Development of a Countergradient Parking System for Gradient Liquid

Aug 8, 2008 - A gradient HPLC approach in combination with a countergradient system for online biochemical detection (BCD) to screen for inhibitors of...
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Anal. Chem. 2008, 80, 6764–6772

Development of a Countergradient Parking System for Gradient Liquid Chromatography with Online Biochemical Detection of Serine Protease Inhibitors Nils Helge Schebb,† Ferry Heus,‡ Thorsten Saenger,† Uwe Karst,† Hubertus Irth,‡ and Jeroen Kool*,‡ Westfa¨lische Wilhelms-Universita¨t Mu¨nster, Institut fu¨r Anorganische and Analytische Chemie, Corrensstrasse 30, 48149 Mu¨nster, Germany, and Vrije Universiteit Amsterdam, Faculty of Sciences, Department of Chemistry and Pharmaceutical Sciences, Section Analytical Chemistry and Applied Spectroscopy, De Boelelaan 1083, NL-1081 HV Amsterdam, The Netherlands A gradient HPLC approach in combination with a countergradient system for online biochemical detection (BCD) to screen for inhibitors of serine proteases is described. For gradient separations, this novel countergradient system was developed to produce a biocompatible constant solvent composition in the BCD. The countergradient system is based on retaining complete gradients in an additional preparative HPLC column, followed by subsequent and reversible elution to the separation column effluent. Major advantages compared with existing countergradient systems are that no additional LC pumps are needed and enhanced stability. The developed countergradient system was systematically characterized applying different gradient programs. Inhibitors eluting in a postcolumn continuous flow analysis interfere with the enzymatic release of fluorescent 7-amino-4-methylcoumarin (AMC) from an AMC-labeled peptide. The inhibitory activity of eluting substances is sensitively detected as the degree of reduced fluorescence intensity. This biochemical detection system (BCD) for proteases was validated with three known inhibitors of the benzamidine type. Their IC50 values were in good accordance with the results of conventional plate reader assays. Finally, a small library of protease inhibitors was successfully screened with the combination of the BCD and the countergradient system. The screening of synthetic and natural product libraries is a common starting point in drug discovery, with the main goal to identify promising pharmacologically active compounds.1 While common high-throughput screening (HTS) techniques are highly efficient in the screening of individual substances, screening of complex mixtures is more demanding, as separation steps have to be applied in combination with the activity determination.2,3 Typical examples for complex mixtures in drug discovery are * To whom correspondence should be addressed. E-mail: [email protected]. † Westfa¨lische Wilhelms-Universita¨t Mu ¨ nster. ‡ Vrije Universiteit Amsterdam. (1) Smith, A. Nature 2002, 418, 453–459. (2) van Rhee, A. M.; Stocker, J.; Printzenhoff, D.; Creech, C.; Wagoner, P. K.; Spear, K. L. J. Comb. Chem. 2001, 3, 267–277.

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natural products4 or their metabolites after biotransformation.5 In all cases, inactive sample constituents are present together with an unknown number of pharmacologically active compounds. The main difficulties encountered are (I) the correlation of biological activity with chemical/spectrometric analysis data for the rapid identification of active compounds and (II) the interferences of matrix components with the assay readout. During recent years, high-resolution screening (HRS) technologies that facilitate the determination and identification of bioactive compounds in complex mixtures have been described.6-14 Here, the separation of complex samples and the detection of the biological activity of the compounds are combined in one step. These methods are based on HPLC separation hyphenated to an online biochemical detection (BCD) system. In more advanced systems, mass spectrometry (MS) is used in parallel to the BCD allowing the simultaneous measurement of MS and MS/MS spectra of biologically active compounds.6,9-11,13,14 (3) Phillipson, D. W.; Milgram, K. E.; Yanovsky, A. I.; Rusnak, L. S.; Haggerty, D. A.; Farrell, W. P.; Greig, M. J.; Xiong, X.; Proefke, M. L. J. Comb. Chem. 2002, 4, 591–599. (4) Strege, M. A. J. Chromatogr., B: Biomed. Sci. Appl. 1999, 725, 67–78. (5) Fura, A.; Shu, Y. Z.; Zhu, M.; Hanson, R. L.; Roongta, V.; Humphreys, W. G. J. Med. Chem. 2004, 47, 4339–4351. (6) van Elswijk, D. A.; Diefenbach, O.; van den Berg, S.; Irth, H.; Tjaden, U. R.; van der Greef, J. J. Chromatogr., A 2003, 1020, 45–58. (7) De Boer, A. R.; Letzel, T.; van Elswijk, D. A.; Lingeman, H.; Niessen, W. M. A.; Irth, H. Anal. Chem. 2004, 76, 3155–3161. (8) de Jong, C. F.; Derks, R. J.; Bruyneel, B.; Niessen, W.; Irth, H. J. Chromatogr., A 2006, 1112, 303–310. (9) Hirata, J.; Ariese, F.; Gooijer, C.; Irth, H. Anal. Chim. Acta 2003, 478, 1–10. (10) Kool, J.; Ramautar, R.; van Liempd, S. M.; Beckman, J.; de Kanter, F. J.; Meerman, J. H.; Schenk, T.; Irth, H.; Commandeur, J. N.; Vermeulen, N. P. J. Med. Chem. 2006, 49, 3287–3292. (11) Schenk, T.; Breel, J.; Koevoets, P.; van den Berg, S.; Hogenboom, A. C.; Irth, H.; Tjaden, U. R.; van der Greef, J. J. Biomol. Screen. 2003, 8, 421– 429. (12) Kool, J.; Eggink, M.; van Rossum, H.; van Liempd, S. M.; van Elswijk, D. A.; Irth, H.; Commandeur, J. N.; Meerman, J. H.; Vermeulen, N. P. J. Biomol. Screen. 2007, 12, 396–405. (13) Kool, J.; van Liempd, S. M.; Ramautar, R.; Schenk, T.; Meerman, J. H.; Irth, H.; Commandeur, J. N.; Vermeulen, N. P. J. Biomol. Screen. 2005, 10, 427–436. (14) Irth, H.; Oosterkamp, A. J.; van der Welle, W.; Tjaden, U. R.; van der Greef, J. J. Chromatogr. 1993, 633, 65–72. 10.1021/ac801035e CCC: $40.75  2008 American Chemical Society Published on Web 08/08/2008

