
Address correspondence to Lars P. Jordheim, INSERM U590, Laboratoire de Cytologie Analy-
tique, Faculte´ de Me´decine Rockefeller, Universite´ Claude Bernard Lyon I, 69008 Lyon, France. E-mail:
jordheim@yahoo.com
289

290
L. P. Jordheim et al.
of untreated patients and 32–48% of pretreated patients, clinical resistance
exists and limits the efficacy of this agent.[1] Different hypothesis are studied
as the mechanism to fludarabine resistance as well as to other clinically used
nucleoside analogues.[2]
Several studies have shown an implication of the cytoplasmic IMP-
selective 5ꢁ-nucleotidase (cN-II) in the in vitro and in vivo resistance to nucleo-
side analogues.[3−6] A weak dephosphorylation of the monophosphorylated
derivative of cladribine by purified cN-II was observed in vitro.[7] In addi-
tion, cN-II interacts with the antiviral nucleoside analogues acyclovir and
carbovir.[8,9] However, whether cN-II dephosphorylates fludarabine induc-
ing thereby drug resistance to this agent is not known. Given the widespread
use of fludarabine in CLL and the preference of cN-II towards purine deriva-
tives, we studied the possible enzymatic conversion of F-ara-AMP into F-ara-A
by cN-II by using two non radioactive methods, one based on high perfor-
mance liquid chromatography (HPLC) and one based on nuclear magnetic
resonance (NMR). We show that cN-II is able to convert F-ara-AMP into its
nonphosphorylated form 9-β-D-arabinofuranosyl-2-fluoro-Adenine (F-ara-A)
and is thus likely to be involved in resistance to the cytotoxic effect of this
drug.
MATERIALS AND METHODS
Reagents
F-ara-AMP (fludarabine, FludaraꢀR) was from Schering Laboratories (Lys-
les-Lannoy, France). F-ara-A, NaCl, Tris, KCl, ethylene-bis (2-aminoethyl-
ether)-N,N,Nꢁ,Nꢁ-tetraacetic acid (EGTA), phenylmethanesulfonyl fluoride
(PMSF), MgCl2, 2-mercaptoethanol, lysozyme, DTT, D2O, and imidazole
were purchased from Sigma Aldrich (Saint-Quentin, France) and isopropyl
β-D-l-thiogalactopyranoside (IPTG) and LB Broth base from Invitrogen
(Cergy Pontoise, France).
Purification of cN-II
The pET24b vector containing the C-terminal His-tagged and N-terminal
deleted (30 amino acids) cN-II (cN-II-ꢀN) was a generous gift from Dr.
J. Spychala (Chapel Hill, North Carolina). Bacterial expression and pro-
tein purification were performed as previously described with only minor
modifications.[10] The fraction containing cN-II-ꢀN was analyzed for purity
by silver staining of a polyacrylamide gel according to the manufacturer’s
instructions (Amersham, Orsay, France) and protein concentration was de-
termined using the Bradford assay (Pierce, Montluc¸on, France).

