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

9-Deazaguanine derivatives connected by a linker to difluoromethylene phosphonic acid are slow-binding picomolar inhibitors of trimeric purine nucleoside phosphorylase Katarzyna Breer1, Ljubica Glavasˇ -Obrovac2, Mirjana Suver2, Sadao Hikishima3, Mariko Hashimoto3, Tsutomu Yokomatsu3, Beata Wielgus-Kutrowska1, Lucyna Magnowska1 and Agnieszka Bzowska1

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: abzowska@biogeo.uw.edu.pl

(Received 4 October 2009, revised 14 January 2010, accepted 29 January 2010)

is 147–389 pm). Stopped-flow experiments,

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 ± 13 pm (pH 7.0, 25 (cid:2)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) · 105 m)1Æs)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 together with the above data, are most consistent with a two-step binding mechanism: E + I M (EI) M (EI)*. The rate constants for reversible enzyme ⁄ inhibitor complex formation (EI), and for the conformational change (EI) M (EI)*, are kon1 = (17.46 ± 0.05) · 105 m)1Æs)1, koff1 = (0.021 ± 0.003) s)1, kon2 = (1.22 ± 0.08) s)1 and koff2 = (0.024 ± 0.005) s)1, respectively. This leads to inhibition constants for the first (EI) and second (EI)* complexes of i = 237 pm Ki = 12.1 nM (68% confidence interval is 8.7–15.5 nm) and K (cid:2) (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.

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Introduction

immunity), as shown by Giblett et al.

of

the

bond

purine

glycosidic

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

i

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 [2]. PNP catalyzes the reversible phosphorolytic cleavage of nucleosides: b-purine nucleoside + orthophosphate = purine base + 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 pKa 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].

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 (m7Guo) 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 ) were determined from initial velocity data with variable concentrations of both the inhibitor and the second substrate (m7Guo). 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 inhibitory activities of 9-(5¢,5¢-difluoro- comparison,

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).

To logically extend the above findings, we have synthesized a series of multisubstrate analogue inhi- bitors of PNP, namely 9-deazaguanine derivatives to difluoromethylene phos- connected by a linker phonic acid [10,11]. All of these 9-deazaguanine derivatives are potent inhibitors of calf spleen and inhibition human erythrocyte PNP, with apparent constants as low as approximately 5 nm; for example, 9-(5¢,5¢-difluoro-5¢-phosphonopentyl)-9-deazagua- for 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, It, is comparable with the total enzyme concentration, Et, which is in the nanomolar range, and under such conditions steady-state assumptions may not hold.

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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 lower than observed for DFPP-G (Table 1). i 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].

observed

those

i = 5.3 nm compared to 8.1 nm; Table 1),

Except for 9-(5¢,5¢-difluoro-5¢-phosphonohexyl)-9-de- azaguanine (homo-DFPP-DG) versus human erythro- even better binding cyte PNP, which exhibits for DGPP-DG properties than (K app the other derivatives with shorter and longer linkers exhibited weaker inhibitory effects.

Fig. 2. Inhibition of human erythrocyte PNP by DFPP-DG. m7Guo was a variable substrate. s, 8.4 lM; •, 12.8 lM; h, 25.2 lM;

, 210 lM.

Time-dependence of inhibition

[15] and a immucillin H [3],

5¢-phosphonopentyl)-guanine (DFPP-G) transition state analogue inhibitor, are also included.

in the nanomolar

,

i

i

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- inhibition (see the initial logues exhibit some 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 the of

the initial velocity studies

[18]. Therefore,

All compounds were found to be very potent inhibi- tors of m7Guo phosphorolysis, with apparent inhibi- tion constants, K app range. Inhibition is competitive versus nucleoside (m7Guo), and the apparent inhibition constants, K app , decrease with decreasing phosphate (fixed substrate) concentra- tion (Table 1). This indicates that the inhibitors bind

is an apparent inhibition constant observed by the classical

i

i

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 initial velocity method, whereas K eq 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 mM Hepes buffer (pH 7.0) at 25 (cid:2)C, with m7Guo 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 mM phosphate and various concentrations of inhibitor and, after a given time interval (0.5–120 min), activity was determined with 60 lM m7Guo (in 50 mM Hepes buffer, pH 7.0, at 25 (cid:2)C).

