Evaluation of capillary chromatographic supports for immobilized human purine nucleoside phosphorylase in frontal affinity chromatography studies

Journal of Chromatography A, 1338 (2014) 77–84

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Evaluation of capillary chromatographic supports for immobilized human purine nucleoside phosphorylase in frontal affinity chromatography studies Marcela Cristina de Moraes a,1 , Caterina Temporini b,1 , Enrica Calleri b , Giovanna Bruni c , Rodrigo Gay Ducati d , Diógenes Santiago Santos d , Carmen Lucia Cardoso e , Quezia Bezerra Cass a , Gabriella Massolini b,∗ a

Departamento de Química, Universidade Federal de São Carlos, Cx Postal 676, São Carlos 13565-905, São Paulo, Brazil Department of Drug Sciences, University of Pavia, 27100 Pavia, Italy c Department of Chemistry, University of Pavia, 27100 Pavia, Italy d Instituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia Molecular e Funcional, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, Rio Grande do Sul, Brazil e Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto 14040-901, São Paulo, Brazil b

a r t i c l e

i n f o

Article history: Received 21 October 2013 Received in revised form 25 January 2014 Accepted 19 February 2014 Available online 28 February 2014 Keywords: HPAC FAC-MS Immobilized human purine nucleoside phosphorylase Ligand affinity determination Monolithic supports

a b s t r a c t The aim of this work was to optimize the preparation of a capillary human purine nucleoside phosphorylase (HsPNP) immobilized enzyme reactor (IMER) for characterization and affinity screening studies of new inhibitors by frontal affinity chromatography coupled to mass spectrometry (FAC-MS). For this purpose two monolithic supports, a Chromolith Speed Rod (0.1 mm I.D. × 5 cm) and a methacrylate-based monolithic epoxy polymeric capillary column (0.25 mm I.D. × 5 cm) with epoxy reactive groups were considered and compared to an IMER previously developed using an open fused silica capillary. Each HsPNP-IMER was characterized in terms of catalytic activity using Inosine as standard substrate. Furthermore, they were also explored for affinity ranking experiments. Kd determination was carried out with the based fused silica HsPNP-IMER and the results are herein discussed. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Human purine nucleoside phosphorylase (HsPNP) is a crucial enzyme of the purine salvage pathway. This enzyme catalyzes the reversible conversion of (deoxy)ribonucleosides, in the presence of inorganic phosphate (Pi ), to the corresponding purine bases and (deoxy)ribose-1-phosphate. HsPNP deficiency is a rare autosomal recessive metabolic disorder which results in severe combined immunodeficiency and depletion of T-cells as a consequence of the failure to degrade deoxyguanosine and its conversion to deoxyguanosine triphosphate. HsPNP deficiency leads to T-lymphocytopenia, usually with no apparent effects on B-lymphocyte function. These symptoms suggest possible chemotherapeutic applications of potent inhibitors of HsPNP, as

∗ Corresponding author. Tel.: +39 0382 987383; fax: +39 0382 422975. E-mail address: [email protected] (G. Massolini). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.chroma.2014.02.057 0021-9673/© 2014 Elsevier B.V. All rights reserved.

selective immunosuppressive agents, to treat T-cell leukemia or T-cell-mediated autoimmune diseases, such as lupus erythematosus and rheumatoid arthritis [1–4]. Moreover, since HsPNP is highly active in the human blood and tissues, nucleoside analogs employed as potential chemotherapeutic agents may be degraded before significant doses reach the target cells. For this reason, PNP inhibitors can also be used in order to avoid anticancer and antiviral drug cleavage. The potential of HsPNP in different clinical applications has consequently stimulated the development of fast and efficient methods capable to identify and characterize new HsPNP inhibitors. To meet this need, we prepared a fused silica capillary based on immobilized HsPNP for function-based inhibitors studies in a bidimensional LC-UV platform [5]. This approach has proven to be a valuable label-free approach to assess the enzyme activity in presence of inhibitory compounds, nevertheless as for many of the functionbased methods, multiple injections of various levels of substrate and inhibitors are required to allow the Ki determination. More recently, PNP from Schistosomamansoni, a promising target for the