cN-II Dephosphorylates Fludarabine Monophosphate
Sample Preparation for HPLC Assays
291
For HPLC studies, purified enzyme (75 ng/µL) was incubated for 8 h at
37◦C in a final volume of 15 µL containing 10 mM MgCl2, 500 mM NaCl,
50 mM imidazole pH 6.5, and 0.2–3.5 mM F-ara-AMP. The pH of this solution
was 6.7 at 25◦C.
Sample Preparation for NMR Assays
For the kinetic studies, F-ara-AMP was introduced in NMR tubes at a
final concentration of 0.5 to 7.0 mM in solutions containing purified enzyme
(500 ng/µL), 10 mM MgCl2, 500 mM NaCl, 130 mM imidazole, 7.7 mM Tris,
1 mM DTT, and 5% D2O. The pH of the solution was 8.3 at 25◦C (without
correction for D2O). Prior to F-ara-AMP addition, each sample was incubated
at 37◦C 0.1◦C for 10–15 min. NMR acquisitions were started immediately
after addition of F-ara-AMP.
HPLC Experiments
Identification of F-ara-A and F-ara-AMP and quantification of F-ara-A
were performed using a validated HPLC method with diode array UV de-
tection (Thermo electron chromatogram system). The column used was
a C18 Symmetry Shield column (5 µm particle size, 250 × 4.6 mm, Wa-
ters). The mobile phase was constituted of acetonitrile and a potassium di-
hydrogenophosphate (50 mM) buffer with octane sulfonic acid (250 mM)
adjusted to pH 7.0 (5:95, v/v). The separation of compounds was achieved
at 30◦C with an isocratic solvent delivery system at a flow rate of 1.0 mL/min.
Detection was performed at 272 nm. Chromatographic peak purity and iden-
tification of compounds were verified using the spectral analysis function of
Chromquest software (Thermo electron).
NMR Experiments
NMR spectra were acquired on a Bruker Avance DRX500 spectrometer,
1
equipped with a 5 mm H/13C/15N triple resonance probe head with
1
z-field gradients. F-ara-AMP H assignments were verified from 1D-proton,
homonuclear multiple bond coherence (HMBC) and phase-sensitive
double quantum filtered correlated spectroscopy (DQF-COSY) experiments
1
performed at 5◦C on a 7.0 mM F-ara-AMP solution. The H chemical
shifts were calibrated using the water signal as a reference (4.96 ppm
at 5◦C). 1D-proton experiments were acquired over a sweep width of
6,000 Hz into 6k complex datapoints. The acquisition time was 1 s and the
relaxation delay was 2.6 s. For water suppression a W5 scheme was used with
WATERGATE pulse-train delays of 400 µs.[11] The free induction decays
were weighted by an exponential function (line broadening of 3 Hz) prior

292
L. P. Jordheim et al.
to Fourier transformation. Gradient-selected HMBC[12] were acquired with
sweep-widths of 16,000 × 4,500 Hz and 64 × 1024 complex points, using
an 80 ms delay for the evolution of long-range 13C-1H couplings. Phase-
sensitive DQF-COSY spectra[13,14] were acquired with a water-suppression
sequence using excitation sculpting.[15] For this purpose, a 2,900 µs, 180◦
rectangular-shaped pulse was employed. The spectra were acquired over
a 4,500 Hz sweep-width in both dimensions, using 128 × 1,024 complex
points. Typical 2D-spectra were recorded with a 0.23 s acquisition time and
a 2 s relaxation delay. For kinetic studies, 128 scan 1D-proton experiments
1
were acquired as described above at 37.0◦C. The H chemical shifts were
recalibrated using the water signal as a reference (4.66 ppm at 37◦C).
The data were processed and analysed using XWINNMR (Bruker Analyti
GmgH).
Calculation of Reaction Rates and Kinetics Parameters
from HPLC Data
The concentration of F-ara-A formed was calculated using the height
of the corresponding peak for each initial F-ara-AMP concentration. The
velocity of F-ara-A production was calculated from F-ara-A concentrations
after 8-h incubations (F-ara-A production was linear over this timespan).
Results are given as a mean value of triplicate experiments. Km and Vmax
values were derived from Lineweaver-Burk plots obtained with MicrosoftꢀR
Excel.
Calculation of Reaction Rates and Kinetic Parameters
from NMR Data
Initial rates of F-ara-A production were derived from the increase
rates of a characteristic peak integral corresponding to the H8 proton
in F-ara-A. The increase of this peak was followed by 8-min steps over
the first 80 min of the reaction, and a linear equation was fitted to the
data (MicrosoftꢀR Excel). Integral values for the H8 peaks were correlated
to concentration values, using a reference sample of 2.50 mM F-ara-
AMP under the same conditions as for the other studies. Background
F-ara-A production was assessed from a control experiment using a
3.5 mM F-ara-AMP sample, incubated at 37.0◦C during 80 min without
cN-II-ꢀN.
Kinetic parameters were derived from the initial rates of F-ara-A pro-
duction, at different concentrations of substrate, using the Lineweaver-Burk
representation. All experiments were duplicated. Standard errors were esti-
mated from experimental conditions.

