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: abzowska@biogeo.uw.edu.pl (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 à 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 à 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 [...]... Yokomatsu T (2007) Synthesis and biological evaluation 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 Yatsu T, Hashimoto M, Hikishima S, Magnowska M, Bzowska A & Yokomatsu T (2008) 9-Deazaguanine derivatives: synthesis and inhibitory properties as multisubstrate analogue inhibitors of. .. of purine nucleoside phosphorylase by phosphonoalkylpurines Nucleosides Nucleotides 8, 1039–1040 Halazy S, Ehrhard A & Danzin C (1991) 9-(Difluorophosphonoalkyl)guanines as a new class of multisubstrate analogue inhibitors of purine nucleoside phosphorylase J Am Chem Soc 113, 315–317 Naesens L, Snoeck R, Andrei G, Balzarini J, Neyts J & De Clercq E (1997) HPMPC (cidofovir), PMEA (adefovir) and related...K Breer et al observed for the total ligand concentration [L], and [Eact] the total concentration of enzyme binding sites Experimental data were fitted to the above equation, using nonlinear regression analysis, obtaining values for four fitted parameters: Kd, [Eact], fL and df = (fE + fL ) fEL) To derive the above equation, it was also necessary to assume that binding of the ligand to each subunit... were assessed by a nonparametric Kruskal–Wallis test (P < 0.05) Statistical analyses were performed using statistica software, version 8.0 (StatSoft, Inc, Tulsa, OK, USA) 4 5 6 7 8 9 10 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. .. J Control Release 107, 143–157 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 Stoeckler JD, Agarwal RP, Agarwal KC & Parks RE Jr (1978) Purine nucleoside phosphorylase from human erythrocytes... of Acyclovir and its metabolites on purine nucleoside phosphorylase J Biol Chem 259, 4065–4069 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-ribosyltransferases Biochemistry 42, 5694–5705 Nakamura CE, Chu S-H, Stoeckler JD & Parks RE Jr (1989)... absorption measurements Concentrations of the ligand varied in the range 0.05–6.4 lm 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 concentrations in the mixture, which are half of the initial values) The measurements were performed at 25 °C at pH... simultaneously analysed a few data sets for various protein concentrations Because the confidence intervals for nonlinear 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 calculated The 68% confidence intervals ranges, based on this analysis,... Minato K, 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 The following supplementary material is available: Doc S1 The dynafit 4.0 script used for a model determination analysis... related acyclic nucleosides phosphonate analogues: a review of their pharmacology and clinical potential in treatment of viral infections Antivir Chem Chemother 8, 1–23 de Clercq E, Andrei G, Balzarini J, Hatse S, Liekens S, Naesens L, Neyts J & Snoeck R (1999) Antitumor potential of acyclic nucleoside phosphonates Nucleosides Nucleotides 18, 759–771 Hikishima S, Hashimoto M, Magnowska L, Bzowska A & Yokomatsu . 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. 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