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development of antischistosomal agents, was immobilized and used for screening of inhibitors [6]. In recent years, the use of a multidimensional detector like mass spectrometer in screening methods of multiple ligand mixtures has become popular in order to overcome the low throughput of established methods. Within the wide range of immobilized targets used (receptors, proteins), there are some interesting reports on the use of immobilized enzyme-reactors (IMERs) coupled to MS for studying the target enzyme activity and inhibition [7–9]. In the screening of inhibitors for HsPNP with IMERs-MS approach, a critical point is that the enzyme activity is dependent upon the presence of Pi as a second substrate, which is incompatible with MS/MS. Assuming that the prerequisite for inhibitory activity in a compound is its affinity for the target, we moved to an affinity-based screening method and selected frontal affinity chromatography coupled to mass spectrometry (FAC-MS) to demonstrate its prediction power on the selection of new potential HsPNP inhibitors. FAC-MS is based on the continuous infusion of potential ligands through an affinity column containing an immobilized target, usually a receptor or an enzyme [10,11]. During their elution, analytes interact with the immobilized protein according to their affinity for the target. The equilibrium is reached when all the binding sites are saturated and this event is experimentally visualized by the formation of a sigmoidal curve in the chromatographic profile, with a plateau corresponding to the saturation of the stationary phase. Ranking experiments can be carried out in a single run by FAC-MS by infusing equimolar mixtures of analytes, and individual breakthrough curves are monitored by MS at their specific m/z values [12]. When FAC is performed with progressive infusions of increasing concentrations of a given analyte, its accurate Kd value for the target can also be measured. To develop a reliable affinity assay for FAC-MS studies, the conformational structure and the activity of the immobilized enzyme should be preserved and, in this context, the binding chemistry used for enzyme immobilization and the physico-chemical properties of the chromatographic supports are important tasks [13]. The binding chemistry via epoxy groups is very convenient for enzyme immobilization together with the use of monolithic materials [14,15]. In order to set up a new affinity screening method for HsPNP inhibitors, the previously described open tubular (OT) system was first prepared and tested [5]. Hence, new silica and polymeric monolithic supports were considered. In particular, the HsPNP was immobilized onto two monolithic capillary materials based on epoxy-silica (silica monolithic capillary, or SMC) and epoxy-polymers (polymeric monolithic capillary, or PMC) [14,15]. The properties of the HsPNP-IMERs, including the OT one, were explored for both affinity ranking of selected inhibitors and Kd determination.

Hypoxanthine (Hypo) were purchased from Sigma (St. Louis, USA). The monolithic epoxy silica capillary column Chromolith Speed Rod (0.1 mm I.D. × 5 cm) was provided by Merck as a research sample (Darmstadt, Germany). The monolithic epoxy polymeric capillary column (0.25 mm I.D. × 5 cm) was furnished by Prof. Gasparrini’s research group (University La Sapienza, Rome, Italy) [15]. HsPNP expression and purification were conducted as reported elsewhere [16]. The fourth-generation Immucillin derivative (compound 1 in Table 1) was synthesized as previously described [17]. Compound 2 was synthesized by researchers at Bio Cryst Pharmaceutical Inc. [6], while compound 3 was donated by Prof. Arlene G. Correa’s research group [18]. 2.2. Apparatus Enzyme immobilization on OT capillary and on silica and polymeric monolithic capillaries was accomplished using a syringepump 341B (Sage Instruments, Boston, USA). 2.3. Chromatographic systems Different chromatographic systems were used for kinetic studies and for affinity studies by FAC-MS. All the chromatographic experiments were carried out at 25 ◦ C. 2.3.1. On-line HsPNP-IMER chromatographic system for kinetic studies For the kinetic studies, a bidimensional chromatographic system was assembled as illustrated in Fig. 1 [5]. An Agilent 1100 chromatographic pump (Palo Alto, USA) with a Rheodyne sample valve (20 ␮L loop) (System 1) was used in the first dimension. The flow rate used for the HsPNP-IMER was 0.05 mL/min. The mobile phase was 100 mM Tris–HCl, pH 7.0. At the second dimension, a home-packed analytical octyl silica column (Luna Phenomenex® , 10 ␮m, 10 cm × 0.46 mm I.D.) was used at an Agilent 1100 liquid chromatographic pump equipped with a Hewlett Packard HP 1100 variable-wavelength detector, and a HP 1100 thermostat (System 2). The mobile phase used for the determination of the hydrolysis percentage consisted of 1% (v/v) solution of triethylamine, pH 6.0 (acidified with acetic acid)/MeOH (95:5, v/v) delivered at 0.5 mL/min. Pumps 1 and 2 could be used independently (valve in position A) or the eluent from Pump 1 could be directed onto the analytical column through a HP six-port switching valve (valve in position B). The samples were loaded on first dimension at valve position A. Loading eluent was delivered by the pump 1 at a flow rate of 0.05 mL/min for the first 1.2 min. After the valve had been switched to position B, the analytes (product and unreacted substrate) were flushed for 7.3 min directly to the analytical column. The valve was then switched back to its original position for separation with eluent delivered by pump 2 (11.5 min). The total analysis time was 20 min.