cN-II Dephosphorylates Fludarabine Monophosphate
293
RESULTS
Purification of cN-II-∆N
We obtained approximately 5 mg of purified cN-II-ꢀN from 1 l of bacteria
culture using the purification method described in the Methods section.
Silver staining of SDS-PAGE showed that the protein fraction obtained was
of high purity (data not shown). The identity of the protein was verified using
a monoclonal antibody against cN-II (generated in our laboratory, data not
shown). The purified protein was stable for over 3 months when stored at
4◦C.
HPLC Study of F-ara-AMP Dephosphosphorylation by cN-II-∆N
As shown in Figure 1, the HPLC method described above was useful
to study the dephosphorylation of F-ara-AMP into the non-phosphorylated
derivative F-ara-A in presence of purified cN-II-ꢀN. Under these conditions,
retention times were 5.6 and 14.9 min for F-ara-AMP and F-ara-A, respectively.
No significant degradation of F-ara-AMP into F-ara-A was observed in the
absence of cN-II-ꢀN.
Based on a kinetic study, further experiments were performed using 8-h
incubations of different concentrations of F-ara-AMP and purified cN-II-ꢀN.
The Lineweaver-Burk plot obtained with the HPLC experiments showed a Km
of 4 mM and a Vmax of 35 nmol/min/mg for F-ara-AMP dephosphorylation
by cN-II-ꢀN (Figure 2).
NMR Study of F-ara-AMP Dephosphosphorylation by cN-II-∆N
F-ara-AMP proton chemical shifts were assigned from the experiments
mentioned in the Methods section. At 5◦C, the 1H resonance assignment is
the following: H1ꢁ, 6.24 ppm (d); H2ꢁ, 4.55 ppm (t); H3ꢁ, 4.42 ppm (t); H4ꢁ,
4.10 ppm (m); H5ꢁ and H5ꢁꢁ, 4.10 and 4.03 ppm (m); H8, 8.49 ppm (s).
FIGURE 1 HPLC analysis of dephosphorylation of F-ara-AMP to F-ara-A. F-ara-AMP (3.5 mM) was incu-
bated without (A) or with (B) cN-II-ꢀN (75 ng/µL) at 37◦C for 8 h.

294
L. P. Jordheim et al.
FIGURE 2 Lineweaver-Burk plot for the conversion of F-ara-AMP to F-ara-A by cN-II-ꢀN with different
initial concentrations of F-ara-AMP using HPLC detection. The figure shows the velocity of the conversion
of F-ara-AMP into F-ara-A by cN-II-ꢀN using 75 ng/µL cN-II-ꢀN at 37◦C for 8 h. The figure shows the
mean of 3 separate experiments with standard errors. Determination coefficient = 0.99.
For the kinetic study, 31P NMR experiments could have been used to over-
come problems arising from the buffer conditions (water and imidazole).
However, 31P NMR is inherently less sensitive than 1H-NMR and quantitative
analysis by 31P NMR was not as reliable as by 1H-NMR, in the concentration
and time conditions of our experiments. Therefore, 1H-NMR was used to fol-
low the reaction with maximum sensitivity. The H8 NMR signals in F-ara-AMP
and F-ara-A appeared as sensitive probes to follow the kinetic reaction. The
1
H8 peaks are well resolved and give sharp singlets in the H-NMR spectra
(Figure 3). Moreover, the H8 chemical shifts difference between the sub-
strate and the product is −0.18 ppm, a value large enough to allow good sig-
nal separation and detection. It was then necessary to work at pH 8.3 because
FIGURE 3 Evolution of F-ara-AMP (o) and F-ara-A (∗) H8 NMR peaks over time. In this example, the
initial F-ara-AMP concentration was 3.8 mM.

