Báo cáo khoa học: 9-Deazaguanine derivatives connected by a linker to difluoromethylene phosphonic acid are slow-binding picomolar inhibitors of trimeric purine nucleoside phosphorylase potx

9-Deazaguanine derivatives connected by a linker to
difluoromethylene phosphonic acid are slow-binding
picomolar inhibitors of trimeric purine nucleoside
phosphorylase
Katarzyna Breer
1
, Ljubica Glavas
ˇ
-Obrovac
2
, Mirjana Suver
2
, Sadao Hikishima
3
, Mariko Hashimoto
3
,
Tsutomu Yokomatsu
3
, Beata Wielgus-Kutrowska
1
, Lucyna Magnowska
1
and Agnieszka Bzowska
1
1 Department of Biophysics, Institute of Experimental Physics, Warsaw University, Poland
2 University Hospital Osijek and School of Medicine, University of J. J. Strossmayer in Osijek, Croatia
3 School of Pharmacy, Tokyo University of Pharmacy and Life Science, Japan
Keywords
9-deazaguanine; multisubstrate analogue
inhibitors; purine nucleoside phosphorylase;

slow-binding inhibitors; tight-binding
inhibitors
Correspondence
A. Bzowska, Department of Biophysics,
Institute of Experimental Physics, Warsaw
University,
_
Zwirki i Wigury 93, 02-089
Warsaw, Poland
Fax: +48 22 554 0771
Tel: +48 22 554 0789
E-mail:
(Received 4 October 2009, revised 14
January 2010, accepted 29 January 2010)
doi:10.1111/j.1742-4658.2010.07598.x
Genetic deficiency of purine nucleoside phosphorylase (PNP; EC 2.4.2.1)
activity leads to a severe selective disorder of T-cell function. Therefore,
potent inhibitors of mammalian PNP are expected to act as selective
immunosuppressive agents against, for example, T-cell cancers and some
autoimmune diseases. 9-(5¢,5¢-difluoro-5¢-phosphonopentyl)-9-deazaguanine
(DFPP-DG) was found to be a slow- and tight-binding inhibitor of mamma-
lian PNP. The inhibition constant at equilibrium (1 mm phosphate concen-
tration) with calf spleen PNP was shown to be K
eq
i
=85±13pm (pH 7.0,
25 °C), whereas the apparent inhibition constant determined by classical
methods was two orders of magnitude higher (K
app
i

= 4.4 ± 0.6 nm). The
rate constant for formation of the enzyme ⁄ inhibitor reversible complex is
(8.4 ± 0.5) · 10
5
m
)1
Æs
)1
, which is a value that is too low to be diffusion-
controlled. The picomolar binding of DFPP-DG was confirmed by fluorimet-
ric titration, which led to a dissociation constant of 254 pm (68% confidence
interval is 147–389 pm). Stopped-flow experiments, together with the
above data, are most consistent with a two-step binding mechanism:
E+IM (EI) M (EI)*. The rate constants for reversible enzyme ⁄ inhibitor
complex formation (EI), and for the conformational change (EI) M (EI)*, are
k
on1
= (17.46 ± 0.05) · 10
5
m
)1
Æs
)1
, k
off1
= (0.021 ± 0.003) s
)1
, k
on2
=

(1.22 ± 0.08) s
)1
and k
off2
= (0.024 ± 0.005) s
)1
, respectively. This leads
to inhibition constants for the first (EI) and second (EI)* complexes of
K
i
= 12.1 nM (68% confidence interval is 8.7–15.5 nm) and K
Ã
i
= 237 pm
(68% confidence interval is 123–401 pm), respectively. At a concentration of
10
)4
m, DFPP-DG exhibits weak, but statistically significant, inhibition of
the growth of cell lines sensible to inhibition of PNP activity, such as human
adult T-cell leukaemia and lymphoma (Jurkat, HuT78 and CCRF-CEM).
Similar inhibitory activities of the tested compound were noted on the growth
of lymphocytes collected from patients with Hashimoto’s thyroiditis and
Graves’ disease. The observed weak cytotoxicity may be a result of poor
membrane permeability.
Abbreviations
6C-DFPP-DG, 9-(5¢,5¢-difluoro-5¢-phosphonoheptyl)-9-deazaguanine; DFPP-DG, 9-(5¢,5¢-difluoro-5¢-phosphonopentyl)-9-deazaguanine;
DFPP-G, 9-(5¢,5¢-difluoro-5¢-phosphonopentyl)-guanine; homo-DFPP-DG, 9-(5¢,5¢-difluoro-5¢-phosphonohexyl)-9-deazaguanine;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; PNP, purine nucleoside phosphorylase.
FEBS Journal 277 (2010) 1747–1760 ª 2010 The Authors Journal compilation ª 2010 FEBS 1747
Introduction

Potent membrane-permeable inhibitors of mammalian
purine nucleoside phosphorylase (PNP; EC 2.4.2.1) are
expected to act as selective immunosuppressive agents
against T-cell cancers, host-versus-graft reaction in
organ transplantation, and against some autoimmune
diseases [1]. This is because a genetic lack of PNP
activity leads to a severe selective disorder of T-cell
function with normal or even elevated B-cell function
(humoral immunity), as shown by Giblett et al. [2].
PNP catalyzes the reversible phosphorolytic cleavage
of the glycosidic bond of purine nucleosides:
b-purine nucleoside + orthophosph ate = purine b ase +
a-d-pentose-1-phosphate. The best inhibitors reported
to date are either transition state analogues, immucil-
lins, which bear features of the proposed transition
state (i.e. positive charge on the pentose moiety and
N7 of the base protonated) [3], or multisubstrate ana-
logue inhibitors capable of competing simultaneously
for both the nucleoside and phosphate-binding sites
[4]. However, in contrast to immucillins, which show a
pK
a
for pentose protonation at neutral pH (pK = 6.9)
[5], multisubstrate analogue inhibitors are anions, or
even a mixture of mono- and di-anions at neutral pH,
and, as charged molecules, do not readily penetrate cell
membranes. They also have short plasma lifetimes
because of a susceptibility to phosphatases. Hence,
they are not promising candidates as in vivo inhibitors.
This has stimulated the synthesis of some mimics with

the terminal phosphate being replaced by a phospho-
nate [6] or a difluorometylene phosphonate [7], which
confer metabolic stability. Moreover, some phospho-
nates appear to be capable of slowly traversing the cell
membrane, conceivably via an endocytosis-like process
[8,9].
To logically extend the above findings, we have
synthesized a series of multisubstrate analogue inhi-
bitors of PNP, namely 9-deazaguanine derivatives
connected by a linker to difluoromethylene phos-
phonic acid [10,11]. All of these 9-deazaguanine
derivatives are potent inhibitors of calf spleen and
human erythrocyte PNP, with apparent inhibition
constants as low as approximately 5 nm; for example,
for 9-(5¢,5¢-difluoro-5¢-phosphonopentyl)-9-deazagua-
nine (DFPP-DG) [10]. Up to now, however, only
apparent inhibition constants were reported. It should
be noted that, for tight-binding ligands, the inhibitor
concentration usually used in the course of classical
experiments, I
t
, is comparable with the total enzyme
concentration, E
t
, which is in the nanomolar range,
and under such conditions steady-state assumptions
may not hold.
In the present study, we employed such an approach
and report the true inhibition constants, and also the
dissociation constants for binding, of DFPP-DG and

