Thermodynamics and kinetics of inhibitor binding to human equilibrative nucleoside transporter subtype-1

G Model BCP 12388 No. of Pages 9

Biochemical Pharmacology xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm

Thermodynamics and kinetics of inhibitor binding to human equilibrative nucleoside transporter subtype-1 Shahid Rehan, Yashwanth Ashok, Rahul Nanekar, Veli-Pekka Jaakola* Oulu Biocenter and Faculty of Biochemistry and Molecular Medicine, University of Oulu, P.O. Box 3000, FI-90014 Oulu, Finland

A R T I C L E I N F O

A B S T R A C T

Article history: Received 4 August 2015 Accepted 24 September 2015 Available online xxx

Many nucleoside transport inhibitors are in clinical use as anti-cancer, vasodilator and cardioprotective drugs. However, little is known about the binding energetics of these inhibitors to nucleoside transporters (NTs) due to their low endogenous expression levels and difficulties in the biophysical characterization of purified protein with ligands. Here, we present kinetics and thermodynamic analyses of inhibitor binding to the human equilibrative nucleoside transporter-1 (hENT1), also known as SLC29A1. Using a radioligand binding assay, we obtained equilibrium binding and kinetic rate constants of well-known NT inhibitors – [3H]nitrobenzylmercaptopurine ribonucleoside ([3H]NBMPR), dilazep, and dipyridamole – and the native permeant, adenosine, to hENT1. We observed that the equilibrium binding affinities for all inhibitors decreased whereas, the kinetic rate constants increased with increasing temperature. Furthermore, we found that binding is enthalpy driven and thus, an exothermic reaction, implying that the transporter does not discriminate between its inhibitors and substrates thermodynamically. This predominantly enthalpy-driven binding by four chemically distinct ligands suggests that the transporter may not tolerate diversity in the type of interactions that lead to high affinity binding. Consistent with this, the measured activation energy of [3H]NBMPR association was relatively large (20 kcal mol
1) suggesting a conformational change upon inhibitor binding. For all three inhibitors the enthalpy (DH ) and entropy (DS ) contributions to the reaction energetics were determined by van’t Hoff analysis to be roughly similar (25–75% DG ). Gains in enthalpy with increasing polar surface area of inhibitors suggest that the binding is favored by electrostatic or polar interactions between the ligands and the transporter. ã 2015 Elsevier Inc. All rights reserved.

Chemical compounds studied in this article: NBMPR (PubChem CID: 5129) Dilazep (PubChem CID: 29976) Dipyridamole (PubChem CID: 3108) Adenosine (PubChem CID: 60961) Keywords: Nucleoside transporter Anti-cancer Thermodynamic Conformational change Activation energy

1. Introduction Nucleosides are nonlipophilic molecules which are substrates for DNA and RNA synthesis [2] and are also involved in cell signaling pathways, enzymes regulation and metabolism [3]. Nucleoside analogs (NA) are synthetic, chemically modified therapeutic molecules that mimic physiological nucleosides in terms of uptake and metabolism. NA’s therapeutic benefits stem from their ability to incorporate into newly synthesized DNA or

Abbreviation: hENT1, human equilibrative nucleoside transporter-1; NBMPR, nitrobenzylmercaptopurine ribonucleoside; ITC, isothermal titration calorimetry; PMSF, phenylmethanesulfonylfluoride; dpm, disintegrations per minute. * Corresponding author. Current address: Center for Proteomic Chemistry, Novartis Institute for Biomedical Research, Virchov 16-2.249.04, CH-4056 Basel, Switzerland. Fax: +41 61 324 8001. E-mail address: veli-pekka.jaakola@oulu.fi (V.-P. Jaakola).

RNA molecules, resulting in chain termination and eventually cell death, an ability which is used to inhibit cell growth in cancer [4]. Transport of nucleoside analogue drugs into the cell is a preliminary step toward their bioavailability and conversion into active drug. Their cellular uptake occurs via two distinct families of nucleoside transporter proteins (NTs); SLC28 and SLC29 [5]. Human equilibrative nucleoside transporter subtype-1 (hENT1), also called SLC29A1, is a member of the SLC29 family of NTs, and is the major plasma membrane NT. hENT1 is expressed in all tissue types. Cellular hENT1 protein levels have been shown to approximately double between G1 and G2-M phases of the cell cycle [6] indicating that hENT1 expression is cell cycle-dependent and high cellular proliferation rates are associated with high hENT1 expression [7,8]. This effect of elevated hENT1 expression on cellular proliferation appears to manifest as disease; for example, elevated hENT1 expression has been found in a variety of cancers including pancreatic adenocarcinoma [9] and breast cancer [10]. Because of the correlation between hENT1

http://dx.doi.org/10.1016/j.bcp.2015.09.019 0006-2952/ ã 2015 Elsevier Inc. All rights reserved.

Please cite this article in press as: S. Rehan, et al., Thermodynamics and kinetics of inhibitor binding to human equilibrative nucleoside transporter subtype-1, Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.09.019

G Model BCP 12388 No. of Pages 9

2

S. Rehan et al. / Biochemical Pharmacology xxx (2015) xxx–xxx

expression and disease, nucleoside transport inhibitors that decrease hENT1 activity present an alternative route in treatment of cancer as well as other diseases such as viral infection. Although, several nucleoside transport inhibitors are already in clinical use, the development of new inhibitors with better efficiency and efficacy is always needed due to drug resistance, poor oral bioavailability, long term toxicity and individualized response towards a particular drug [4,11]. This study lays the initial groundwork for structure-based design and development of new NT inhibitors by examining the kinetics and thermodynamics of inhibitor-NT binding. Thermodynamic characterization; quantitative determination of changes in all thermodynamic parameters, such as enthalpy (DH), entropy (DS) and Gibb’s free energy (DG) provides information about the energetic forces driving the binding reaction and is an important factor for optimizing the molecular interactions involved in inhibitor binding. Such information is undeniably significant for structure-based drug development [12]. However, the only direct method for measuring these critical parameters is isothermal titration calorimetry (ITC), which requires purified protein in sufficient quantities [13]. ITC analysis of integral membrane proteins, particularly receptors and transporters, is also hampered by their low endogenous expression levels, difficulties with heterologous expression, and a tendency towards aggregation after purification in detergents [14]. Alternative methods for determining thermodynamic parameters of cell surface receptors [15,16] and membrane transporters [17] take advantage of radiolabeled ligand to measure kinetic parameters across different temperatures and then extract thermodynamic parameters using a van’t Hoff analysis. Here we report the use of radioligand binding assay to describe the kinetics and thermodynamics of binding of clinically relevant inhibitors—nitrobenzylmercaptopurine ribonucleoside (NBMPR), dilazep and dipyridamole (Fig. 1) to hENT1. This work is intended to inform and accelerate future structure-based drug design of new NT inhibitors.