[nM]

)1Æs)1]

Compound

Phosphate concentration [mM]

K app i human PNP

K app [nM] i calf PNP

K eq [pM] i calf PNP

k [M calf PNP

720 ± 130

(4.5 ± 0.7) · 106

1 0.025

10.8 ± 0.7 – –

85 ± 13

(8.4 ± 0.5) · 105

8.1 ± 0.6 1.0 ± 0.2 5.3 ± 0.4 13 ± 1

DFPP-G DFPP-G DFPP-DG DFPP-DG DFPP-DG Homo-DFPP-DGb 6C-DFPP-DG Immucillin H Immucillin H

50 1 0.025 1 1 1 50

6.9 ± 0.7a 2.7 ± 0.2 28 ± 5 4.4 ± 0.6 1.0 ± 0.2 5.7 ± 0.6 21 ± 2 19 ± 2 41 ± 8c

–

23 ± 5d

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.

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M

)1Æs)1. Right: Determination of the inhibition constant at equilibrium, K eq

i

Fig. 3. Left: Time-dependence of inhibition of calf spleen PNP by DFPP-DG. PNP (2.3 nM subunits), DFPP-DG (s) 0 nM, (*) 1.0 nM, ()) 2.0 nM 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) · 105 , for interaction of DFPP-DG with calf PNP. Constants were obtained by fitting equation [2] to the equilibrium velocities, vs, obtained from experi- ments depicted in the upper panel. The K eq

value obtained from these data is 85 ± 13 pM.

i

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, vs were determined as shown in Fig. 3. From the set of vs for various inhibitor concentrations, the inhibition constant at equilibrium, K eq , was determined i by fitting Eqn (2) to the vs[I] ⁄ kc dependence, and was found to be K eq i = 85 ± 13 pm, 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

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 (8.4 ± 0.5) · 105 m)1Æs)1 for resulted in a value of 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 108 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).

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- to tions were used, does not unequivocally point binding as a two-step mechanism. It may simply be the observation of a process of achieving equilibrium

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

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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).

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

immucillin

constant

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 immucillins with interaction of trimeric PNPs) is also observed with DFPP-DG, but is not as well defined. In the initial phase of the reaction ((cid:3)10 min), 12.3 nm of immucillin H does not cause any inhibition of inosine phosphorolysis, by contrast to DFPP-DG. Ki 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 being for K eq i = 23 ± 5 pm [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).

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).

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 it is important to fit these two slow-onset binding, models to a set of progress curves using software based on numerically solving systems of differential equa-

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 identical affinity to ligand by ligand molecule but 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).

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560

540

520

) .

500

.

U A

(

480

460

440

l

e c n e c s e r o u F

420

400

380

0.0

0.1

0.2

0.3

0.4

0.5

0.6

4 3 2 1 0 –1 –2 –3

0.0

0.1

0.2

0.5

0.6

0.4 0.3 [DFPP-DG] (µM)

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, Kd1 (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, Kd = 3Kd1 (i.e. Kd = 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 ± 13 pm 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.

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.

150

560

145

540

140

520

) .

135

500

.

U A

130

480

125

460

120

440

115

l

420

110

( e c n e c s e r o u F

400

105

100

380

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14

0.0

0.1

0.2

0.3

0.4

0.5

0.6

1.0

0.2

0.5

0.1

0.0

0.0

–0.5

–0.1

–1.0

–1.2

–1.3

–1.5

0.0

0.1

0.2

0.5

0.6

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 [DFPP-DG] (µM)

0.4 0.3 [DFPP-DG] (µM)

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 lM (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 pM (68% confidence int- erval is 147–389 pM). The molar fluores- cence for protein complexes with one, two and three ligand particles are: fPL1 = 414.4 ± 20.7, fPL2 = 800.0 ± 30.8, fPL3 = 1070.3 ± 36.6 AU (left) and fPL1 = 414.7 ± 4.8, fPL2 = 725.9 ± 6.6, fPL3 = 1002.6 ± 7.5 AU (right).

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Stopped-flow measurements

constants derived from by other methods. Rate this model are: kon1 = (17.46 ± 0.05) · 105 m)1Æs)1, koff1 = (0.021 ± 0.003) s)1 for the (EI) complex and kon2 = (1.22 ± 0.08) s)1, koff2 = (0.024 ± 0.005) s)1 for the (EI)* complex, leading to inhibition constants for the (EI) and (EI)* complexes Ki = 12.1 nm (68% i = 237 pm confidence interval is 8.7–15.5 nm) and K (cid:2) (68% confidence interval is 123–401 pm), respectively. in excellent agreement with The second value is 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

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, kon1 = (16.6 ± 0.1) · 105 m)1Æs)1, koff1 = (0.0013 ± 0.0001) s)1, is an order of magnitude higher than the values derived from other methods (koff1 ⁄ kon1 = 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

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) · 106 m)1Æs)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

lymphocytes,

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 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 them from features, which separate non-T-cell malignancies [21].