2. Materials and methods 2.1. Reagents and chemicals Methanol used for the mobile phase and LC–MS analysis was HPLC grade (Carlo Elba Reagents Milan, Italy or J.T. Baker, Phillipsburg, USA). Buffer components and all chemical materials used were of analytical grade and supplied by Merck (Darmstadt, Germany), Sigma–Aldrich (St. Louis, USA) or Synth (São Paulo, Brazil). Water was purified in a Milli-Q system (Millipore, São Paulo, Brazil) or Direct-Q system (Millipore, Bedford, USA). The silica-fused capillary (0.375 mm O.D. × 0.10 mm I.D. × 5 cm) used for enzyme immobilization and IMERs preparation was acquired from Polymicro Technologies (Phoenix, USA). Inosine (Ino) and

2.3.2. FAC-MS studies Frontal analysis were carried out with a chromatographic system composed by a syringe pump delivering the mobile phase (ammonium acetate 5 mM, pH 7.4) through the capillary at 5 ␮L/min for OTC and PMC, and 1 ␮L/min for SMC. To improve the sensitivity, the eluent from the capillary was mixed with an organic make-up flow (methanol/water 1:1) pumped at 10 ␮L/min with an LC pump (Surveyor, Thermo Finnigan, San Jose, USA) by a teeunion before MS/MS. Detection of the ligands was made by a Linear Trap Quadrupole (LTQ) mass spectrometer with electro-spray ionization as ion source (Thermo Finnigan, San Jose, USA). The system was controlled by Xcalibur software 1.4 (Thermo Finnigan, San Jose, USA).

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Table 1 Chemical structure and inhibitory characteristics of HsPNP inhibitors used in this study. Name (m/z)

Structure

Ki a

IC50 b

Kd (nM)e (R2 )

64.3 ± 0.2 nMc

119.5 ± 11.5 nM

176.67 ± 27.55 (R2 = 0.986)

0.56 ± 0.08 ␮Md

1.83 ± 0.20 ␮M

39.34 ± 2.25 (R2 = 0.818)

No inhibitory activityd

–

O H N

HN N Compound 1 (253.3)

OH

N

OH O H N

HN Compound 2 (257.3)

H2N

N OH

O H N

N

Compound 3 (287.3)

H2N

N –

O

F a b c d e

Ki is the dissociation constant which indicates the concentration needed to inhibit the enzyme. IC50 is the inhibitor concentration at which the enzymatic activity is inhibited by 50%. Ref. [5]. Determined as reported in Session 2.6. Determined by FAC-MS as reported in Section 2.7.2.

The mass spectrometer was operated in the MRM mode under positive ionization, with simultaneous detection of m/z 287.3 → 191, m/z 254.3 → 163, m/z 253.3 → 106 for compounds 1, 2 and 3, respectively. 2.4. HsPNP-IMERs preparations 2.4.1. HsPNP-OTC preparation The enzyme immobilization on OT capillary was carried out using glutaraldehyde as spacer, as previously described for the HsPNPs [5,6]. 2.4.2. HsPNP-SMC preparation The selected monolithic silica support comprises a silica gel skeleton containing mesopores of 13 nm and macropores of 2 ␮m diameter and column porosity higher than 80% [19]. The specific surface area was 323 m2 /g and, as previously reported, the modification of the silica-rods was carried out with 3-glycidoxypropyltrimethoxysilane; the surface coverage (˛epoxy ) was found to be 2.9 ␮mol epoxy groups per m2 unmodified silica [14]. The immobilization of HsPNP in epoxy silica-based monolithic capillaries was carried out following an in situ covalent