2. Materials and methods 2.1. Materials SLC29A1 antibody produced in rabbit (HPA012384) and adenosine was purchased from Sigma, USA; goat anti-rabbit IgG-horseradish peroxidase (RPN4301) and PVDF membrane was purchased from AmershamTM, UK. ECL Plus Western detection reagents were from Thermo Fisher, USA. Insect cell culture media was from Lonza, Switzerland. [3H]NBMPR with a specific activity of 8.43 Ci/mmol was purchased from Moravek Biochemicals, USA. NBMPR, dilazep and dipyridamole were purchased from Tocris Bioscience, UK. GF/B glass filter 96 well plates were purchased from Millipore, USA. 2.2. Generation of recombinant hENT1 for insect cell expression Full-length hENT1 (NCBI gene ID: 2030) was PCR amplified and cloned into a pFastBac1 vector (Invitrogen, USA) using gene specific primers containing sites for SphI and KpnI restriction enzymes (New England Biolabs, USA). This construct was transformed into DH10Bac competent cells (Invitrogen, USA) to create a recombinant bacmid capable of expressing hENT1 in Sf9 cells. Sf9 insect cells transfection was carried out according to Invitrogen’s protocol. Recombinant baculovirus was passaged three times to obtain high titer virus and protein expression was initiated by infecting 1.5  106 cells/ml of Sf9 cells at MOI (multiplicity of infection) of 1–2 for 48 h. Cells were harvested by centrifugation at 1000g for 5 min and stored at
70  C until further analysis. 2.3. Preparation of Sf9 membranes expressing hENT1 Sf9 membranes were prepared as described previously [18]. Briefly, frozen cell pellets were resuspended in 2 insect cell lysis buffer (10 mM HEPES pH 7.4, 10 mM MgCl2, 200 mM NaCl, 10% glycerol, 5 mM EDTA, 1 mM PMSF) and lysed using a dounce homogenizer. Cell lysate was ultracentrifuged at 235,000g for 1 h

Fig. 1. Chemical structures of hENT1 inhibitors. Inset; Ki values of nucleoside transport inhibitors of pharmacologically relevant hENTs subtypes.

Please cite this article in press as: S. Rehan, et al., Thermodynamics and kinetics of inhibitor binding to human equilibrative nucleoside transporter subtype-1, Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.09.019

G Model BCP 12388 No. of Pages 9

S. Rehan et al. / Biochemical Pharmacology xxx (2015) xxx–xxx

to isolate the membrane fraction. Isolated membranes were resuspended in solubilization buffer (50 mM HEPES pH 7.4, 800 mM NaCl, 10% glycerol) and stored at
70  C until further analysis. 2.4. Western blotting The presence of recombinant hENT1 transporter in isolated membranes was confirmed by Western blotting analysis. Briefly, 10 mg of total isolated membrane was loaded on 12% SDS-PAGE gel. After electrophoresis, protein was transferred by electroblotting on a PVDF membrane under semi dry conditions, followed by overnight blocking at 4  C in blocking buffer (10 mM Tris pH 7.5, 100 mM NaCl, 0.01% Tween 20 and 2.5% BSA). Membrane was incubated with 1:10000 dilution of SLC29A1 primary antibody for 1 h at room temperature and washed three times for 1 h. Goat antirabbit IgG-horseradish peroxidase was used as secondary antibody at 1:40000 dilutions. Membrane was washed and protein was detected using ECL plus Western detection reagents, following manufacturer’s protocol. 2.5. Incubation conditions for [3H]NBMPR saturation binding studies All buffers, ligands and membranes were pre-incubated for two hours at 0  C, 4  C, 14  C, 22  C, 30  C and 37  C unless stated otherwise. In all the experiments, care was taken so that total binding never exceeded 10% of the [3H]NBMPR present in the assay. Dissociation constants (Kd) and total binding sites (Bmax) were calculated from saturation binding experiments at different temperatures as described previously [1]. Briefly, membranes isolated from Sf9 cells expressing hENT1 were diluted in Tris buffer (10 mM Tris–HCl, pH 7.5) to a final volume of 2 ml. Binding was initiated by incubating membranes (10 mg/well) with increasing

3

concentrations of [3H]NBMPR (from 0.1 to 15 nM) for 40–60 min at the indicated temperatures. The incubation time for each temperature condition was empirically selected with the aim of reaching equilibrium binding as conducted previously [1]. Nonspecific binding was assessed using 10 mM of dipyridamol. Unbound ligand was removed by placing the reactions into wells of 0.2 mm 96-well glass filter plates (Millipore) and washing with ice cold Tris buffer under vacuum. The filter plates were dried at room temperature before addition of 30 ml of scintillation liquid. Radioactivity was measured using a MicroBeta Trilux scintillation counter (PerkinElmer). The amount of [3H]NBMPR specifically bound to Sf9 membranes was calculated as the difference between amount of [3H]NBMPR that bound in the presence and absence of 10 mM dipyridamole. Kd and Bmax were calculated by fitting experimental data to a non-linear regression one site-specific binding equation using Graphpad Prism software. 2.6. Incubation conditions for competition binding studies For competition binding experiments, increasing concentrations of either dilazep or dipyridamole were pre-incubated with [3H]NBMPR at a concentration of 2–3 times the Kd for 40–60 min at the indicated temperatures. The incubation time for competition binding at each temperature is selected based on experimental observation that confirmed the displacement of 75% or above. At the end of the preincubation time, membranes were added and incubated for the indicated time. Bound and unbound [3H]NBMPR were separated by filtration under vacuum as described above. Apparent inhibition constants (Ki values) were calculated according to Cheng and Prusoff equation below [19]. Ki ¼