some analogues with trimeric PNPs. To examine these
analogues as possible candidates for in vivo PNP inhib-
itors, we also determined some of their biological
properties. In particular, cytotoxic activities of DFPP-
DG against human lymphocytes from healthy subjects
and patients with autoimmune thyroid diseases (i.e.
Hashimoto’s thyroiditis and Graves’ disease), as well
as against a panel of human leukaemia and lymphoma
cell lines, were determined.
Results and Discussion
Apparent inhibition constants
Structures of new compounds embraced in the present
study are shown in Fig. 1. Apparent inhibition con-
stants versus two mammalian purine nucleoside phos-
phorylases, from calf spleen and human erythrocytes,
with 7-methylguanosine (m
7
Guo) as a variable sub-
strate, were determined using methods described previ-
ously for other inhibitors of trimeric PNPs [12,13].
With fixed concentrations of one substrate (i.e. inor-
ganic phosphate), apparent inhibition constants (K
app
i
)
were determined from initial velocity data with variable
concentrations of both the inhibitor and the second
substrate (m
7
Guo). Dixon plots displayed a competitive

mode of inhibition, as shown in Fig. 2 for DFPP-DG
and human erythrocyte PNP. Data sets were analysed,
and apparent inhibition constants calculated, with the
use of the weighted least-squares nonlinear regression
software leonora [14], as summarized in Table 1. For
comparison, inhibitory activities of 9-(5¢,5¢-difluoro-

Fig. 1. Structure of DFPP-DG and analogues: n = 1, DFPP-DG;
n = 2, homo-DFPP-DG; n = 3, 6C-DFPP-DG (left); and the structure
of immucillin H (right).
Inhibitors of purine nucleoside phosphorylase K. Breer et al.
1748 FEBS Journal 277 (2010) 1747–1760 ª 2010 The Authors Journal compilation ª 2010 FEBS
5¢-phosphonopentyl)-guanine (DFPP-G) [15] and a
transition state analogue inhibitor, immucillin H [3],
are also included.
All compounds were found to be very potent inhibi-
tors of m
7
Guo phosphorolysis, with apparent inhibi-
tion constants, K
app
i
, in the nanomolar range.
Inhibition is competitive versus nucleoside (m
7
Guo),
and the apparent inhibition constants, K
app
i
, decrease

with decreasing phosphate (fixed substrate) concentra-
tion (Table 1). This indicates that the inhibitors bind
to both nucleoside- and phosphate-binding sites, and
hence act as multisubstrate analogue inhibitors.
As predicted by previous structural studies [16],
DFPP-DG allows more favourable interactions with
the base-binding site of calf spleen and human erythro-
cyte PNPs compared to DFPP-G, and therefore yields
a K
app
i
lower than observed for DFPP-G (Table 1).
However, the effect is not large as a result of enthalpy-
entropy compensation. The gain in enthalpic contribu-
tion to the Gibbs binding energy, when compared with
DFPP-G binding, is balanced by an entropic effect
[17].
Except for 9-(5¢,5¢-difluoro-5¢-phosphonohexyl)-9-de-
azaguanine (homo-DFPP-DG) versus human erythro-
cyte PNP, which exhibits even better binding
properties than those observed for DGPP-DG
(K
app
i
= 5.3 nm compared to 8.1 nm; Table 1), the
other derivatives with shorter and longer linkers
exhibited weaker inhibitory effects.
Time-dependence of inhibition
The inhibition constants shown in Table 1 should be
treated as apparent values because the reaction rates

observed in the presence of DFPP-DG and its ana-
logues exhibit some initial inhibition (see the initial
velocity experiments), which increases as a function of
time (Fig. 3, left). This may be a result of low enzyme
and inhibitor concentrations (both in the nanomolar
range), leading to slow-binding inhibition because the
equilibrium may not be attained in the time-scale
of the initial velocity studies [18]. Therefore, the
Table 1. Inhibitory properties of DFPP-G, DFPP-DG and their analogues versus calf-spleen and human erythrocyte PNPs, and rates of associ-
ation (k) of some of the analogues with calf spleen PNP. K
app
i
is an apparent inhibition constant observed by the classical initial velocity
method, whereas K
eq
i
is an equilibrium inhibition constant determined after the slow-binding inhibitor is allowed to equilibrate with the
enzyme (see Materials and methods). For classical inhibitory studies, all reactions were carried out in 50 m
M Hepes buffer (pH 7.0) at 25 °C,
with m
7
Guo as variable substrate, in the presence of a fixed concentration of phosphate, as indicated. For equilibrium studies, and for deter-
mination of the association rate-constant, the enzyme was incubated with 1 m
M phosphate and various concentrations of inhibitor and, after
a given time interval (0.5–120 min), activity was determined with 60 l
M m
7
Guo (in 50 mM Hepes buffer, pH 7.0, at 25 °C).
Compound
Phosphate

concentration [m
M]
K
app
i
[nM]
human PNP
K
app
i
[nM]
calf PNP
K
eq
i
[pM]
calf PNP
k [M
)1
Æs
)1
]
calf PNP
DFPP-G 1 10.8 ± 0.7 6.9 ± 0.7
a
720 ± 130 (4.5 ± 0.7) · 10
6
DFPP-G 0.025 – 2.7 ± 0.2
DFPP-DG 50 – 28 ± 5
DFPP-DG 1 8.1 ± 0.6 4.4 ± 0.6 85 ± 13 (8.4 ± 0.5) · 10

5
DFPP-DG 0.025 1.0 ± 0.2 1.0 ± 0.2
Homo-DFPP-DG
b
1 5.3 ± 0.4 5.7 ± 0.6
6C-DFPP-DG 1 13 ± 1 21 ± 2
Immucillin H 1 19 ± 2
Immucillin H 50 – 41 ± 8
c
23 ± 5
d
a
Data from Iwanow et al. [15].
b
Poor solubility.
c
From Miles et al. [3], with the constant for the first reversible step.
d
From Miles et al. [3],
with the constant in equilibrium.
Fig. 2. Inhibition of human erythrocyte PNP by DFPP-DG. m
7
Guo
was a variable substrate. s, 8.4 l
M; •, 12.8 lM; h, 25.2 lM;
, 210 lM.
K. Breer et al. Inhibitors of purine nucleoside phosphorylase
FEBS Journal 277 (2010) 1747–1760 ª 2010 The Authors Journal compilation ª 2010 FEBS 1749
inhibition constant for binding of DFPP-DG to calf
spleen PNP was also determined at equilibrium, as

described in the Materials and methods.
The approach to equilibrium was followed by mea-
suring the velocity observed after various times of
incubation (0.5–120 min), and for various inhibitor
concentrations (in the range 0.5–20 nm) steady-state
velocities, v
s
were determined as shown in Fig. 3. From
the set of v
s
for various inhibitor concentrations, the
inhibition constant at equilibrium, K
eq
i
, was determined
by fitting Eqn (2) to the v
s
[I] ⁄ k
c
dependence, and was
found to be K
eq
i
=85±13pm, and hence two orders
of magnitude lower than the apparent inhibition con-
stant determined in the standard initial velocity experi-
ment, K
app
i
= 4.4 nm (see above).