immobilization protocol with some modifications [14]. The epoxy silica-based monolithic column was washed at 1 ␮L/min with H2 O for 30 min and after conditioned with a “grafting solution” consisting of 50 mM phosphate and 1.9 M ammonium sulfate buffer pH 8.0, for 90 min. After that, the purified HsPNP enzyme (0.5 mg) solubilized in the grafting solution was eluted through the capillary at a flow rate of 1 ␮L/min. After 2 h, the ends of the capillary were dipped in the enzyme solution and incubated overnight at 4 ◦ C. Therefore, the unbound enzyme was removed by washing with 50 mM phosphate buffer pH 7.0, at 1 ␮L/min for 60 min. Then, the HsPNP-IMER was rinsed with 100 mM glycine solution in phosphate buffer pH 7.0, to quench all unreacted epoxide functionalities at 1 ␮L/min for 60 min. Finally, the epoxy silica-based monolithic HsPNP-IMER was washed with 50 mM phosphate buffer pH 7.0 at a flow rate of 1 ␮L/min for 30 min and stored in the same solution at 4 ◦ C.

2.4.3. HsPNP-PMC preparation The polymeric monolithic support was a medium density monoliths prepared using glycidyl methacrylate (GMA) as reactive monomer, glycerylmonomethacrylate (GlyMA) and acrylamide (AMD) as hydrophilic co-monomers and diethylene glycol dimethacrylate (DEGDMA) as crosslinker (monomers/crosslinker

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POSITION A Waste

IMER

Pump 1 System 1

2

3

1

INJ 4

Pump 2

1

INJ

6

5

Analytical Column

System 2

Detector

POSITION B Waste 1

IMER

Pump 1 System 1

2

3

INJ

1

4

Pump 2

1

INJ

6 5

System 2

Analytical Column

Detector

Fig. 1. Schematic representation of the multidimensional system used for kinetic studies. The bioreactor and the analytical column are uncoupled in position A, while in position 2 they are in-line connected.

ratio 12.5/12.5/75, v/v), following a previously developed protocol [15]. For enzyme immobilization, the epoxy polymeric-based monolithic column was washed at 50 ␮L/min with H2 O for 10 min and then conditioned with a grafting solution consisting of 50 mM phosphate and 1.9 M ammonium sulfate buffer pH 8.0, at the same flow rate for 30 min. Afterward, the purified HsPNP enzyme (0.5 mg) solubilized in the grafting solution was eluted through the capillary at a flow rate of 5 ␮L/min. After 2 h, the ends of the capillary were dipped in the enzyme solution and incubated overnight at 4 ◦ C. Then, the unbound enzyme was removed by washing with 50 mM phosphate buffer pH 7.0, at 50 ␮L/min for 10 min. Then, the HsPNPIMER was rinsed with 100 mM glycine solution in phosphate buffer pH 7.0, to quench all unreacted epoxide functionalities at 50 ␮L/min for 10 min. Finally, the epoxy silica-based monolithic HsPNP-IMER was washed with 50 mM phosphate buffer pH 7.0 at a flow rate of 50 ␮L/min for 10 min and stored in the same solution at 4 ◦ C. 2.5. HsPNP-IMERs characterization 2.5.1. Morphologic characterization by scanning electron microscopy (SEM) The morphologic characterization of the three capillaries was made by scanning electron microscopy. SEM micrographs were collected with a Zeiss EVO MA10 (Carl Zeiss, Oberkochen, Germany) on gold sputtered samples. The two images are reported in Fig. 2. SEM images taken on the three reactors clearly show the typical microglobular structure of monoliths, together with micron-sized openspaces. 2.5.2. Kinetic studies The HsPNP-IMERs activity was directly evaluated by quantification of produced Hypo using the multidimensional chromatography system showed at Fig. 1. A linear correlation (y = 3.9256x − 0.1141; r = 0.9939) was established for the injected concentration versus the peak area using Hypo solutions with concentrations ranging from 2.5 to 1000 ␮M. In the kinetic studies,