IC50 ð1 þ ½L=K d Þ

Fig. 2. Expression of hENT1 in Sf9 insect cells. (a) Graphical representation of the expression construct used in this study. (b) Western blotting analysis of hENT1 using SLC29A1 antibodies. (c) Effect of membrane concentrations on total and non-specific binding of 2.5 nM [3H]NBMPR to hENT1 transporter. Various concentrations of Sf9 membrane expressing hENT1 were titrated with 2.5 nM [3H]NBMPR at 22  C using similar incubation conditions as described in Section 2. Non-specific binding was determined with 10 mM dipyridamole. (d) Non-specific binding expressed as% of total binding at different membrane concentrations per well.

Please cite this article in press as: S. Rehan, et al., Thermodynamics and kinetics of inhibitor binding to human equilibrative nucleoside transporter subtype-1, Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.09.019

G Model BCP 12388 No. of Pages 9

4

S. Rehan et al. / Biochemical Pharmacology xxx (2015) xxx–xxx

where Ki is the equilibrium discosiaton constant for unlabeled inhibitor, IC50 is the concentration causing 50% inhibition, L is the concentration of the radioligand and Kd is the equilibrium dissociation constant for the radioligand. 2.7. Incubation conditions for kinetic studies Sf9 membranes were incubated with [3H]NBMPR at a concentration of 2–3 times the Kd for 60–80 min (until equilibrium is reached). Dissociation was initiated by adding 104-fold excess of unlabeled NBMPR. Fractions of the assay mixture were taken at different time points and filtered under vacuum as described above. The dissociation rate constant (k
1) was determined by fitting the experimental data to a non-liner one phase exponential decay equation using Graphpad prism. For association experiments, [3H]NBMPR was incubated with membranes, with fractions of the assay mixture collected at different time points and processed as above. The association rate constant (k + 1) was calculated by fitting the experimental data to a non-linear one phase exponential association equation using Graphpad Prism. 2.8. Thermodynamic data analysis Thermodynamic parameters such as Gibb’s free energy (DG ), enthalpy (DH ) and entropy (DS ) were calculated using a van’t Hoff analysis and the equation: 



and competition binding studies, determined across various temperatures (0  C, 4  C, 14  C, 22  C, 30  C and 37  C). The excitation energy of [3H]NBMPR binding to the hENT1 transporter was estimated using the Arrhenius equation: lnðk þ 1Þ ¼

lnA
Ea RT

where (k + 1) is the association rate constant of the reaction; A is the Arrhenius constant; and Ea is the excitation energy of the inhibitor–transporter binding reaction. The change in Gibbs free energy represents the temperature dependence of the enthalpy and under the assumption that entropy is constant at all temperatures, it can be estimated from 

DG ¼
RTðlnKAÞ 

DH ¼
RTlnKA þ DS



Heat capacities of inhibitor binding were calculated from the slopes of T vs DH plots using the equation: " #  dðDH Þ Cp ¼ T

3. Results 3.1. hENT1 expression in Sf9 cells



DG ¼ DH
T DS ¼ RTlnK A The standard enthalpy of binding can be calculated from the slope,
DH /R and the standard entropy of binding from the intercept, DS /R or as DS = (DH
DG )/T with T = 298.15 K and 1.987 kcal mol
1. KA is the association binding constant of the inhibitors (i.e., 1/Kd). These analyses were carried out by saturation

Baculovirus mediated expression of hENT1 in Sf9 cells resulted in plasma membrane insertion of the transporter, as confirmed by Western blotting analysis of isolated membrane fractions. Using the hENT1-specific antibody, a major band migrating at the expected hENT1 size of 45–50 kDa is clearly visible while two other bands at 80 kDa and 110 kDa are also detected (Fig. 2b).

Fig. 3. Representative saturation binding isotherm of [3H]NBMPR binding to hENT1. Panels (a) and (c) represent saturation analysis of binding of [3H]NBMPR to isolated Sf9 membranes expressing hENT1 at 0  C and 22  C, respectively. hENT1 membranes (10 mg/well) were incubated for 1 h with increasing concentrations (0.125–10 nM) of [3H] NBMPR in the absence (total binding) and presence of 10 mM dipyridamole (non-specific binding). Each point is the average of at least three measurements and each experiment was repeated at least four times under same experimental conditions. Scatchard transformation of the saturation binding data for [3H]NBMPR at 0  C (b) and 22  C (d) are also shown.

Please cite this article in press as: S. Rehan, et al., Thermodynamics and kinetics of inhibitor binding to human equilibrative nucleoside transporter subtype-1, Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.09.019