Slow-onset binding, slow binding or binding
limited by diffusion
Time-dependence of inhibition was previously reported
for the transition-state inhibitors, immucillins [3], and
was interpreted as a slow-onset (i.e. two-step binding)
mechanism. For such a mechanism, binding involves
the rapid formation of the enzyme ⁄ inhibitor collision
complex, followed by a slow conformational change,
leading to a more tightly bound enzyme ⁄ inhibitor com-
plex: E + I M (EI) M (EI)*. However, it should be
noted that the presence of a slow-onset phase, espe-
cially when nanomolar enzyme and ligand concentra-
tions were used, does not unequivocally point to
binding as a two-step mechanism. It may simply be the
observation of a process of achieving equilibrium
between ingredients (Figs S1 and S2, data simulated
assuming one-step and two-steps mechanisms). The
question then arises as to whether the one- or two-step
mechanism also applies to binding of DFPP-DG and
analogues to trimeric PNPs. The data presented in
Fig. 3 (left) suggest only that equilibrium is reached
more rapidly with higher DFPP-DG concentrations, in
agreement with both mechanisms. The rate of the
exponential decay (Eqn 1; see Materials and methods)
increases linearly with increasing inhibitor concentra-
tion (Fig. 3, insert). This is usually considered as an
indicator for a mechanism involving two molecules
(i.e. E + I M E I), and not the conformational change
of the (EI) complex, (EI) M (EI)*. However, the simu-
lated data according to a two-step mechanism show

that linearity may be observed in the case of more
complicated binding patterns [19]. The rate constant
derived form the data shown in the insert to Fig. 3
resulted in a value of (8.4 ± 0.5) · 10
5
m
)1
Æs
)1
for
complex formation between PNP and DFPP-DG,
which is too small to be classified as a diffusion-con-
trolled encounter rate, and which is approximately 10
8
or higher [20]. However, to confirm that complex
formation is not limited by diffusion, a control experi-
ment was performed. The reaction mixture containing
the enzyme (2.3 nm) and the inhibitor (3.0 nm) was
continuously mixed. The rate measured in this case did
not differ from the rate measured without mixing
(Fig. S3).
To confirm that DFPP-DG is a slow-binding inhibi-
tor of trimeric PNP, we conducted an experiment with
calf spleen PNP and DFPP-DG, using continuous

Fig. 3. Left: Time-dependence of inhibition of calf spleen PNP by DFPP-DG. PNP (2.3 nM subunits), DFPP-DG (s)0nM, (*) 1.0 nM,())
2.0 n
M or (•) 3.0 nM and only one PNP substrate (1 mM phosphate) Data for several other inhibitor concentrations were collected, but are
not shown. The insert shows the dependence of the observed rate constants on DFPP-DG concentration, with an exponential decay fitted,
leading to an association rate constant of (8.4 ± 0.5) · 10

5
M
)1
Æs
)1
. Right: Determination of the inhibition constant at equilibrium, K
eq
i
, for
interaction of DFPP-DG with calf PNP. Constants were obtained by fitting equation [2] to the equilibrium velocities, v
s
, obtained from experi-
ments depicted in the upper panel. The K
eq
i
value obtained from these data is 85 ± 13 pM.
Inhibitors of purine nucleoside phosphorylase K. Breer et al.
1750 FEBS Journal 277 (2010) 1747–1760 ª 2010 The Authors Journal compilation ª 2010 FEBS
monitoring with saturating substrate concentration,
according to Morrison and Walsh [18]. This was previ-
ously performed with inosine as a substrate and immu-
cillin as an inhibitor (at pH 7.7) to characterize the
slow-onset binding observed with such transition state
inhibitors [3]. Therefore, as a control, we performed
the same experiment with immucillin H, both at the
same pH 7.7 (not 7.0). The data presented in Fig. 4
clearly show that the slow-onset phase (i.e. the charac-
teristic feature of interaction of immucillins with
trimeric PNPs) is also observed with DFPP-DG, but is
not as well defined. In the initial phase of the reaction

($10 min), 12.3 nm of immucillin H does not cause
any inhibition of inosine phosphorolysis, by contrast
to DFPP-DG. K
i
for the rapidly reversible complex
observed for immucillin H is 41 ± 8 nm (Table 1) [3].
However, over time, immucillin inhibits more and
more strongly, and finally the equilibrium for the slow-
onset step is attained (Fig. 4, left) with the equilibrium
dissociation constant for immucillin being
K
eq
i
=23±5pm [3]. This is not so with DFPP-DG
as an inhibitor. In this case, an almost linear depen-
dence of uric acid formation [the final product of the
couple assay for inosine as a PNP substrate) versus
time is observed over the whole course of the experi-
ment (Fig. 4, right; but see also below).
In the progress curve method, the inhibitor competes
with a high excess of substrate for the active sites of
the enzyme; therefore, the slow-onset phase of the
reaction may not always be observed [18]. This is
shown in the left panel of Fig. 4, where, in the case of
immucillin H, a change of pH from 7.7 to 7.0 is such
that equilibrium for the slow-onset phase is not
reached in the course of the experiment. Hence, to dis-
tinguish between one-step slow-binding and two-step
slow-onset binding, it is important to fit these two
models to a set of progress curves using software based

on numerically solving systems of differential equa-
tions (e.g. dynafit; BioKin, Ltd, Watertown, MA,
USA). However, some problems may arise. We used
dynafit, version 4.0 to simulate sets of progress
curves described by both mechanisms. Only in the case
of one-step binding were we able to reconstruct param-
eters used for simulations (Docs S1 and S2; Figs S1
and S2).
Confirmation of picomolar binding constant by
titration experiments
To confirm strong binding of DFPP-DG by calf spleen
PNP, the dissociation constant for this complex was
determined directly. Classical fluorimetric approaches
were employed but only provided confirmation that
one ligand molecule is bound per enzyme monomer
and that binding is strong because the binding curve
displayed the typical stoichiometric character, which
means that the binding process was rapidly stopped
when the ligand concentration added was equal to the
PNP subunit concentration (Fig. 5). A classical data
evaluation (i.e. fitting of the well-known Eqn (5)
derived under assumptions described in the Materials
and methods, separately for each titration, resulted in
plots of residuals showing unequivocally that the used
model does not properly describe the experimental
data (Fig. 5, lower panel).
Therefore, an approach using dynafit software was
employed. Three various models were tested (see Mate-
rials and methods): assuming non-identical changes of
fluorescence upon binding of the first, second and third