DFPP-DG moderately affects growth of

and T-cell

(CCRF-CEM)

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 lM (black), 0.2 lM (grey) and 0.1 lM (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.

several leukaemia and lymphoma cell lines, especially T-cell leukaemias (Jurkat and MOLT), acute lymphoblastic leukaemia 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).

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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

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).

Percentage inhibition

DFPP-DG concentration

M

M

M

M

10)7

10)6

10)5

10)4

Cell line

Bood CCRF-CEM Jurkat MOLT-4 HuT78 K562 Raji HL-60 Hashimoto’s thyroiditis Graves’ disease

1.5 – 7.5 1.9 6.4 – 2.8 – 10.9 1.1

12.0 6.4 8.4 6.7 7.0 – 6.5 – 8.6 10.0

16.4 10.5 5.9 7.7 11.9* – 5.4 3.4 20.8* 15.3

13.8 49.4* 33.7* 33.3 25.4* 8.1 – 21.1 29.8* 28.6*

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.

compounds with properties

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 leukaemia autoimmune thyroid disorders and T-cell 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

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 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

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Inhibitors of purine nucleoside phosphorylase

Standard enzymatic procedures

Kinetic studies, if not otherwise indicated, were conducted at 25 (cid:2)C in 50 mm 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.

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) ((cid:3)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), Na2HPO4, NaH2PO4, m7Guo 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 (cid:2)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).

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 (cid:2)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 kobs = 300 nm and the molar extinction coefficient differ- ence between inosine and uric acid is De300 nm = 9600 m)1Æcm)1 [12].

for

e(261 nm) = 9540 m)1Æcm)1 and Concentrations of all substrates and inhibitors were determined spectrophotometrically using the extinction coefficients (at pH 7.0): e(260 nm) = 8500 m)1Æcm)1 for m7Guo (pKa (cid:3) 7.0), e(249 nm) = 12 300 m)1Æcm)1 for ino- sine (pKa 8.9), e(273 nm) = 10 100 m)1Æcm)1 for DFPP- DG (pKa 4.9) and homo-DFPP-DG, e(273 nm) = 9000 m)1Æcm)1 the DFPP-DG analogue with 6-carbon linker [9-(5¢,5¢-difluoro-5¢-phosphono-heptyl)-9-deazaguanine for (6C-DFPP-DG)], immucillin H [3].

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 m7Guo 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].

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.

Phosphorolysis of m7Guo was examined spectrophotomet- rically by a direct method [35]. The observation wavelength, kobs = 260 nm, corresponds to the maximal difference between extinction coefficients of nucleoside substrate, m7Guo, and the respective purine base, 7-methylguanine: De = 4600 m)1Æcm)1 at 260 nm at pH 7.0 for the mixture of cationic and zwitterionic forms of m7Guo [12,35].

Instrumentation

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 (cid:2)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, m7Guo or inosine). In the case of inosine phosphorolysis, xanthine oxidase was also present ((cid:3)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. 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 (Norwalk, CT, USA), using LS-50 spectrofluorimeter 4 · 10 mm cuvettes, with continuous mixing of the solu- tion. Stopped-flow kinetic measurements were

Initial rate procedures were employed in all kinetic studies. In the case of inhibition studies, for each combination of the initial substrate concentration, co, and the inhibitor concen- tration [I], the rates were determined at least twice. The initial velocities, vo, 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. 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. incubator CO2

Time-dependence of inhibition: progress curves

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Time-dependence of inhibition was measured using two inosine was the substrate and approaches. In the first, (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.

K. Breer et al.

Inhibitors of purine nucleoside phosphorylase

where [S] is the concentration of m7Guo (60 lm), and Km and kc are constants.

Fluorimetric titrations

titrations were conducted essentially

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

Fluorescence 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 (cid:2)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- 0.03 mL of the tein, assuming a one-step process for each binding site, is:

ka1 P þ L , kd1

PL1 Kd1 ¼ kd1=ka1

ð3Þ

PL2 Kd2 ¼ kd2=ka2

ka2 PL1 þ L , kd2

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 (cid:2)C in 50 mm 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– second substrate, m7Guo 120 min), (2000 lm), was mixed with 0.97 mL of the incubated solu- tion. The final concentration of m7Guo 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, vo(t, [I]), were measured.