G Model BCP 12388 No. of Pages 9

S. Rehan et al. / Biochemical Pharmacology xxx (2015) xxx–xxx

3.2. Effect of incubation temperature on [3H]NBMPR specific binding At [3H]NBMPR concentration of 2.5 nM, specific binding increased as the concentrations of hENT1-expressing Sf9 membranes increased, although concentrations above 10 mg/well resulted in significantly increased non-specific binding of up to 50% (Fig. 2c and d). With 10 mg/well of hENT1-membranes and 2.5 nM [3H]NBMPR, incubation temperature had no effect on specific binding: 0  C = 91  2%, 4  C = 94  2%, 14  C = 90  6%, 22  C = 89  5%, 30  C = 95  5%, 37  C = 87  7%; n = 8 (specific binding expressed as a percentage of total binding). Even though, we were able to detect specific [3H]NBMPR binding with as little as 0.5 mg/well of hENT1 membranes, we used 10 mg/well in all subsequent experiments for more optimal total vs non-specific binding signal (dpm values). Under these conditions, total binding never exceeded the 10% of added [3H]NBMPR in the assay. 3.3. Saturation and competition binding experiments To obtain the data for van’t Hoff analysis of thermodynamics of the inhibitor binding, we conducted saturation binding studies of [3H]NBMPR with Sf9 membranes expressing hENT1 across multiple temperatures (0  C, 4  C, 14  C, 22  C, 30  C and 37  C). Fig. 3 shows a representative saturation binding experiment of [3H] NBMPR with hENT1 membranes at 0  C and 22  C. Four experiments gave similar results yielding Kd values of 0.20  0.02 nM and 0.66  0.09 nM at 0  C and 22  C, respectively (Table 1). Scatchard transformation of the saturation binding data for [3H] NBMPR at 0  C (Fig. 3b) and 22  C (Fig. 3d) are also shown. Within the concentration range tested, all Scatchard plots were linear (R2  0.9) and analogously, non-linear regression analysis of the saturation experiments suggests a one-site binding model. We calculated inhibitory constants (Ki) through competition binding experiments using labeled [3H]NBMPR and unlabeled dilazep or dipyridamole (Table 1). Representative data for competition studies at 0  C, 22  C and 37  C are shown in Fig. 4. For both dilazep and dipyrimadole, these studies support a onesite binding model at all temperatures except 37  C where the data support a two binding site model (Fig. 4c and f). The affinity of hENT1 membranes for both [3H]NBMPR and unlabeled inhibitors varied with temperature (Table 1). We observed nearly a 10-fold increase in the Kd of [3H]NBMPR and a 5-fold increase in the Ki of both dilazep and dipyridamole when temperature was increased from 0  C to 30  C. Similarly, adenosine affinity also decreased with an increase in temperature, suggesting comparable binding thermodynamics for the native permeant of hENT1 (Table 1). A complete list of equilibrium biding and kinetic rate parameters—Kd, Bmax, Ki and k + 1/k
1 calculated for the tested

5

hENT1 inhibitors at different temperatures are listed in Table 1. While both Kd and Ki are temperature dependent, Bmax seems independent of temperature, suggesting identical transporter populations. 3.4. Kinetics of hENT1 inhibitors For all temperatures tested (4  C, 22  C, 30  C), dissociation rate constants for [3H]NBMPR fell within a broad range (k
1 = 3– 36 s
1), with corresponding half-lives of 60–900 s. As expected, association and dissociation rates for [3H]NBMPR binding increased with temperature over the range tested (Fig. 5). [3H] NBMPR showed a significant tendency towards slower association (k + 1) and dissociation (k
1) rate constants as affinity increased (Table 1). The kinetics of [3H]NBMPR at 0  C was quite unusual as association was somewhat similar to 4  C but dissociation was exceptionally slow (data not shown). On the other hand, data at 37  C is unreliable due to very fast kinetics which exceeds the sensitivity range of the instrumentation and methods used. Therefore, data sets from both 0  C and 37  C are excluded from our kinetics analysis. 3.5. Calculation of thermodynamic parameters We have used the affinity data obtained at 0  C and 30  C to construct van’t Hoff plots in order to better understand the mode of inhibitor binding to hENT1 (Fig. 6a and Table 2). Since ligandreceptor binding typically possesses a linear van’t Hoff plot (Lee, 1994), the slope of the van’t Hoff plot (ln K vs 1/T) can reveal whether binding is endothermic (negative slope) or exothermic (positive slope). In our experiments, the van’t Hoff analysis showed a linear relationship between the natural logarithm of the association constant (ln KA =
ln Kd) and 1/T over the range of temperatures tested for all three inhibitors—[3H]NBMPR, dilazep and dipyridamole. For each inhibitor tested, enthalpy (DH ) and entropy (DS ) were calculated from the slopes and y-intercepts of the van’t Hoff plot based on the 95% confidence interval in all cases (Fig. 6a and Table 2). The slopes of van’t Hoff plots for all inhibitors were positive, supporting an exothermic binding reaction model and therefore, inhibitor binding that is essentially enthalpy driven. The relative contributions of entropic and enthalpic changes for all inhibitors were roughly similar (25–75% of DG ; Table 2). Consistent with this analysis, the Gibb’s free energies and equilibrium binding affinities increased linearly with temperature, suggesting that DH has temperature dependence. Assuming that the entropy of binding is independent of temperature, the enthalpy of binding at every temperature tested was found to vary linearly when calculated using the equation: DH =
RT(ln KA) + TDS (Fig. 6b).

Table 1 Equilibrium binding affinities and kinetic rate constants of clinically relevant hENT1 inhibitors and physiological permeant adenosine, observed at different temperatures. Temperature ( C) Inhibitor/ligand 3

0

4

14

22

30

37

[ H]NBMPR

Kd (nM) Bmax (pmol/mg) k + 1 (M
1 min
1) k
1 (min
1) Half-life (t1/2) min Kd (nM) k
1/k + 1

0.20  0.02 139  4 – – – –

0.36  0.07 146  6 2.52  107 0.01 14.6 0.39

0.43  0.05 141  9 – – – –

0.66  0.05 166  9 4.31 108 0.22 3.0 0.51

1.0  0.10 148  8 4.95  108 0.60 1.2 1.2

2.3  0.20 143  3 – – – –

Dilazep Dipyridamole Adenosine

Ki (nM) Ki (nM) Ki (mM)

1.6  0.05 13  0.04 0.10  0.02

2.4  0.04 23.9  0.10

3.1  0.05 24.4  0.10

4.3  0.05 27.3  0.03 0.32  0.05

4.8  0.02 40.2  0.05 0.52  0.02

– –

Please cite this article in press as: S. Rehan, et al., Thermodynamics and kinetics of inhibitor binding to human equilibrative nucleoside transporter subtype-1, Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.09.019