ligand molecule but identical affinity to ligand by
subunits, then non-identical affinities (allosteric behav-
iour) but identical changes of fluorescence and, finally,
non-identical changes of fluorescence and affinities.
The fit based on the assumption that monomers bind
the ligand with identical affinities, but with different
fluorescent responses, was the most accurate (Fig. 6).
Time (min) Time (min)
Fig. 4. Slow-onset binding of immucillin H
by calf spleen PNP (left, pH 7.7, if not other-
wise indicated) and the similar, but less well
defined, slow-onset phase for binding of
DFPP-DG (right, pH 7.7).
K. Breer et al. Inhibitors of purine nucleoside phosphorylase
FEBS Journal 277 (2010) 1747–1760 ª 2010 The Authors Journal compilation ª 2010 FEBS 1751
We fitted simultaneously more than one data set,
obtained with various protein concentrations, but trea-
ted molar fluorescence parameters for different forms
of the PNP ⁄ DFPP-DG complexes as the independent
adjustable parameters for each curve as a control. We
obtained comparable values of fluorescent increments
upon binding for both curves, which confirms that the
used model properly describes the experimental data.
From this fit, the first dissociation constant, K
d1
(see
Materials and methods) was found to be 84.6 pm (68%
confidence intervals is 49.3–129.4 pm), which corre-
sponds to a classical dissociation constant three-fold
higher, K

d
=3K
d1
(i.e. K
d
= 254 pm) (68% confidence
intervals is 147–389 pm). This value is somewhat higher
than the one obtained from inhibition at equilibrium,
K
eq
i
=85±13pm but, according to the 90% confi-
dence intervals, the data are in agreement (Tables S1
and S2). It should be recalled that additions of the
ligand in the fluorimetric titrations were made every
40 s. It could be argued that, as a result of slow bind-
ing, equilibrium may not be fully achieved. However,
the concentrations used for the titrations were a few
orders of magnitude higher than in the kinetic
approach. Furthermore, we did not observe any change
in signal when data were collected for an additional
40 s, which means that the formation of the first com-
plex is completed during only 40 s. These facts taken
together suggest a two-step binding mechanism for
DFPP-DG rather than one-step binding. Both methods
confirm that DFPP-DG binds as strongly as the transi-
tion state analogue inhibitors, immucillins.
0.00.10.20.30.40.50.6
380
400

420
440
460
480
500
520
540
560
0.00.10.20.30.40.50.6
–3
–2
–1
0
1
2
3
4
Fluorescence (A.U.)
[DFPP-DG] (µ
M
)
Fig. 5. Fluorimetric titration of calf spleen PNP (0.4 lM binding
monomers; see Materials and methods) with DFPP-DG. Data show
that binding is stoichiometric and, hence, with a very low dissocia-
tion constant (and much lower than the enzyme concentration) (i.e.
the binding process stops rapidly when the added ligand concentra-
tion is equal to the concentration of the active binding sites). The
classical approach was employed to analyse the data (see Materials
and methods); however, the residual plot (lower panel) shown
indicates that this method is not correct.

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14
0.0
0.1
0.2
0.0 0.1 0.2 0.3 0.4 0.5 0.6
380
400
420
440
460
480
500
520
540
560
0.0 0.1 0.2 0.3 0.4 0.5 0.6
–1.5
–1.0
–0.5
–1.3
–1.2
–0.1
0.0
0.5
1.0
0.00 0.02 0.04 0.06 0. 08 0.10 0.12 0.14
100
105
110
115

120
125
130
135
140
145
150
[DFPP-DG] (µ
M
) [DFPP-DG] (µ
M
)
Fluorescence (A.U.)
Fig. 6. Fluorimetric titrations of calf spleen
PNP with DFPP-DG (upper panels) with resi-
dual plots (lower panels) for the best model
fitted (see Results and Discussion). Protein
concentrations (in terms of binding mono-
mers; see Materials and methods) were 0
1 l
M (left) and 0.4 lM (right). Data were
analysed simultaneously, using
DYNAFIT soft-
ware as described in the Materials and met-
hods. The dissociation constant obtained
from this fit is 254 p
M (68% confidence int-
erval is 147–389 p
M). The molar fluores-
cence for protein complexes with one, two

and three ligand particles are:
f
PL1
= 414.4 ± 20.7, f
PL2
= 800.0 ± 30.8,
f
PL3
= 1070.3 ± 36.6 AU (left) and
f
PL1
= 414.7 ± 4.8, f
PL2
= 725.9 ± 6.6,
f
PL3
= 1002.6 ± 7.5 AU (right).
Inhibitors of purine nucleoside phosphorylase K. Breer et al.
1752 FEBS Journal 277 (2010) 1747–1760 ª 2010 The Authors Journal compilation ª 2010 FEBS
Stopped-flow measurements
To finally resolve the one- or two-step binding prob-
lem, we conducted a series of stopped-flow experiments
(Fig. 7). Kinetic traces were analysed using dynafit
software. Various models were considered. Data may
be adequately well described by the one-step model,
although the dissociation constant calculated from the
rate constants obtained in this case, k
on1
= (16.6 ±
0.1) · 10

5
m
)1
Æs
)1
, k
off1
= (0.0013 ± 0.0001) s
)1
,isan
order of magnitude higher than the values derived
from other methods (k
off1
⁄ k
on1
= 783 pm compared to
85 and 254 pm; see above). The two-step model with
similar fluorescence properties of both enzyme-ligand
complexes, (EI) and (EI)* (2.12 AU and 2.15 AU
respectively), gave a slightly lower sum of squares, but
also much better agreement with the results obtained
by other methods. Rate constants derived from
this model are: k
on1
= (17.46 ± 0.05) · 10
5
m
)1
Æs
)1

,
k
off1
= (0.021 ± 0.003) s
)1
for the (EI) complex and
k
on2
= (1.22 ± 0.08) s
)1
, k
off2
= (0.024 ± 0.005) s
)1
for the (EI)* complex, leading to inhibition constants
for the (EI) and (EI)* complexes K
i
= 12.1 nm (68%
confidence interval is 8.7–15.5 nm) and K
Ã
i
= 237 pm
(68% confidence interval is 123–401 pm), respectively.
The second value is in excellent agreement with
the steady-state titration experiments (see above). We
conclude that DFPP-DG binding with calf PNP
follows a two-step binding model.
DFPP-DG analogues
DFPP-DG analogues also bind slowly with trimeric
PNPs. Moreover, slow binding is not limited to com-

pounds with the 9-deazaguanine aglycone because the
same slow-binding effect was observed also for DFPP-
G. Hence, it appears that the 9-deaza feature is not
responsible for the slow-binding phenomenon. For
DFPP-G, the rate constant for EI complex formation
is (4.5 ± 0.7) · 10
6
m
)1
Æs
)1
, and the difference between
the apparent and equilibrium inhibition constants is
only approximately ten-fold (6.9 nm compared to
0.79 nm; Table 1), and much less pronounced than
with DFPP-DG.
Cytotoxic activities
Tight binding of DFPP-DG to PNP led us to check its
possible inhibitory potential on the growth of human
normal cells and cell lines derived from haematological
malignancies. Cells selected for testing were human
normal lymphocytes, lymphocytes of patients with
autoimmune thyroid diseases, and a panel of lym-
phoma and leukaemia cells from B- and T-cells. T-cell
malignancies have specific biochemical, immunological
and clinical features, which separate them from
non-T-cell malignancies [21].
DFPP-DG moderately affects growth of several
leukaemia and lymphoma cell lines, especially T-cell
leukaemias (Jurkat and MOLT), acute lymphoblastic