solutions containing different concentrations of Ino ranging from 5 to 4000 ␮M and 5 mM phosphate were injected in duplicate in the HsPNP-IMERs, using as mobile phase 50 mM Tris–HCl, pH 7.0. The kinetic parameters were derived by non-linear regression using Prism 4 software (Graph Pad Software Inc., San Diego, USA). 2.6. Inhibitors characterization The inhibitory activity of compounds 1, 2 and 3, reported in Table 1, was evaluated on the OT capillary. The inhibitory activity was evaluated at 200 ␮M, as required in screening assays when the IC50 is not known [6,20–23], by injecting 20 ␮L of a sample containing 382.5 ␮M Ino, 5 mM phosphate buffer, pH 7.4, and 200 ␮M of the assessed compound. The percentage inhibition for each compound was calculated by comparing the Hypo concentration obtained with that obtained in the absence of the target compound. The following expression was employed: %I = 100 − (Ci /C0 × 100), where Ci is the Hypo concentration obtained in the presence of the compounds, and C0 is the Hypo concentration calculated in the absence of the evaluated compounds. Compounds 1 and 2 were identified as HsPNP inhibitors and inhibition curves were constructed for these compounds by plotting the percentage inhibition versus the inhibitor concentration utilized in the assay 5–500 ␮M. The linear regression parameters were calculated, and the IC50 was extrapolated. The Ki value for compound 1 is already published [5]. Under the same experimental conditions of compound 1, Ki value for compound 2 was determined using three different concentrations (0.45, 0.9 and 2.7 ␮M) with four different Ino concentrations (80, 200, 400 and 1600 ␮M). 2.7. FAC-MS assays 2.7.1. Ranking experiments In the FAC-ESI-MS/MS ranking experiments, a mixture of three compounds at 100 nM each with different inhibitory capacities (IC50 ) against the HsPNP in 5 mM ammonium acetate pH 7.4

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rate of 5 ␮L/min using a syringe pump. The make-up flow consisted of methanol/water (1:1) and was mixed with the effluent postcolumn at 10 ␮L/min. The combined flow rate of 15 ␮L/min was introduced in the ESI source. The individual breakthrough curves for each compound were obtained monitoring the specific transition state: m/z 253.3 → 106 (compound 1); 257.3 → 163 (compound 2) and 287.3 → 191 (compound 3). The breakthrough times are the results of a single determination carried out on the freshly prepared capillary. Breakthrough volumes were determined with a polynomial equation of degree 3 (y = ax3 + bx2 + cx + d) to fit the chromatogram data, and the inflection point was obtained by the second derivative. All data were processed with Microsoft Excel and Origin 11.0 software. 2.7.2. Measurements of binding constants The dissociation constant (Kd ) values for the inhibitors were calculated using the OTC following a previously described approach [12], in which the Kd and the binding sites of the immobilized enzyme (Bt ) can be calculated using the following equation: (V − V0 )−1 = [A] · Bt−1 +

K  d

Bt

(1)

where [A] is the ligand concentration, V is the retention volume of the ligand, V0 is the retention volume of the void marker. From the plot (V − V0 )−1 versus [A], the Bt and Kd can be obtained. For Kd determination, four increasing concentrations of these ligands (80–200 nM for compound 1 and 75–150 nM for compound 2) were infused onto the HsPNP-IMERs until a typical sigmoidal profile was obtained. The injection of increasing concentration of the selected ligands resulted in frontal traces with reduced saturation volumes. Each chromatographic profile was analyzed with a polynomial equation to derive the inflection point corresponding to the breakthrough volume. The investigated concentration ranges were assessed starting from the lowest detectable concentration for the two compounds in ESI-MS/MS, up to the highest concentration allowing saturation of the binding sites. The saturation was experimentally confirmed when two consequential concentration levels resulted in the same breakthrough volumes. The experimental error was determined by using Sigma Plot 12.0 software (Systat Software 290). 3. Results and discussion

Fig. 2. SEM images of the three supports: (A) open tubular fused silica capillary; (B) polymeric-based monolithic capillary; and (C) epoxy silica-based monolithic capillary.