G Model BCP 12388 No. of Pages 9

6

S. Rehan et al. / Biochemical Pharmacology xxx (2015) xxx–xxx

We used the slopes of the DH vs T plots, to calculate the heat capacities (Cp) of NBMPR, dilazep, and dipyridamole binding to the transporter. Cps for all three inhibitors fall in the narrow range of 0.01 to 0.02 kcal mol
1 (Fig. 6b). To calculate the binding activation energy (Ea), we used the Arrhenius equation (Fig. 6c). We found a linear correlation between association rate constants and temperatures, and an Ea for [3H]NBMPR–hENT1 binding (calculated from the slope of the Arrhenius plot) of 20 kcal mol
1. The measured enthalpy and entropy contributions for all inhibitors tested were inversely correlated with each other (Fig. 6d). 4. Discussion NT inhibition has a well-established role in the treatment of many diseases including cancer, HIV, and cardiovascular diseases [11,20,21]. While several ENTs inhibitors such as dilazep, dipyridamole and draflazine are currently in clinical use, the development of novel inhibitors with better efficiency and efficacy is always needed due to drug resistance, poor oral bioavailability and long term toxicity of a particular inhibitor. Unfortunately, the absence of high resolution molecular models of hENT1 and other mammalian nucleoside transporters preclude structure-based drug design of molecules that block their nucleoside reuptake activities. Due to challenges in biophysical characterization of the isolated transporter, there is no data available that deals with the nature and relative contributions of interactions between inhibitors and hENT1, and that can account for transporter conformational transitions induced by the ligand. In addition, the usual ligand binding experiments of hENT1 with [3H]NBMPR are typically conducted at a single temperature allowing one to calculate only the DG of the equilibrium but not its two components i.e., DH and DS [22,23]. Current information from non-human NT homologs has been equally uninformative for structure-based drug design studies. The only crystal structure solved of a member of the SLC28 family of

NTs is from Vibrio cholerae [24], but is irrelevant to hENT1 due to differences in membrane topology, poor sequence homology, different substrate specificities, and a proton dependent transport mechanism. NTs are believed to share some common features with MFS (major facilitator superfamily) transporters. However, our attempts of building a robust homology model for hENT1 based on the structure of the glycerol transporter GlpT (a member of MFS family) failed to give any significant information about the nature and binding residue contributions of nucleoside binding pocket (data not shown). Without a structural model in hand, understanding the thermodynamic forces driving inhibitor binding to hENT1 becomes critical for structure-based drug design attempts. We report here, for the first time, the thermodynamics of well characterized hENT1 inhibitors using radioligand binding assays and recombinant hENT1 expressed in the membranes of Sf9 cells. In order to use ligand binding assays to determine the thermodynamic parameters of ligand–receptor/transporter interaction, four conditions should be met [25]. The binding reaction must reach equilibrium, binding should be measured for single class or subtype of the receptor, multiple temperatures should be used, and the receptor/transporter must be in its pharmacologically relevant state during all experimental conditions tested. We have attempted to meet all four conditions by conducting saturation and competition binding experiments at six different temperatures. To avoid ambiguity in our experiments, we first made sure that Sf9 membranes alone (untransfected with recombinant hENT1 baculovirus) show no traces of [3H]NBMPR binding at all temperatures tested. Thus, all subsequent data was purely associated with recombinant hENT1 and not the indigenous nucleoside transporters of insect cells (data not shown). The Kd of [3H]NBMPR and Ki of non-radiolabeled inhibitors with hENT1-expressing Sf9 membranes at 22  C correspond well with existing data from the literature [26], and found to have similar temperature

Fig. 4. Representative competition binding analysis hENT1 inhibitors. Competition binding analysis of dialzep (a–c) and dipyridamole (d–f) to hENT1 transporter at 0  C 22  C and 37  C, respectively. Results are shown as the percentages of [3H]NBMPR bound as function of logarithm of the concentration of dilazep or diypridamole. The amount of [3H]NBMPR that bound in the absence of inhibitors was taken as 100% binding. Each data point is the average of at least three measurements and each experiment was repeated at least four times under same experimental conditions.

Please cite this article in press as: S. Rehan, et al., Thermodynamics and kinetics of inhibitor binding to human equilibrative nucleoside transporter subtype-1, Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.09.019

G Model BCP 12388 No. of Pages 9

S. Rehan et al. / Biochemical Pharmacology xxx (2015) xxx–xxx

7

affinity binding site [28]. Competition binding experiments of [3H] NBMPR with dipyridamole have found a pseudo-Hill coefficient that was not equal to unity, signifying the presence of cooperativity or multiple sites [29]. In another study, differences in the rate of competitive dissociation of NBMPR by nucleosides and inhibitors such as dilazep and dipyridamole were found, again suggesting the presence of multiple sites or co-operativity [29,30]. However, it is still disputed whether the transporter is a monomer with a single binding site or allosteric sites or exists as an oligomer with one site on each subunit. To date no experimental evidence has been published that clearly supports either of these possibilities. Indeed, the presence of a higher molecular weight band between 80 and 110 kDa on Western blots [31] and on SDS-PAGE gels of purified protein [18] supports the existence of oligomeric states. However, hENT1 show great diversity in size on SDS-PAGE due to varying degree of glycosylation [1]. Since, we only find evidence for more than one binding site at 37  C and 37  C is closer to ambient human body temperature; we propose that hENT1 may exist as an oligomer in its native membrane, but that oligomerization is not essential for the functioning of the protein. Furthermore, although NBMPR, dilazep and dipyridamole competed with adenosine for binding, it is still not clear whether nucleoside and inhibitor bind to the same pocket or share residues of closely located but distinct binding pockets. Such information remains to be elucidated through comprehensive mutagenesis studies of the transporter, or by the challenging task of ligandtransporter co-crystallization. 4.2. The thermodynamic and kinetic analysis

Fig. 5. Kinetic analysis of [3H]NBMPR binding to hENT1 at different temperatures. Association and dissociation of [3H]NBMPR binding with Sf9 membranes expressing hENT1 at 4  C (a), 22  C (b) and 30  C (c). Data shown represent the time course of association and dissociation of [3H]NBMPR binding to hENT1 transporter in the absence and presence of unlabeled NBMPR, respectively. Each point is the average of six measurement obtained from three independent experiments under similar conditions. Association and dissociation rate constants were calculated as described in Section 2.