cN-II Dephosphorylates Fludarabine Monophosphate
295
FIGURE 4 Lineweaver-Burk plot for the conversion of F-ara-AMP to F-ara-A by cN-II-ꢀN with different
initial concentrations of F-ara-AMP using NMR detection. The figure shows the velocity of the conversion
of F-ara-AMP into F-ara-A by cN-II-ꢀN using 500 ng/µL cN-II-ꢀN at 37◦C. The figure simultaneously
shows two separate experiments. Determination coefficient = 0.97.
at this pH, the H8 peaks were not hidden by imidazole resonances as is the
case at pH 6.5, owing to a strong pH-dependent shift. Under these condi-
tions, during the reaction course in the presence of 0.5 µg/µL cN-II-ꢀN
and at the time points and substrate concentrations studied, the H8 F-ara-A
peak increased linearly and the H8 F-ara-AMP peak decreased concomitantly
(Figure 3). An 80-min control experiment using a 3.5 mM F-ara-AMP sample
without cN-II-ꢀN evidenced neither F-ara-AMP degradation nor F-ara-A pro-
duction. Following the F-ara-A-H8 peak intensity increase by 8-min steps over
the first 80 min of the reaction, kinetic parameters were derived as described
in the Methods section. A Km of 10 4 mM was obtained for the system cN-II-
ꢀN/F-ara-AMP (Figure 4). The Vmax of the F-ara-AMP to F-ara-A conversion
was 34 9 nmol/min/mg cN-II-ꢀN.
DISCUSSION
Fludarabine is a widely used nucleoside analogue in the treatment
of hematological malignancies such as refractory chronic lymphocytic
leukemia. Because of low solubility, this compound is administered as a
monophosphorylated derivative that is dephosphorylated by the ectoplas-
mic 5ꢁ-nucleotidase eN (CD73) to form F-ara-A. This latter compound enters
the cell by the mean of nucleoside transporters, and is phosphorylated in-
tracellularly by deoxycytidine kinase (dCK) and other kinases to form the
active triphosphorylated form of the drug, F-ara-ATP. Conversely, the first
phosphorylation step is likely to be reversed by the activity of cytoplasmic
5ꢁ-nucleotidases such as cN-II. Because of the preference of cN-II for purine
analogues, we studied the dephosphorylation of F-ara-AMP by this enzyme.
Using two different methods we show that the cytoplasmic 5ꢁ-nucleotidase
cN-II is able to catalyze the conversion of F-ara-AMP to F-ara-A. The Vmax
values derived from HPLC and NMR methods were statistically identical and

296
L. P. Jordheim et al.
the Km were reasonably similar. The small differences observed in Km may be
attributed to the methods used or to the differences in buffer conditions.
The enzymatic characteristics found in this study (Km = 4–10 mM and
Vmax = 34–35 nmol/min/mg) should be compared to those obtained during
similar studies with related compounds. Inhibition studies of purified cN-II
from rat liver with 5ꢁ-deoxy-5ꢁ-isobuthylthio derivatives of adenosine and
inosine, showed K in the same order of magnitude as the Km of F-ara-AMP
i
found in our study.[16] Skladanowski et al. determined kinetic parameters
for different nucleotides using purified cN-II from rabbit heart.[17] Their
results (AMP: Km = 18.4 mM, Vmax = 22.6 µmol/min/mg protein, IMP:
Km = 2.8 mM, Vmax = 50.2 µmol/min/mg protein, GMP: Km = 7.2 mM,
Vmax = 82.5 µmol/min/mg protein, XMP: Km = 5.3 mM, Vmax = 37.1
µmol/min/mg protein) suggest Km constants in the same millimolar range
as for F-ara-AMP with our protein preparation but the maximum velocity
in our case is 1,000-fold slower than what was observed with rabbit heart
cN-II. Similarly, Spychala et al. found a Vmax of 21.2 µmol/min/mg for the
dephosphorylation of IMP, the preferred substrate of cN-II, using the same
protein as in this study.[10] The dephosphorylation of F-ara-AMP described
here is about 1,000-fold slower than that observed with IMP. Nevertheless,
cN-II in our protein preparation is able to dephosphorylate F-ara-AMP.
Whether the dephosphorylation of F-ara-AMP by cN-II would still occur in
a cellular environment remains uncertain. If such an enzymatic reaction
was possible, cN-II could be involved in the resistance to fludarabine.
The NMR method we used is well suited for the study of interactions
between our purified protein and substrates or molecules developed to
be enzyme inhibitors. Further studies will show whether such molecules
have inhibitory effects on the dephosphorylating reaction of F-ara-AMP by
cN-II.
In conclusion, we show for the first time that the cytoplasmic 5ꢁ-
nucleotidase cN-II is able to dephosphorylate a clinically cytotoxic nucleo-
side analogue. It is therefore possible that this enzyme is involved in clinical
resistance to fludarabine in patients with chronic lymphocytic leukemia.
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