was continuously infused on the HsPNP-IMER. The standard mixture used in all ranking experiments was constituted by a potent inhibitor (compound 1, IC50 = 40.6 nM), a moderate inhibitor (compound 2, IC50 = 1.83 ␮M), and a non-inhibitor (compound 3, used as a void marker). Before each analysis, the HsPNP-IMER was equilibrated with 5 mM ammonium acetate buffer pH 7.4 for 15 min at a flow rate of 5 ␮L/min. The compound mixture was infused at a flow

In a previous paper [5] we reported the preparation of an HsPNPIMER using open tubular fused silica capillary (OTC) as the support for inhibition studies by zonal chromatography. In the herein work, we describe the application of the HsPNP-OTC IMER in FAC-MS assays and the comparison with the results obtained with two newly prepared capillary HsPNP-IMERs: a silica-monolith capillary (HsPNP-SMC) and a polymeric-monolith capillary (HsPNP-PMC). The preliminary evaluation of the kinetic parameters of the three IMERs using Ino as standard substrate gives information about the altered or unaltered behavior of the enzyme in the immobilized form and on the effect of the supports on the enzyme activity. Hence, the performances of HsPNP-IMERs were evaluated for fast affinity studies in order to develop a new method for HsPNP ligand screening. 3.1. Characterization of HsPNP-IMERs 3.1.1. Supports selection and HsPNP immobilization The first task to develop a reliable affinity assay by FAC-MS is the choice of the right chromatographic support in terms of its physico-chemical properties, and the binding chemistry for enzyme immobilization. OTC offers the advantage of a low back

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Fig. 3. Michaelis–Menten hyperbola for the HsPNP immobilized on OTC (A), PMC (B) and SMC (C).

pressure and reduced specific interactions with ligands, since the enzyme is directly bound to the internal surface of the capillary. Bulk supports, on the other hand, although producing some backpressure (depending on the porosity and morphology) can bind a higher amount of protein furnishing higher breakthrough time differences in FAC applications. Chemistry, dimensions and reactivity of the considered supports are given in Table 2. The same amount of enzyme (0.5 mg) was used to prepare the three IMERs. The immobilization protocols are detailed in Section 2.4. 3.1.2. Kinetic assays The first comparative study of the three IMERs required the kinetic characterization using Ino as standard substrate. To monitor the enzyme activity on-column, the formed Hypo was quantified by the bi-dimensional chromatographic method previously developed [5]. The Ino kinetic curves were obtained by varying Ino concentration at a fixed Pi concentration (5 mM). Within the substrate concentration range explored to reach saturation with OTC, PMC and SMC IMERs (5–4000 ␮M, 5–4000 ␮M, 5–3000 ␮M, respectively) the curves were best fitted to a Michaelis–Menten hyperbolic function (Fig. 3). Non-linear regression analysis allowed the estimation of KM and Vmax values for the substrate (Table 3). The kinetic studies demonstrated that the HsPNP immobilized onto the two new selected monolith materials activated with epoxy groups retained the catalytic activity. However, the apparent affinities (KM ) of the substrate for the enzyme were strongly affected, when compared with the OT support, being the KM values 927.3 ␮M and 1357.4 ␮M, for PMC and SMC, respectively, corresponding to 7- and 10-fold higher than the free enzyme’s KM value [5]. OTC, instead, gives a KM value comparable to that of the free solution. One possible explanation could arise from the substrate diffusion mechanism of the substrate in the monolithic supports, influencing its concentration in the active site. This effect is more pronounced with the SMC, where the protein binds to epoxy groups on the

silica skeleton. The influence of immobilization process on the kinetic performances of enzymes compared to the soluble form was already observed and discussed by Lundahl [23–25], supporting our data. On the other hand, IMERs prepared with the PMC and SMC supports demonstrated higher Hypo production capacity compared to the OTC, suggesting (as expected) the presence of a higher amount of immobilized enzyme due to the higher available surface area. Taking into account that the mechanical resistance of the solid supports to the mobile phase flowing imposed different operating conditions (i.e. 50 ␮L/min for OTC and PMC, 1 ␮L/min for SMC), the contact times (CT) between enzyme and substrate in the three IMERs were significantly different (7.3 min and 30 min, respectively). A more realistic evaluation of the kinetic performances, which include this parameter, can be carried out looking at the number of enzyme active units/column (U), which is the number of ␮moles of product obtained under the effective enzyme/substrate contact time. Using this normalized parameter, it clearly emerged that the PMC is the best support in terms of catalytic efficiency (123.64 ␮molemax /min), and the high number of enzyme active units.