dependence as observed previously [23], signifying wild type-like functional expression. 4.1. Number of ligand binding sites Our studies support the presence of two binding sites at higher temperatures (37  C) but not lower temperatures. We interpret this to mean that the transporter may exist in a low affinity state with respect to the second binding site, and that higher temperatures modulate the binding of nucleoside at this site. Such behavior is well reported for some GPCRs, including the adenosine A1 receptor [27], but not for the nucleoside transporters. This finding that hENT1 may have two ligand binding sites is in agreement with previous studies. Studies by Gati and Paterson support the presence of two permanent ligand recognition sites on hENT1—one high affinity site for NBMPR and other substrates, and a second low affinity site that may allosterically modulate the high

From measured equilibrium binding affinities at different temperatures (0  C, 4  C, 14  C, 22  C and 30  C) we have calculated the relative contributions of entropic and enthalpic changes to reaction energetics by van’t Hoff analysis. We found that the binding of all inhibitors and adenosine seems to be enthalpically favored i.e., an exothermic reaction. This further implies that the transporter does not discriminate between inhibitors and substrates from a thermodynamic point of view. However, experimental verification is still needed on whether other inhibitors and nucleosides follow similar binding thermodynamics. Nevertheless, our findings clearly imply that the affinity of hENT1 for [3H]NBMPR, dilazep, dipyridamole and adenosine varied with temperature. The relative contributions of entropic and enthalpic changes for all inhibitors were roughly similar (25–75% of the total binding energy). Likewise, the inverse correlation between entropy and enthalpy changes for all inhibitors were consistent with the more general phenomenon of entropy–enthalpy compensation of protein–ligand interaction [32]. In the entropy–enthalpy compensation phenomenon, a gain in enthalpy from protein–ligand bond strengthening (e.g., van der waals interaction) is counteracted by concomitant losses in entropy due to conformational changes and solvation effects [33,34]. A gain in enthalpy with increasing polar surface area of inhibitors suggests that the binding is favored by electrostatic or polar interactions between the ligand and the transporter. Pharmacophore modeling and computational analysis of hENT1 has also indicated the presence of highly positively charged residues in the ligand binding pocket which interact with the negatively charges moieties of NBMPR [35]. Furthermore, the manifestation of a single binding mode (enthalpy driven) by three chemically distinct inhibitors and adenosine may suggest that the binding pocket of hENT1 will not tolerate diversity in the types of interactions that result in high affinity binding. This limits the theoretically exploitable space for new hENT1 inhibitors and emphasizes the importance of hENT1 crystallization studies for structure-guided drug designing.

Please cite this article in press as: S. Rehan, et al., Thermodynamics and kinetics of inhibitor binding to human equilibrative nucleoside transporter subtype-1, Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.09.019

G Model BCP 12388 No. of Pages 9

8

S. Rehan et al. / Biochemical Pharmacology xxx (2015) xxx–xxx

Fig. 6. Thermodynamic analysis of inhibitor binding to hENT1 transporter. van’t Hoff plot analysis of inhibitors binding to hENT1 (a). Each point plotted represents the association constant, KA (i.e., 1/Kd or 1/Ki) determined by saturation or competition binding experiments at controlled temperatures (0  C, 4  C, 14  C, 22  C and 30  C). Each experiment was repeated at least four times at the indicated temperature under the same experimental conditions. (b) Estimated change in Gibbs free energy with temperature. (c) Arrhenius plot showing the effect of temperature on the association rate constants of [3H]NBMPR calculated at 4  C, 22  C and 30  C. The energy of activation (Ea) for inhibitor–transporter binding was determined from slope of the best-fit line obtained from Arrhenius equation (see Section 2). Each data point plotted shows a single estimate of k + 1 at respective temperature. (d) Inverse correlation between entropy (
TDS ) and enthalpy (DH ) for all inhibitors tested demonstrating entropy–enthalpy compensation phenomenon. Values of enthalpy and entropy were determined using van’t Hoff plot analysis (See Table 2).

The effect of temperature on the kinetics of [3H]NBMPR binding to hENT1 was assessed in dissociation and association binding experiments. The association and dissociation rate constants observed at 22  C were similar to the literature data [36]. We have found a linear relationship between ln KA and 1/T in all experiments, presumably reflecting a linear relationship between temperature and enthalpy that results from constant Cp across all experimental conditions. This implies a lack of substantial conformational changes upon inhibitor binding [37]. However, the effect of temperature on the forward kinetic constant of [3H]NBMPR binding yielded an Ea value of 20 kcal mol
1. This value is more typical of protein–ligand interactions in which the heat capacity changes with temperature [17,38]. Although high Ea and temperature dependence of Cp are incompatible, they can coexist if small, self-cancelling effects contribute to DCp. This is likely to be the actual scenario in our system and may imply that the nucleoside/inhibitor binding pocket of the hENT1 transporter is very small relative to the size of the protein. Furthermore, the effects of non-polar and polar interactions between transporter and ligand are also likely to cancel out each other’s impact on DCp. Similar studies on other membrane proteins with comparable DG values for both entropy- and enthalpy-

driven binding indicate minimal ligand-induced conformational changes [12]. In such studies, van’t Hoff plots were linear, predicting a similar temperature independence of heat capacities, possibly due to similar cancellation effects and relatively small ligand binding pockets compared to the overall protein structure. The overall thermodynamic data obtained in our study correlated well with similar studies carried out previously for other membrane proteins, including ion channels, transporters and membrane-bound receptors binding to small molecule agonist and antagonist [12]. While further studies are needed to confirm our findings – optimally through ITC or similar methods using purified proteins – the similarities we found in the van’t Hoff analysis on multiple inhibitors diminishes the influence of experimental constraints and representational bias. The direct measurements of binding energetics of clinically relevant NT inhibitors remain elusive due to difficulties related to biophysical characterization of purified NTs. To our knowledge, this is the first study that reports the thermodynamics of well-known inhibitors of the NT hENT1. We have shown that the affinity of NBMPR, dilazep and dipyridamole to hENT1 decreases with an increase in temperature within the ranges tested, suggesting that inhibitor binding is essentially enthalpy driven, hence an exothermic reaction. We observed that the equilibrium binding