leukaemia (CCRF-CEM) and T-cell lymphoma
(HuT78). Some differences were observed between
the effects on the growth of tumor cells sensible to
inhibition of PNP activity, such as human adult T-cell
leukemia and lymphoma (Jurkat, MOLT, HuT78,
CCRF-CEM) and other leukaemia and lymphoma
cells of B-cell, or non-T- and non-B-cell lineages
(K562, Raji, HL-60). However, the effects were detect-
able only at the highest concentration applied, 10
)4
m
(Table 2).
Fig. 7. Set of stopped-flow kinetic traces obtained after mixing of
PNP with DFPP-DG. Concentrations of PNP subunits in the
stopped-slow spectrometer, 0.4 l
M (black), 0.2 lM (grey) and
0.1 l
M (light grey), and the concentration of DFPP-DG (in lM) are
given for each trace. Data were analysed simultaneously using
DYNAFIT software (see Materials and methods) and the curves fitted
are also shown.
K. Breer et al. Inhibitors of purine nucleoside phosphorylase
FEBS Journal 277 (2010) 1747–1760 ª 2010 The Authors Journal compilation ª 2010 FEBS 1753
Hashimoto’s thyroiditis and Graves’ disease are
T-cell mediated autoimmune thyroid diseases [22–25].
Regarding the known features of autoimmunity to the
thyroid gland, we expected significant inhibitory effects
of DFPP-DG on lymphocytes collected from patients
suffering from human autoimmune thyroid disorders,
relative to normal lymphocytes. DFPP-DG, at 10

)4
m,
exhibited modest, but statistically significant, inhibitory
effects (almost 30%) on lymphocytes from patients
suffering from Hashimoto’s thyroiditis and Graves’
disease.
The reason behind the modest cytotoxic properties of
DFPP-DG observed in vivo, despite its excellent inhibi-
tory properties versus trimeric PNP, lies most probably
in the poor penetration capability of this compound
through cell membranes. Some phosphonates appear to
be capable of slowly traversing the cell membrane [8,9].
However, DFPP-DG is a difluorometylene phospho-
nate. It is known that fluorination of alkylphospho-
nates yields compounds with properties suitably
resembling phosphate esters [7,26], and, in turn, this
leads to optimized interactions of such analogues with
the phosphate-binding site residues in the PNP active
site [16,27]. Because the physical properties of DFPP-
DG are rather similar to those of phosphates, it is not
unusual that this compound is not readily taken up by
the cells. To demonstrate this, we plan to mark DFPP-
DG with a fluorescent dye so that we can follow its
entry into cells and its intracellular localization. If we
confirm that the poor uptake is, in fact, responsible for
the mild cytotoxic effects observed, we plan to synthe-
size a pro-drug of DFPP-DG. Alternatively, we also
plan to employ one of the recently developed drug-
delivery systems [28,29], to improve the cell penetration
of this excellent PNP inhibitor.

Conclusions
DFPP-DG and some analogues show inhibition and
dissociation constants versus trimeric purine nucleoside
phosphorylases in the picomolar range. Similarly to
immucillins – transition state analogue inhibitors [3],
the compounds described in the present study exhibit
slow-onset binding pattern as well. Stopped-flow exper-
iments together with data obtained by other methods
are consistent with two-step binding mechanism, and
hence similar to that observed in the case of immucil-
lins. DFPP-DG shows moderate inhibitory effects on
the growth of lymphocytes from patients with human
autoimmune thyroid disorders and T-cell leukaemia
and lymphoma cells, but only at a concentration of
10
)4
m. Because DFPP-DG is a phosphonate and car-
ries a negative charge, the inefficient transport of the
inhibitor into cells is most probably responsible for the
mild cytotoxic effects. Although some phosphonates
appear to be capable of slowly traversing the cell mem-
brane, conceivably via an endocytosis-like process, this
is not likely the case with DFPP-DG. For that reason,
future studies will be directed toward the synthesis of a
pro-drug of DFPP-DG to improve its cell penetration.
The problem of the poor uptake of the compound by
cells may, in principle, also be overcome by use of one
of the recently developed drug-delivery systems [28,29].
One of these approaches is based on use of the cross-
linked cationic polymer network (Nanogel) for intra-

cellular delivery of negatively charged drugs, and
shown to be successful with the cytotoxic 5¢-phosphate
of 5-fluoroadnenosine arabinoside, fludarabine [30],
and 5¢-triphosphates of cytarabine (araCTP), gemcita-
bine (dFdCTP) and floxuridine (FdUTP) [31]. We
also plan to mark DFPP-DG with a fluorescence
dye to follow its entry into cells and its intracellular
localization in an effort to explain the observed mild
cytotoxic effects.
Materials and methods
Reagents
Commercially available PNP from calf spleen (Sigma,
St Louis, MO, USA), as a suspension in 3.2 m ammonium
Table 2. Cytotoxic effects of DFPP-DG towards various cell types.
Exponentially growing cells were treated with different concentration
of DFPP-DG for 72 h periods. Cytotoxicity was analysed by the MTT
survival assay. All experiments were performed at least three times.
Cell lines: acute lymphoblastic leukemia (CCRF-CEM), T-cell leukemia
(Jurkat and MOLT-4), T-cell lymphoma (HuT78), acute myeloid leuke-
mia (HL-60), Burkitt’s lymphoma (RAJI) and chronic myeloid
leukemia in blasts crisis (K562). Human blood lymphocytes from
healthy donors, from patients with Graves’ disease and from patients
with Hashimoto’s disease. –, no effect. *Statistically significant
change (P < 0.05).
Cell line
Percentage inhibition
DFPP-DG concentration
10
)7
M 10

)6
M 10
)5
M 10
)4
M
Bood 1.5 12.0 16.4 13.8
CCRF-CEM – 6.4 10.5 49.4*
Jurkat 7.5 8.4 5.9 33.7*
MOLT-4 1.9 6.7 7.7 33.3
HuT78 6.4 7.0 11.9* 25.4*
K562 – – – 8.1
Raji 2.8 6.5 5.4 –
HL-60 – – 3.4 21.1
Hashimoto’s thyroiditis 10.9 8.6 20.8* 29.8*
Graves’ disease 1.1 10.0 15.3 28.6*
Inhibitors of purine nucleoside phosphorylase K. Breer et al.
1754 FEBS Journal 277 (2010) 1747–1760 ª 2010 The Authors Journal compilation ª 2010 FEBS
sulphate, with specific activity versus inosine of approxi-
mately 15–22 UÆmg
)1
, was desalted as described previously
[12]. Lyophylized human erythrocyte PNP (from Sigma)
was dissolved in 20 mm Hepes buffer (pH 7.0) ($0.5 mg in
100 lL of buffer). The specific activity of this enzyme ver-
sus inosine was approximately 8 UÆmg
)1
. Inosine, NaCl ⁄ Pi,
Hepes (ultra pure), Na
2