3.2. Screening studies by FAC-MS The capacity of the prepared HsPNP-IMERs to screen compounds based on their relative affinity order was then explored. A mixture containing two inhibitors (compounds 2 and 3, Table 1) and one non-inhibitor compound (compound 1, Table 1) 100 nM each, was continuously infused through the HsPNP-IMERs. Analyte detection was carried out in positive ion mode by ESI-MS/MS. The HsPNPIMER prepared using the SMC demonstrated a high back pressure, making possible the analysis only at 1 ␮L/min flow rate. In these conditions, the method sensibility was affected and compound 1

Table 2 Characteristics of the supports used for HsPNP immobilization. HsPNP-OTC

HsPNP-PMC

HsPNP-SMC

Epoxy groups

Epoxy groups

Material

Aldehyde groups after activation by glutaraldehyde Fused silica

Monolithic silica

Dimensions (L × I.D.) Porosity mmol epoxy/g monolith

50 mm × 0.1 mm – –

Monolithic polymer based on methacrylate, glycerylmonomethacrylate and acrylamide 50 mm × 0.25 mm 0.72a 0.81a

Reactive groups

a b

Porosity was determined as reported in [15]. Porosity was determined as reported in [14].

50 mm × 0.1 mm 0.80b 7.6b

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Table 3 Kinetic parameters for the IMERs prepared with the open tubular fused silica capillary (OTC), epoxy silica-based monolith SMC- and epoxy polymeric-based monolith PMC. System

KM (␮M)

Free in solution OTC PMC SMC

133.2 254.6 927.3 1357.4

a b

± ± ± ±

KM 14.9b 29.2 53.2 129.1

immobilized/ KM free

– 1.91 6.96 10.2

Highest hypo production capacity (␮M) – 84.77 902.6 594.2

U (␮mol max/CTa )

KM /U

11.61 123.64 19.81

21.93 7.5 68.52

Contact time (min). Data from previously described work [5].

was not detected, for these reasons, the SMC system was discharged for further screening experiments. In HsPNP-OTC the breakthrough times were of 3.19 min, 4.60 min and 5.09 min for compounds 3, 1 and 2, respectively. The ranking experiments carried out on HsPNP-PMC resulted in the same elution order as in HsPNP-OTC, suggesting a specific binding of the three analytes for the immobilized enzyme. The ranking profiles are reported in Fig. 4A and B. Interestingly, PMC showed the best breakthrough time differences, with a separation window of approximately 28 min, with breakthrough times of 13.4 min, 36.00 min and 41.7 min for compounds 3, 1 and 2, respectively, while the difference in the breakthrough times on OTC was only 2 min. This result can be ascribed to the higher amount of immobilized enzyme on epoxy-polymeric monolith support rather than on the OT one, in agreement with the kinetic data previously reported.

Fig. 4. Extracted breakthrough curves for compounds 1–3 infused into HsPNP OTC (A), and PMC monolith (B) IMERs. Red lines correspond to compound 3; black lines correspond to compound 1, green lines correspond to compound 2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