Table 2 Thermodynamic parameters of hENT1 inhibitors. Inhibitor 3

[ H]NBMPR Dilazep Dipyridamole

Slope

y-Intercept

DS (Kcal mol
1 K
1)

DH (Kcal mol
1)


TDS at 25  C

DG (Kcal mol
1)

3437  513 2906  401 3714  694

4.9  2 7.7  1 4.5  1

0.01 0.01 0.01


6.8  1.0
5.8  1.0
7.4  1.3


2.9
4.6
2.7


9.7
10.4
10.1

Please cite this article in press as: S. Rehan, et al., Thermodynamics and kinetics of inhibitor binding to human equilibrative nucleoside transporter subtype-1, Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.09.019

G Model BCP 12388 No. of Pages 9

S. Rehan et al. / Biochemical Pharmacology xxx (2015) xxx–xxx

affinities for all inhibitors decreased as temperature increased, whereas the kinetic rate constants increased as temperature increased. Consistent with this, the measured activation energy of [3H]NBMPR association was relatively large (20 kcal mol
1) suggesting a conformational change upon inhibitor binding. This study provides the basis for dissecting thermodynamic forces governing the binding of inhibitors and ligands to the hENT1 transporter. Furthermore, this data is certainly an invaluable tool for screening of novel active drugs and conducting pharmacological characterization of NTs subtypes. Indeed, similar studies with nucleosides, nucleoside analogs and inhibitors of other NTs would further assist us in understanding the protein–drug interactions. Acknowledgements This work was supported by Biocenter Oulu/University of Oulu; Academy of Finland (#132138), Sigrid Juselius Foundation and FP7 Marie Curie European Reintegration Grant (FP7-PEOPLE-2009-RG, #249081). References [1] M.F. Vickers, R.S. Mani, M. Sundaram, D.L. Hogue, J.D. Young, S.A. Baldwin, et al., Functional production and reconstitution of the human equilibrative nucleoside transporter (hENT1) in Saccharomyces cerevisiae. Interaction of inhibitors of nucleoside transport with recombinant hENT1 and a glycosylation-defective derivative (hENT1/N48Q), Biochem. J. 339 (Pt. 1) (1999) 21–32. [2] D.R. Rauchwerger, P.S. Firby, D.W. Hedley, M.J. Moore, Equilibrative-sensitive nucleoside transporter and its role in gemcitabine sensitivity, Cancer Res. 60 (2000) 6075–6079. [3] J.E. Foster, S.F. Holmes, D.A. Erie, Allosteric binding of nucleoside triphosphates to RNA polymerase regulates transcription elongation, Cell 106 (2001) 243–252. [4] L.P. Jordheim, D. Durantel, F. Zoulim, C. Dumontet, Advances in the development of nucleoside and nucleotide analogues for cancer and viral diseases, Nat. Rev. Drug Discov. 12 (2013) 447–464. [5] S.A. Baldwin, P.R. Beal, S.Y. Yao, A.E. King, C.E. Cass, J.D. Young, The equilibrative nucleoside transporter family, SLC29, Pflugers Arch. 447 (2004) 735–743. [6] J. Pressacco, J.S. Wiley, G.P. Jamieson, C. Erlichman, D.W. Hedley, Modulation of the equilibrative nucleoside transporter by inhibitors of DNA synthesis, Br. J. Cancer 72 (1995) 939–942. [7] J.S. Wiley, J.S. Cebon, G.P. Jamieson, J. Szer, J. Gibson, R.K. Woodruff, et al., Assessment of proliferative responses to granulocyte-macrophage colonystimulating factor (GM-CSF) in acute myeloid leukaemia using a fluorescent ligand for the nucleoside transporter, Leukemia 8 (1994) 181–185. [8] C.L. Smith, L.M. Pilarski, M.L. Egerton, J.S. Wiley, Nucleoside transport and proliferative rate in human thymocytes and lymphocytes, Blood 74 (1989) 2038–2042. [9] W. Greenhalf, P. Ghaneh, J.P. Neoptolemos, D.H. Palmer, T.F. Cox, R.F. Lamb, et al., Pancreatic cancer hENT1 expression and survival from gemcitabine in patients from the ESPAC-3 trial, J. Natl. Cancer Inst. 106 (1) (2014) djt347. [10] J. Lane, T.A. Martin, C. McGuigan, M.D. Mason, W.G. Jiang, The differential expression of hCNT1 and hENT1 i n breast cancer and the possible impact on breast cancer therapy, J. Exp. Ther. Oncol. 8 (2010) 203–210. [11] C.R. Boyer, P.L. Karjian, G.M. Wahl, M. Pegram, S.T. Neuteboom, Nucleoside transport inhibitors, dipyridamole and p-nitrobenzylthioinosine, selectively potentiate the antitumor activity of NB1011, Anticancer Drugs 13 (2002) 29–36. [12] P.A. Borea, A. Dalpiaz, K. Varani, P. Gilli, G. Gilli, Can thermodynamic measurements of receptor binding yield information on drug affinity and efficacy? Biochem. Pharmacol. 60 (2000) 1549–1556. [13] G. Siligardi, R. Hussain, S.G. Patching, M.K. Phillips-Jones, Ligand- and drugbinding studies of membrane proteins revealed through circular dichroism spectroscopy, Biochim. Biophys. Acta 1838 (2014) 34–42. [14] K. Rajarathnam, J. Rosgen, Isothermal titration calorimetry of membrane proteins—progress and challenges, Biochim. Biophys. Acta 1838 (2014) 69–77.