PL3 Kd3 ¼ kd3=ka3

ka3 PL2 þ L , kd3

For each inhibitor concentration, the velocity at equilib- rium [i.e. at infinite time; vo(¥, [I])] (later referred to as vs[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], vo(t, [I]):

ð1Þ m0ðt; ½I(cid:4)Þ ¼ A expð(cid:5)ktÞ þ ms½I(cid:4) 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 ¼ ½P(cid:4) fP þ ½L(cid:4) fL þ ½PL1(cid:4) fPL1 þ ½PL2(cid:4) fPL2 þ ½PL3(cid:4) fPL3 ð4Þ

Fluorimetric titration data were

it may assumed hence, that be

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, Kd; hence, the appropriate equation is [36]:

In separate experiments, kc was determined as the initial velocity obtained at time t = 0 (i.e. no incubation) with a saturating concentration of m7Guo (120 lm) and in the absence of inhibitor [i.e. vo(0; [0]) = kc]. It was also found that 120 min of incubation has no influence on enzyme vo(0; activity; [0]) = vo(120 min; [0]). The Michaelis constant was deter- mined as previously described [12], and the value obtained, Km = 17 lm, 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]:

Parameters fE, fL and fEL, 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

(cid:3)

ð2Þ

(cid:2) ms½I(cid:4)=kc ¼ ½S(cid:4)= Kmð1 þ ½I(cid:4)=K eq

i þ ½S(cid:4)

q

0

1

þ

þ

(cid:5)

ð5Þ

@

Fð½L(cid:4)Þ ¼ F0 (cid:5) ðfE þ f L (cid:5) fELÞ

A þ ½L(cid:4)f L

[L] 2

½Eact(cid:4) 2

Kd 2

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ð½L(cid:4) (cid:5) ½Eact(cid:4) þ KdÞ2 þ 4½Eact(cid:4)Kd 2

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Inhibitors of purine nucleoside phosphorylase

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).

observed for the total ligand concentration [L], and [Eact] 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: Kd, [Eact], fL and df = (fE + fL ) fEL). 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.

the first,

the enzyme,

The measurements were performed at 25 (cid:2)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 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.

rate reaction constants (kdi)

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.

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 second and third ligand molecule (fPL1 „ fPL2 „ fPL3) 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: 3ka1 = 2ka2 = ka3 and kd1 = 2kd2 = 3kd3 between the association (kai) and (and dissociation 3Kd1 = Kd2 = Kd3 ⁄ 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 Kd = 3Kd1 because Kd refers to the protein monomer con- centration, but Kd1 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

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% CO2) CO2 incubator (Shell Lab) at 37 (cid:2)C. The trypan blue dye exclusion method was used to assess cell viability before each experiment.

Cytotoxicity test

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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 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.

K. Breer et al.

Inhibitors of purine nucleoside phosphorylase

state inhibitors for purine nucleoside phosphorylase. Biochemistry 37, 8615–8621. 1 · 10)2 m stock. All working dilutions (10)4–10)7 m) were prepared immediately before an experiment in NaCl ⁄ Pi. 4 Tuttle JV & Krenitsky TA (1984) Effects of Acyclovir

and its metabolites on purine nucleoside phosphorylase. J Biol Chem 259, 4065–4069.

5 Sauve AA, Cahill SM, Zech SG, Basso LA, Lewandowicz A, Santos DS, Grubmeyer C, Evans GB, Furneaux RH, Tyler PC et al. (2003) Ionic states of substrates and transition state analogues at the catalytic sites of N-ribo- syltransferases. Biochemistry 42, 5694–5705. 6 Nakamura CE, Chu S-H, Stoeckler JD & Parks RE Jr

(1989) Inhibition of purine nucleoside phosphorylase by phosphonoalkylpurines. Nucleosides Nucleotides 8, 1039–1040. At day zero, 1 · 105 cellsÆmL)1 were plated onto 96-mi- crowell plates and incubated overnight in a CO2 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 lL of 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 A570 was monitored. All experiments were performed at least in triplicate with three wells each. 7 Halazy S, Ehrhard A & Danzin C (1991) 9-(Dif- The percent of viable cells was determined by the equa- tion: luorophosphonoalkyl)guanines as a new class of multi- substrate analogue inhibitors of purine nucleoside phosphorylase. J Am Chem Soc 113, 315–317. Percentage viable cells ¼ ðACOMPOUND (cid:5) ABLANCK= ACONTROL (cid:5) ABLANCKÞ (cid:6) 100