HPO
4
, NaH
2
PO
4
,m
7
Guo and other
chemicals were products obtained from Sigma or Fluka
(Buchs, Switzerland). Xanthine oxidase from buttermilk, a
suspension in 2.3 m ammonium sulphate (1 UÆmg
)1
at
25 °C) was from Sigma. DFPP-DG and analogues were
prepared as previously described [10,11]. All solutions were
prepared with high-quality MilliQ water (Millipore, Billeri-
ca, MA, USA).
Concentrations of all substrates and inhibitors were
determined spectrophotometrically using the extinction
coefficients (at pH 7.0): e(260 nm) = 8500 m
)1
Æcm
)1
for
m
7
Guo (pK
a
$ 7.0), e(249 nm) = 12 300 m

)1
Æcm
)1
for ino-
sine (pK
a
8.9), e(273 nm) = 10 100 m
)1
Æcm
)1
for DFPP-
DG (pK
a
4.9) and homo-DFPP-DG, e(273 nm) = 9000
m
)1
Æcm
)1
for the DFPP-DG analogue with 6-carbon
linker [9-(5¢,5¢-difluoro-5¢-phosphono-heptyl)-9-deazaguanine
(6C-DFPP-DG)], and e(261 nm) = 9540 m
)1
Æcm
)1
for
immucillin H [3].
Enzyme concentrations were determined from the extinc-
tion coefficient of 9.6 cm
)1
at 280 nm for a 1% solution

[32]. In calculations, the theoretical molecular mass of one
monomer of the calf spleen enzyme, based on its amino
acid sequence, was used; molecular mass = 32 093 Da [33]
(SwissProt entry P55859). Molar concentrations are given
in all experiments in terms of enzyme monomers.
Instrumentation
Kinetic and spectrophotometric measurements were carried
out on a Uvikon 930 (Kontron, Vienna, Austria) spectro-
photometer fitted with a thermostatically controlled cell
compartment, using 10, 5, 2 or 1 mm path-length quartz
cuvettes (Hellma, Mullheim, Germany). A Beckman model
F300 pH-meter (Beckman Coulter, Fullerton, CA, USA)
equipped with a combined semi-microelectrode and temper-
ature sensor, was used for pH determination.
Fluorescence data were recorded on a Perkin-Elmer
LS-50 spectrofluorimeter (Norwalk, CT, USA), using
4 · 10 mm cuvettes, with continuous mixing of the solu-
tion.
Stopped-flow kinetic measurements were run on a
SX.18MV stopped-flow reaction analyzer from Applied
Photophysics Ltd (Leatherhead, UK). The dead time of the
instrument was 1.2 ms.
CO
2
incubator (Shell Lab, Sheldon Manufacturing,
Cornelius, OR, USA) was used for cell culturing and an
ELISA plate reader (Stat fax 2100; Pharmacia Biotech,
Uppsala Sweden) for absorbance measurement in the cyto-
toxic activity measurements.
Standard enzymatic procedures

Kinetic studies, if not otherwise indicated, were conducted
at 25 °Cin50mm Hepes ⁄ NaOH buffer (pH 7.0) in 1 mm
phosphate buffer for determination of inhibition constants,
and in 50 mm phosphate buffer for determination of the
enzyme specific activity.
One unit of PNP is defined as the amount of enzyme that
causes phosphorolysis of 1 lmol of inosine to hypoxanthine
and ribose-1-phosphate per minute under standard condi-
tions (i.e. at 25 °C with 0.5 mm inosine and 50 mm sodium
phosphate buffer, pH 7.0). The standard coupled xanthine
oxidase procedure [32] was used in which hypoxanthine,
liberated in the PNP catalysed reaction, is oxidized to uric
acid by xanthine oxidase. The observation wavelength was
k
obs
= 300 nm and the molar extinction coefficient differ-
ence between inosine and uric acid is De
300 nm
= 9600
m
)1
Æcm
)1
[12].
PNP is known for its nonhyperbolic kinetics. Deviations
from the classical Michaelis–Menten kinetics depend on the
nucleoside substrate and concentration of the co-substrate,
phosphate [12]. Therefore, inhibition type and inhibition
constants were determined, if not otherwise indicated, using
m

7
Guo as the variable substrate because it was shown that,
for this substrate, the classical Michaelis–Menten [34] equa-
tion is sufficient for data analysis [12].
Phosphorolysis of m
7
Guo was examined spectrophotomet-
rically by a direct method [35]. The observation wavelength,
k
obs
= 260 nm, corresponds to the maximal difference
between extinction coefficients of nucleoside substrate,
m
7
Guo, and the respective purine base, 7-methylguanine:
De = 4600 m
)1
Æcm
)1
at 260 nm at pH 7.0 for the mixture of
cationic and zwitterionic forms of m
7
Guo [12,35].
The reaction mixture for the direct method and for the
coupled method had a 1 mL volume in a 10 mm path-length
cuvette at 25 °C. It contained 50 mm Hepes (pH 7.0), with
both substrates of the phosphorolytic reaction (phosphate
buffer of the same pH as the main buffer, and a nucleoside,
m
7

Guo or inosine). In the case of inosine phosphorolysis,
xanthine oxidase was also present ($0.1 UÆmL
)1
). In inhibi-
tion studies, an inhibitor was included in the reaction
mixture. The reaction was started by the addition of PNP.
Initial rate procedures were employed in all kinetic studies.
In the case of inhibition studies, for each combination of the
initial substrate concentration, c
o
, and the inhibitor concen-
tration [I], the rates were determined at least twice. The initial
velocities, v
o
, were measured directly from the computer con-
trolling the spectrophotometer. Linear regression software
(Kontron, Vienna, Austria) was used for determination of
slopes, with their standard errors, of absorbance versus time.
Time-dependence of inhibition: progress curves
Time-dependence of inhibition was measured using two
approaches. In the first, inosine was the substrate and
K. Breer et al. Inhibitors of purine nucleoside phosphorylase
FEBS Journal 277 (2010) 1747–1760 ª 2010 The Authors Journal compilation ª 2010 FEBS 1755
continuous monitoring of uric acid formation was used to
measure the progress curve, as described by Miles et al. [3].
Briefly the enzyme (1.3 nm subunits) was added to the com-
plete reaction mixture (50 mm Hepes ⁄ NaOH buffer, pH
7.7) containing an excess of both substrates (0.71 mm ino-
sine, 50 mm phosphate buffer, pH 7.7) and various inhibi-
tor concentrations. Formation of uric acid, the final