9

[15] A. Dalpiaz, A. Townsend-Nicholson, M.W. Beukers, P.R. Schofield, APIJ, Thermodynamics of full agonist, partial agonist, and antagonist binding to wild-type and mutant adenosine A1 receptors, Biochem. Pharmacol. 56 (1998) 1437–1445. [16] E.A. Harper, S.P. Roberts, S.B. Kalindjian, Thermodynamic analysis of ligands at cholecystokinin CCK2 receptors in rat cerebral cortex, Br. J. Pharmacol. 151 (2007) 1352–1367. [17] R.S. Martin, R.A. Henningsen, A. Suen, S. Apparsundaram, B. Leung, Z. Jia, et al., Kinetic and thermodynamic assessment of binding of serotonin transporter inhibitors, J. Pharmacol. Exp. Ther. 327 (2008) 991–1000. [18] S. Rehan, V.P. Jaakola, Expression, purification and functional characterization of human equilibrative nucleoside transporter subtype-1 (hENT1) protein from Sf9 insect cells, Protein Exp. Purif. 114 (2015) 99–107. [19] Y. Cheng, W.H. Prusoff, Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction, Biochem. Pharmacol. 22 (1973) 3099–3108. [20] J.W. Phillis, M.H. O’Regan, G.A. Walter, Effects of two nucleoside transport inhibitors, dipyridamole and soluflazine, on purine release from the rat cerebral cortex, Brain Res. 481 (1989) 309–316. [21] M. Masuda, C. Chang-Chun, T. Mollhoff, H. Van Belle, W. Flameng, Effects of nucleoside transport inhibition on long-term ex vivo preservation of canine hearts, J. Thorac. Cardiovasc. Surg. 104 (1992) 1610–1617. [22] J.R. Hammond, Kinetic analysis of ligand binding to the Ehrlich cell nucleoside transporter: pharmacological characterization of allosteric interactions with the [3H]nitrobenzylthioinosine binding site, Mol. Pharmacol. 39 (1991) 771–779. [23] H. Boleti, I.R. Coe, S.A. Baldwin, J.D. Young, C.E. Cass, Molecular identification of the equilibrative NBMPR-sensitive (es) nucleoside transporter and demonstration of an equilibrative NBMPR-insensitive (ei) transport activity in human erythroleukemia (K562) cells, Neuropharmacology 36 (1997) 1167–1179. [24] Z.L. Johnson, C.G. Cheong, S.Y. Lee, Crystal structure of a concentrative nucleoside transporter from Vibrio cholerae at 2.4 A, Nature 483 (2012) 489–493. [25] R.B. Raffa, F. Porreca, Thermodynamic analysis of the drug–receptor interaction, Life Sci. 44 (1989) 245–258. [26] C.E. Cass, L.A. Gaudette, A.R. Paterson, Mediated transport of nucleosides in human erythrocytes. Specific binding of the inhibitor nitrobenzylthioinosine to nucleoside transport sites in the erythrocyte membrane, Biochim. Biophys. Acta 345 (1974) 1–10. [27] A. Lorenzen, L. Guerra, H. Vogt, U. Schwabe, Interaction of full and partial agonists of the A1 adenosine receptor with receptor/G protein complexes in rat brain membranes, Mol. Pharmacol. 49 (1996) 915–926. [28] W.P. Gati, A.R. Paterson, Interaction of [3H]dilazep at nucleoside transporterassociated binding sites on S49 mouse lymphoma cells, Mol. Pharmacol. 36 (1989) 134–141. [29] D.A. Griffith, S.M. Jarvis, Nucleoside and nucleobase transport systems of mammalian cells, Biochim. Biophys. Acta 1286 (1996) 153–181. [30] T. Casillas, E.G. Delicado, F. Garcia-Carmona, M.T. Miras-Portugal, Kinetic and allosteric cooperativity in L-adenosine transport in chromaffin cells. A mnemonical transporter, Biochemistry 32 (1993) 14203–14209. [31] J.B. Rose, Z. Naydenova, A. Bang, M. Eguchi, G. Sweeney, D.S. Choi, et al., Equilibrative nucleoside transporter 1 plays an essential role in cardioprotection, Am. J. Physiol. Heart Circ. Physiol. 298 (2010) H771–H777. [32] T.S. Olsson, M.A. Williams, W.R. Pitt, J.E. Ladbury, The thermodynamics of protein–ligand interaction and solvation: insights for ligand design, J. Mol. Biol. 384 (2008) 1002–1017. [33] A.J. Ruben, Y. Kiso, E. Freire, Overcoming roadblocks in lead optimization: a thermodynamic perspective, Chem. Biol. Drug Des. 67 (2006) 2–4. [34] V. Lafont, A.A. Armstrong, H. Ohtaka, Y. Kiso, Amzel L. Mario, E. Freire, Compensating enthalpic and entropic changes hinder binding affinity optimization, Chem. Biol. Drug Des. 69 (2007) 413–422. [35] H.P. de Koning, C.J. Watson, L. Sutcliffe, S.M. Jarvis, Differential regulation of nucleoside and nucleobase transporters in Crithidia fasciculata and Trypanosoma brucei, Mol. Biochem. Parasitol. 106 (2000) 93–107. [36] R. Koren, C.E. Cass, A.R. Paterson, The kinetics of dissociation of the inhibitor of nucleoside transport, nitrobenzylthioinosine, from the high-affinity binding sites of cultured hamster cells, Biochem. J. 216 (1983) 299–308. [37] M.J. Todd, N. Semo, E. Freire, The structural stability of the HIV-1 protease, J. Mol. Biol. 283 (1998) 475–488. [38] J.M. Bradshaw, A. Hudmon, H. Schulman, Chemical quenched flow kinetic studies indicate an intraholoenzyme autophosphorylation mechanism for Ca2 + /calmodulin-dependent protein kinase II, J. Biol. Chem. 277 (2002) 20991–20998.

Please cite this article in press as: S. Rehan, et al., Thermodynamics and kinetics of inhibitor binding to human equilibrative nucleoside transporter subtype-1, Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.09.019