8 Naesens L, Snoeck R, Andrei G, Balzarini J, Neyts J & De Clercq E (1997) HPMPC (cidofovir), PMEA (adefo- vir) and related acyclic nucleosides phosphonate ana- logues: a review of their pharmacology and clinical potential in treatment of viral infections. Antivir Chem Chemother 8, 1–23. where ABLANCK is the absorbance of the medium without cells, but containing cytostatic and MTT, and ACONTROL is the absorbance of cell suspension grown without DFPP- DG. 9 de Clercq E, Andrei G, Balzarini J, Hatse S, Liekens S,

nonparametric Kruskal–Wallis by a Naesens L, Neyts J & Snoeck R (1999) Antitumor potential of acyclic nucleoside phosphonates. Nucleo- sides Nucleotides 18, 759–771.

The Kolmogorov–Smirnov test, a normality distribution test, was applied. The differences between groups were assessed test (P < 0.05). Statistical analyses were performed using stat- istica software, version 8.0 (StatSoft, Inc, Tulsa, OK, USA).

Acknowledgements

10 Hikishima S, Hashimoto M, Magnowska L, Bzowska A & Yokomatsu T (2007) Synthesis and biological evalua- tion of 9-deazaguanine derivatives connected by a linker to difluoromethylene phosphonic acid as multi-substrate analogue inhibitors of PNP. Bioorg Med Chem Lett 17, 4173–4177.

11 Yatsu T, Hashimoto M, Hikishima S, Magnowska M, Bzowska A & Yokomatsu T (2008) 9-Deazaguanine derivatives: synthesis and inhibitory properties as multi- substrate analogue inhibitors of mammalian PNPs. Nucleic Acids Symp Ser (Oxf) 52, 661–662. 12 Bzowska A (2002) Calf spleen purine nucleoside

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.

phosphorylase: complex kinetic mechanism, hydrolysis of 7-methylguanosine, and oligomeric state in solution. Biochim Biophys Acta 1596, 293–317.

References

13 Wielgus-Kutrowska B & Bzowska A (2006) Probing the mechanism of purine nucleoside phosphorylase by steady-state kinetic studies and ligand binding charac- terization determined by fluorimetric titrations. Biochim Biophys Acta 1764, 887–902. 1 Bzowska A, Kulikowska E & Shugar D (2000) Purine nucleoside phosphorylases: properties, functions, and clinical aspects. Pharmacol Ther 88, 349–425.

FEBS Journal 277 (2010) 1747–1760 ª 2010 The Authors Journal compilation ª 2010 FEBS

1758

14 Cornish-Bowden A (1995) Analysis of the Enzyme Kinetic Data. Oxford University Press, New York. 15 Iwanow M, Magnowska L, Yokomatsu T, Shibuya S & Bzowska A (2003) Interactions of potent multi- substrate analogue inhibitors with purine nucleoside phosphorylase from calf spleen-kinetic and spectro- 2 Giblett ER, Ammann AJ, Wara DW, Sandman R & Diamond LK (1975) Nucleoside-phosphorylase defi- ciency in a child with severely defective T-cell immunity and normal B-cell immunity. Lancet 1, 1010–1013. 3 Miles RW, Tyler PC, Furneaux RH, Bagdassarian CK & Schramm VL (1998) One-third-the-sites transition-

K. Breer et al.

Inhibitors of purine nucleoside phosphorylase

fluorimetric studies. Nucleosides Nucleotides Nucleic Acids 22, 1567. delivery systems for antisense oligonucleotides. Colloids and Surfaces: Biointerfaces 16, 291–304. 16 Luic´ M, Koellner G, Yokomatsu T, Shibuya S &

30 Vinogradov SV, Zeman AD, Batrakova EV & Kabanov AV (2005) Polyplex Nanogel formulations for drug delivery of cytotoxic nucleoside analogs. J Control Release 107, 143–157. Bzowska A (2004) Calf spleen purine-nucleoside phos- phorylase: crystal structure of the binary complex with a potent multisubstrate analogue inhibitor. Acta Crystallogr D Biol Crystallogr 60, 1417–1424.

31 Galmarini CM, Warren G, Kohli E, Zeman A, Mitin A & Vinogradov SV (2008) Polymeric nanogels containing the triphosphate form of cytotoxic nucleoside analogues show antitumor activity against breast and colorectal cancer cell lines. Mol Cancer Ther 7, 3373–3380.