product of the coupled PNP and xanthine oxidase reaction
[32], was monitored at 300 nm.
Time-dependence of inhibition: initial velocity
In the progress curve approach, the inhibitor competes with
the substrate for the active sites of the enzyme. With a high
excess of substrate, the slow-onset phase of the reaction
may not always be observed [18]. Therefore, the initial
velocity method was also used. In this approach, the
enzyme (2.3 nm) and inhibitor (concentration range 0.5–
20 nm) were incubated at 25 °Cin50mm Hepes ⁄ NaOH
(pH 7.0) and 1 mm phosphate buffer (pH 7.0). The total
volume was 1.2 mL. After a given time interval, t (0.5–
120 min), 0.03 mL of the second substrate, m
7
Guo
(2000 lm), was mixed with 0.97 mL of the incubated solu-
tion. The final concentration of m
7
Guo was therefore
60 lm, with all other concentrations changed by only 3%,
to allow treatment equal to the initial values. The initial
velocities observed after various incubation times for each
inhibitor concentration, v
o
(t, [I]), were measured.
For each inhibitor concentration, the velocity at equilib-
rium [i.e. at infinite time; v
o
(¥, [I])] (later referred to as
v

s
[I]; steady-state velocity observed in the presence of inhib-
itor at [I] concentration) was determined. This was achieved
by fitting the one-phase exponential decay to each set of
velocities observed with various [I], v
o
(t, [I]):
m
0
ðt; ½IÞ ¼ A expðÀktÞþm
s
½Ið1Þ
In separate experiments, k
c
was determined as the initial
velocity obtained at time t = 0 (i.e. no incubation) with a
saturating concentration of m
7
Guo (120 lm) and in the
absence of inhibitor [i.e. v
o
(0; [0]) = k
c
]. It was also found
that 120 min of incubation has no influence on enzyme
activity; hence, it may be assumed that v
o
(0;
[0]) = v
o

(120 min; [0]). The Michaelis constant was deter-
mined as previously described [12], and the value obtained,
K
m
=17lm, was used in subsequent calculations.
The inhibition constant at equilibrium, K
eq
i
,was finally
determined from Eqn (18), as reported previously for
immucillins [3]:
m
s
½I=k
c
¼½S= K
m
ð1 þ½I=K
eq
i
þ½S
ÈÉ
ð2Þ
where [S] is the concentration of m
7
Guo (60 lm), and K
m
and k
c
are constants.

Fluorimetric titrations
Fluorescence titrations were conducted essentially as
described previously [27] but the protein was not diluted
during experiments because the ligand stock used for titra-
tions was prepared in the buffer and protein solution corre-
sponding to their concentrations in a cuvette. Experiments
were performed in 20 mm Hepes buffer (pH 7.0), in the
presence of 1 mm phosphate at 25 °C. The enzyme subunit
concentrations were either 0.2 or 0.8 lm, as determined
from UV absorption. PNP specific activity was approxi-
mately 15 UÆmg
)1
, which gives approximately 0.1 and
0.4 lm binding monomers because the activity of the fully
active enzyme preparation is 34 UÆmg
)1
, as shown previ-
ously [27]. The rest of the protein is inactive PNP, which,
as shown previously, does not interfere with binding of
ligands by the active enzyme [12,27]. Additions of ligand
were made every 40 s.
The protein-ligand binding model for the trimeric pro-
tein, assuming a one-step process for each binding site, is:
P þ L ,
k
a1
k
d1
PL1 K
d1

¼ k
d1
=k
a1
PL1 þ L ,
k
a2
k
d2
PL2 K
d2
¼ k
d2
=k
a2
ð3Þ
PL2 þ L ,
k
a3
k
d3
PL3 K
d3
¼ k
d3
=k
a3
At any given time, the fluorescence of the solution may
be represented as the sum of the fluorescence of the various
molecular species present in the mixture, free trimeric pro-

tein, P, free ligand, L, and trimeric protein complexed with
one, two or three ligand molecules (PL1, PL2, PL3):
Fluorescence ¼½P f
P
þ½Lf
L
þ½PL1f
PL1
þ½PL2 f
PL2
þ½PL3f
PL3
ð4Þ
Fluorimetric titration data were evaluated by two
approaches. The classical approach assumed that ligand
binds to all three subunits of the trimeric PNP molecule
independently and is described by a single dissociation con-
stant, K
d
; hence, the appropriate equation is [36]:
Parameters f
E
, f
L
and f
EL
, are molar fluorescence coeffi-
cients of free PNP subunit, free ligand and PNP subunit
complexed with the ligand, respectively, [L] is the total con-
centration of the ligand, F ([L]) is the fluorescence intensity

Fð½LÞ ¼ F
0
Àðf
E
þ f
L
À f
EL
Þ
[L]
2
þ
½E
act

2
þ
K
d
2
À
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ð½LÀ½E
act
þK
d
Þ
2
þ 4½E
act

K
d
q
2
0
@
1
A
þ½Lf
L
ð5Þ
Inhibitors of purine nucleoside phosphorylase K. Breer et al.
1756 FEBS Journal 277 (2010) 1747–1760 ª 2010 The Authors Journal compilation ª 2010 FEBS
observed for the total ligand concentration [L], and [E
act
]
the total concentration of enzyme binding sites. Experimen-
tal data were fitted to the above equation, using nonlinear
regression analysis, obtaining values for four fitted parame-
ters: K
d
,[E
act
], f
L
and df =(f
E
+ f
L
) f

EL
). To derive the
above equation, it was also necessary to assume that bind-
ing of the ligand to each subunit yields the same change in
the molar fluorescence coefficient, dF. origin 7 software
(OriginLab Corporation, Northampton, MA, USA) was
used. In the present study, fitting based on this equation is
referred to as classical fitting.
In the second approach, the model referring to PNP as a
trimeric protein was used. A few variations were tested:
assuming non-identical changes of fluorescence upon bind-
ing of the first, second and third ligand molecule
(f
PL1
„ f
PL2
„ f
PL3
) but identical affinity to ligand by
subunits, then non-identical affinities (allosteric behaviour)
but identical changes of fluorescence and, finally, non-iden-
tical changes of fluorescence and affinities. It should be
noted that, if all PNP subunits show identical affinity to
ligand, it implies the following relations: 3k
a1
=2k
a2
= k
a3
and k

d1
=2k
d2
=3k
d3
between the association (k
ai
) and
dissociation (k
di
) reaction rate constants (and
3K
d1
= K
d2
= K
d3
⁄ 3) as a result of the different number
of free binding sites available for ligand at the different
complex formation steps. In this case, we also have
K
d
=3K
d1
because K
d
refers to the protein monomer con-
centration, but K
d1
refers to protein trimers. We used