It must be underlined that in the two IMERs the non-inhibitor compound 3 was the less retained, while the two inhibitors demonstrated significantly higher affinity for the target, although with a reverse trend in respect to the inhibitory potency. To clarify this behavior, the same ranking experiment was carried out on a blank OTC and on an inactive HsPNP-OTC (the capillary with the lower differences in breakthrough times) in order to account for any nonspecific and specific interactions. The breakthrough times obtained in both the blank OTC and the inactive HsPNP-OTC were very close to each other, with RSD values of 4.4% and 3.7%, respectively. These differences cannot be considered significative, thus a specific binding occurs between the analytes and the immobilized enzyme in the active capillaries producing the observed relative elution order. 3.3. Binding constants determination The unexpected affinity order observed during the FAC-MS screening was confirmed by the accurate dissociation constants determination (Kd ) and the number of active binding sites (Bt ) for compound 1 and compound 2. For this experiment, HsPNP-OTC was selected since it gave the most reliable KM value during the kinetic experiments, compared to the soluble enzyme. Furthermore, the low back pressure, mechanical resistance and the analysis time with this IMER were also taken into account. In each experiment, serial concentrations of each ligand were added to the mobile phase and continuously infused onto the column until the typical sigmoidal-like profile, with front and plateau regions, was obtained. The explored concentration ranges are reported at Section 2.7.2. The Bt and Kd values were calculated using Eq. (1), and correlation coefficients (R2 ) were used to determine the correctness of the fit. The Kd and R2 values are reported in Table 1. Bt represents the number of dynamic binding sites for each ligand in the capillary, thus the two values obtained are characteristics of each specific ligand-affinity dynamic interaction measured. The obtained data revealed for compound 2 a higher affinity for HsPNP than that of compound 1 (Table 1), and accordingly the number of dynamic binding sites occupied by compound 2 (1.57 pmol) was lower than those involved in the binding with compound 1 (11.11 nmol), as already demonstrated [26–28]. It is important to note that the order of Kd values for the tested ligands is the same as for the ranking experiment. However, compound 1, the most potent inhibitor, has lower affinity for HsPNP than compound 2, the moderate inhibitor. This is surprising because data concerning agreement between affinity and inhibitory capacity can be found in the literature [29,30]. The observed inversion in affinity order/inhibitory activity can be related to some factors, such as: (i) non-specific interactions between the ligands and the support; (ii) absence of Pi in the mobile phase of the FAC-MS/MS assays; and (iii) unspecific interactions between the ligands and the target (interactions with a region differ from enzyme active site). The assumption (i) is not relevant for this study, because the enzyme is directly immobilized to the inner wall of the OT capillary and the same affinity order was observed for the HsPNP-PMCIMER and the absence of non-specific interactions was confirmed

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comparing the breakthrough times for the three compounds on the HsPNP-OTC, a blank OTC and an inactive HsPNP-OTC. The statement (ii) considers that the absence of Pi in the FACMS/MS studies, due the incompatibility with the MS system, can alter/affect the enzyme structure, resulting in different affinities compared with the inhibition capacities. The Pi acts as a second substrate, and previously, we demonstrate the importance of its concentration in the catalytic efficiency of the PNP-IMERs [5,6]. It is important to say that in the activity studies, the injection of Ino substrate or inhibitors without Pi results in an irreversible decrease of its activity (>90%), and the same outcome was observed after each FAC-MS/MS assays. The absence of Pi can induce the formation of a stable complex between Ino or the ligands and HsPNP, blocking the active site and reducing its activity. The absence of Pi does not affect the discrimination between ligands and non-ligands. The assumption (iii) takes into account the possibility of unspecific interactions between the ligands and the enzyme which is not accounted in the activity assays. The fact that the binding of these compounds in FAC-MS experiments does not necessarily reflect the inhibitory activity was already observed [31]. This may explain the observed result of compound 2. Based on the described experiments, one can infer that both the effects (ii) and (iii) can contribute to the observed inversion in affinity order/inhibitory activity. Nevertheless, the main finding of this study is that the FAC-MS method using HsPNP-OTC IMER allowed fast discrimination of ligands and non-ligands in mixture. 4. Conclusions The selection of the support is a crucial step in FAC-MS/MS assays. In this study, we demonstrated that the chosen support for target immobilization affect the breakthrough time and volume window, besides the back pressure of the capillary of the resultant IMER. All these parameters determinate their usability and feasibility in FAC assays. In our study, the same ranking order was obtained for the three evaluated capillary supports, demonstrating the specificity of the interactions between the ligands and HsPNP. The FAC-MS/MS method is a useful tool to discriminate inhibitors from a compound mixture; but the ranking order is not always parallel to the inhibitory capacity. Therefore, the association of the FAC-MS/MS method herein described, an affinity-based assay for screening inhibitors in mixtures, with the previously described activity-zonal based assay [5], is a reliable approach to readily identify and characterize new PNP inhibitors. Acknowledgments This work was funded by grants from the São Paulo State Research Foundation (FAPESP, process number 2008/04051-0) and the Brazilian Agency for Support and Evaluation of Graduate

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