17 Breer K, Wielgus-Kutrowska B, Hashimoto M, Hikishi- ma S, Yokomatsu T, Szczepanowski R, Bochtler M, Girstun A, Staron´ K & Bzowska A (2008) Thermody- namic studies of interactions of calf spleen PNP with acyclic phosphonate inhibitors. Nucleic Acids Symp Ser (Oxf) 52, 663–664. 32 Stoeckler JD, Agarwal RP, Agarwal KC & Parks RE Jr (1978) Purine nucleoside phosphorylase from human erythrocytes. Methods Enzymol 51, 530–538.

18 Morrison JF & Walsh CT (1988) The behaviour and significance of slow-binding enzyme inhibitors. Adv Enzymol Relat Areas Mol Biol 61, 201–301.

33 Bzowska A, Luic´ M, Schro¨ der W, Shugar D, Saenger W & Koellner G (1995) Calf spleen purine nucleoside phosphorylase: purification, sequence and crystal struc- ture of its complex with an N(7)-acycloguanosine inhib- itor. FEBS Lett 367, 214–218. 19 Kuzmicˇ P (2008) A steady state mathematical model for stepwise ‘slow-binding’ reversible enzyme inhibition. Anal Biochem 380, 5–12. 34 Segel IH (1975) Enzyme Kinetics. John Wiley and Sons, 20 Eigen M & Hammes G (1963) Elementary steps in New York. enzyme reactions (as studied by relaxation spectrome- try). Adv Enzymol Relat Subj Biochem 25, 1–38.

21 Cooper TM (2007) Role of nelarabine in the treatment of T-cell acute lymphoblastic leukemia and T-cell lym- phoblastic lymphoma. Ther Clin Risk Manag 3, 1135– 1141. 35 Kulikowska E, Bzowska A, Wierzchowski J & Shugar D (1986) Properties of two unusual, and fluorescent substrates of purine-nucleoside phosphorylase: 7-meth- ylguanosine and 7-methylinosine. Biochim Biophys Acta 874, 355–366. 36 Eftink MR (1997) Fluorescence methods for studying

equilibrium macromolecule-ligand interactions. Methods Enzymol 278, 221–257. 22 Stassi G & de Maria R (2002) Autoimmune thyroid dis- ease: new models of cell death in autoimmunity. Nat Rev Immunol 2, 195–204.

37 Kuzmicˇ P (1996) Program DYNAFIT for the analysis of enzyme kinetic data: application to HIV proteinase. Anal Biochem 237, 260–273. 38 Horiuchi N, Nakagawa K, Sasaki Y, Minato K, 23 Stefanic M (2008) Recent advances in the immunopatho- genesis and genetics of autoimmune thyroid disorders. In Biochemistry and Immunology Intersections (Markotic A, Glavas-Obrovac Lj, Varljen J & Zanic-Grubisic T eds), pp. 123–150. Research Signpost, Kerala. 24 Weetman AP (2004) Cellular immune responses in

autoimmune thyroid disease. Clin Endocrinol 61, 405– 413. 25 Stefanic M, Papic S, Suver M, Glavas-Obrovac L & Fujiwara Y, Nezu K, Ohe Y & Saijo N (1988) In vitro antitumor activity of mitomycin C derivative (RM-49) and new anticancer antibiotics (FK973) against lung cancer cell lines determined by tetrazolium dye (MTT) assay. Cancer Chemother Pharmacol 22, 246–250.

Supporting information

Karner I (2008) Association of vitamin D receptor gene 3¢-variants with Hashimoto’s thyroiditis in the Croatian population. Int J Immunogenet 35, 125–131.

26 Blackburn GM & Kent DE (1986) Synthesis of alpha- and gamma-fluoroalkylphosphonates. J Chem Soc Perkin Trans I 1, 913–917.

27 Bzowska A, Koellner G, Wielgus-Kutrowska B, Stroh A, Raszewski G, Holy´ A, Steiner T & Frank J (2004) Crystal structure of calf spleen purine nucleoside phos- phorylase with two full trimers in the asymmetric unit: important implications for the mechanism of catalysis. J Mol Biol 342, 1015–1032.

28 Di Paolo A (2004) Liposomal anticancer therapy: phar- macokinetic and clinical aspects. J Chemother 16(Suppl 4), 90–93.

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

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29 Vinogradov SV & Kabanov AV (1999) Poly(ethylene glycol)-polyethylimine NanoGel particles: novel drug

K. Breer et al.

Inhibitors of purine nucleoside phosphorylase

This supplementary material can be found in the

online version of this article.

Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

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.

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