dynafit, version 4 [37] to discriminate between models. We
simultaneously analysed a few data sets for various protein
concentrations. Because the confidence intervals for nonlin-
ear model parameters are, by definition, nonsymmetrical,
and this asymmetry can be neglected for relatively small
formal errors only, for formal errors approaching 50% or
larger, the nonsymmetrical confidence intervals were calcu-
lated. The 68% confidence intervals ranges, based on this
analysis, are given in the text. Script files used for running
the dynafit software model determination analysis, and
the nonsymmetrical confidence intervals for various like-
lihoods are given in Doc. S1 and Table S1, respectively.
The nonsymmetrical confidence intervals were obtained by
putting two question marks next to the initial values of the
dissociation constant in the dynafit script file.
Stopped-flow experiments
Emission of PNP and PNP ⁄ DFPP-DG complexes was
excited at 290 nm (slit width = 0.5 mm = 2.32 nm), and
monitored using a cut-off filter (320 nm). Both extinction
and emission path lengths in the stopped-flow cell were
2 mm. The reactions consisted of mixing equal volumes of
PNP (35 UÆmg
)1
) and the ligand. Concentrations of the
protein solution were 0.2, 0.4 and 0.8 lm (in terms of PNP
monomers), determined by absorption measurements. Con-
centrations of the ligand varied in the range 0.05–6.4 lm.
Concentrations of protein and ligand refer to the situation
prior to mixing in the stopped-flow apparatus (i.e. to the
concentrations in the syringes, and not the final concentra-

tions in the mixture, which are half of the initial values).
The measurements were performed at 25 °C at pH 7.0 in
50 mm Hepes buffer with 1 mm phosphate. One thousand
data points were recorded over the course of each reaction,
using the oversampling option of the instrument, and usu-
ally seven to ten runs were averaged for each concentration
of the reagents. The volume for each reaction was 70 lL.
The data were corrected for the inner filter effect. We used
dynafit, version 4 [37] to analyse the stopped-flow data.
We simultaneously analysed data sets obtained for various
protein and ligand concentrations. Rate constants (two or
four, depending on the model used, one-step or two-step
binding, respectively), active enzyme concentration in each
experiment, and emission of the enzyme, ligand, and
enzyme ⁄ ligand complexes were adjustable parameters. The
nonsymmetrical confidence intervals were calculated using
dynafit software (see above). The confidence intervals for
other than 68% likelihoods are given in Table S2.
Cell culturing
Experiments were carried out on seven human cell lines
derived from leukaemia and lymphoma cells, and on three
primary lymphocyte cultures. Chronic myeloid leukaemia in
blasts crisis (K562], T-cell leukaemia (Jurkat and MOLT),
Burkett’s lymphoma (Raji) and acute myeloid leukaemia
(HL-60) were obtained from American Type Culture Col-
lection (Manassas, VA, USA, USA). Acute T-cell lympho-
blastic leukaemia (CCRF-CEM) and T-cell lymphoma
(HuT78) were purchased from ECACC, Health Protection
Agency (Sailsbury, UK). Lymphocytes from 10 patients
affected by Hashimoto’s thyroiditis and lymphocytes from

10 patients with Graves’ disease, as well as control lympho-
cytes, were isolated from heparinized blood by the standard
method of density-gradient centrifugation over Ficoll-Hyp-
aque reagents (Amersham Pharmacia, Uppsala, Sweden)
according to the manufacturer’s instructions.
Cells were grown in RPMI-1640 medium (Gibco BRL,
Life Technologies, Paisley, UK) supplemented with 10%
fetal bovine serum, streptomycin (100 lgÆmL
)1
) and penicil-
lin G (100 UÆmL
)1
). Cells were cultured in a humidified
(95% air, 5% CO
2
)CO
2
incubator (Shell Lab) at 37 ° C.
The trypan blue dye exclusion method was used to assess
cell viability before each experiment.
Cytotoxicity test
Cytotoxic effect of DFPP-DG on tumour and normal cells
was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-tetrazolium bromide (MTT) assay [38]. The inves-
tigated compound was dissolved in dimethylsulfoxide as a
K. Breer et al. Inhibitors of purine nucleoside phosphorylase
FEBS Journal 277 (2010) 1747–1760 ª 2010 The Authors Journal compilation ª 2010 FEBS 1757
1 · 10
)2
m stock. All working dilutions (10

)4
–10
)7
m) were
prepared immediately before an experiment in NaCl ⁄ Pi.
At day zero, 1 · 10
5
cellsÆmL
)1
were plated onto 96-mi-
crowell plates and incubated overnight in a CO
2
incubator.
After 24 h, the medium was replaced with fresh medium
containing various well-defined concentrations of investi-
gated the compound. Controls were grown under the same
conditions without addition of the test compound. After
72 h of incubation, medium was removed and 40 lLof
MTT (5 mgÆmL
)1
of NaCl ⁄ Pi) was added, followed by the
addition of 10% dimethylsulfoxide with 0.01 molÆL
)1
HCl
to dissolve water-insoluble MTT-formazane crystals. The
plates were transferred to an ELISA plate reader, and A
570
was monitored. All experiments were performed at least in
triplicate with three wells each.
The percent of viable cells was determined by the equa-

tion:
Percentage viable cells ¼ðA
COMPOUND
À A
BLANCK
=
A
CONTROL
À A
BLANCK
ÞÂ100
where A
BLANCK
is the absorbance of the medium without
cells, but containing cytostatic and MTT, and A
CONTROL
is
the absorbance of cell suspension grown without DFPP-
DG.
The Kolmogorov–Smirnov test, a normality distribution
test, was applied. The differences between groups were
assessed by a nonparametric Kruskal–Wallis test
(P < 0.05). Statistical analyses were performed using stat-
istica software, version 8.0 (StatSoft, Inc, Tulsa, OK,
USA).
Acknowledgements
The authors thank Professor Vern L. Schramm for
providing the sample of immucillin H and Professor
David Shugar for careful reading of the manuscript.
This study was supported by the Polish Ministry of

Science and Higher Education N301 003 31 ⁄ 0042, Cro-
atian Ministry of Science, Education and Sports grant
No 219-0982914-2176 and the Ministry of Education,
Culture, Sports, Science and Technology of Japan.
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Supporting information
The following supplementary material is available:
Doc. S1. The dynafit 4.0 script used for a model
determination analysis.
Doc. S2. The dynafit scripts used for one-step and

two-step mechanism data simulations.
Fig. S1. The curves were simulated under the assump-
tion that no inhibitor was present (
), with presence of
0.35 lm (s), 0.5 lm (D) and 1.0 lm (•) inhibitor con-
centration and according to the parameters claimed in
the dynafit script.
Fig. S2. The curves were simulated under the assump-
tion that no inhibitor was present (
), with presence
of 0.1 lm (s), 0.5 lm (D) and 0.2 lm (•) inhibitor
K. Breer et al. Inhibitors of purine nucleoside phosphorylase
FEBS Journal 277 (2010) 1747–1760 ª 2010 The Authors Journal compilation ª 2010 FEBS 1759
concentration and according to the parameters claimed
in the dynafit script.
Fig. S3. Figure shows that the mixing during an exper-
iment has no effect on the rate of association of
DFPP-DG and calf spleen PNP.
Table S1. The dissociation constant with the confi-
dence intervals obtained from the fluorescence titration
curves.
Table S2. The association and dissociation rate con-
stants derived from the stopped-flow data. The two-
step model was used.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
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from supporting information (other than missing files)
should be addressed to the authors.
Inhibitors of purine nucleoside phosphorylase K. Breer et al.
1760 FEBS Journal 277 (2010) 1747–1760 ª 2010 The Authors Journal compilation ª 2010 FEBS