Figure 1. Structures of the used substrate (A) H-D-cyclohexylalanine-Ala-Arg-AMC and the potential inhibitors (B) 4-(2-aminoethyl)benzensulfonylfluoride (SC), (C) N-R-(2-naphthylsulfonylglycyl)-4-amidino-(D,L)-phenylalanine piperidide acetate (TH), (D) 2,9-dihydroxy-1,10dimethoxyaporphine (Boldine, BOL), (E) 4-amidinophenylbenzoate (TRY), and (F) 4-amidinophenylpyruvic acid (SP). The arrow indicates the cleaving site of the substrate by the proteases.

In HPLC-BCD methods, the changing concentration of organic modifier in the eluate is one of the fundamental problems when coupling a BCD system to gradient reversed phase (RP) HPLC. The enzymatic activity is strongly affected by the solvent composition. In particular, organic modifiers like methanol (MeOH) and acetonitrile (ACN) may lead to inactivation by denaturation of the proteins. To render the system applicable to enzymes, which are labile toward organic solvents, a new countergradient system was developed, which requires no additional LC pumps by retaining whole gradients in a preparative HPLC column. Different BCD for both receptor ligand binding10 and enzyme inhibition6-9,11,12,14 assays have been developed successfully in recent years. However, HPLC-BCD methods for the screening for inhibitors have been only described for few enzymes, e.g., acetylcholine esterase (ACh),8 angiotensin converting enzyme (ACE),6 phosphodiesterase,11 glutathion S-transferase, 12 and the proteases subtilisin9 and cathepsin B.7 Besides the application of BCD for the screening of new compounds in HRS systems, it is used in environmental analysis to identify toxic pollutants, e.g., pesticides, by their activity. In this field, the term “effect-directed analysis” is more common and methods have been developed determining pollutants, e.g., by ACh-inhibition15 or reduced luminescence of fibrio fischeri.16 No HRS methods have been described for the proteases. As serine proteases represent a large group of mammal regulatory enzymes, a screening method for complex mixtures would be useful for pharmaceutical and toxicological research, e.g., for the serine proteases of the blood coagulation cascade. As disorders in the blood coagulation lead to serious diseases like thrombosis, these enzymes are important targets for drug development, e.g., as antithrombotic drugs.17 In particular, low molecular weight direct thrombin inhibitors are still in the focus of drug develop(15) Fabel, S.; Niessner, R.; Weller, M. G. J. Chromatogr., A 2005, 1099, 103– 110. (16) Stolper, P.; Fabel, S.; Weller, M. G.; Knopp, D.; Niessner, R. Anal. Bioanal. Chem. 2008, 390, 1181–1187. (17) Huntington, J. A.; Baglin, T. P. Trends Pharmacol. Sci. 2003, 24, 589–595.

ment.17-19 Natural sources, such as snake venoms, are known to interfere with the blood coagulation cascade.20 Recently, we developed an ESI-MS based screening method for serine proteases, allowing us to characterize proteolytic activities in the venom of bothrops moojeni.21 As the presence of active inhibitory compounds in snake venom20 and in plant constituents, e.g., flavonoids, 22 has also been described in the past, we developed a new HPLC-BCD screening method for serine protease inhibitors also suitable for complex mixtures. In order to demonstrate that the complete HPLC-BCD system is capable to work with different serine proteases, it was applied to the mammalian proteases trypsin and thrombin where it was used to screen mixtures of protease inhibitors. EXPERIMENTAL SECTION Chemicals. The inhibitors 4-amidinophenylpyruvic acid (SP), 4-(2aminoethyl)-benzenesulfonylfluoride (SC), 4-amidinophenylbenzoate hydrochloride (TRY), N-R-(2-naphthylsulfonylglycyl)-4amidino-(D,L)-phenylalanine piperidide acetate (TH) and the substrate Pefafluor TH, H-D-cyclohexylalanine-Ala-Arg-7-amino-4methylcoumarin, were a kind gift of Pentapharm (Basel, Switzerland). The chemical structures of the inhibitors and the substrate are displayed in Figure 1. Methanol (MeOH, 99.9%) and 99.95% acetonitrile (ACN) were from Biosolve (Valkenswaard, The Netherlands). Polyethyleneglycol 6000 (PEG) was obtained from (Fluka, Zwijndrecht, The Netherlands), formic acid (FA) from Riedel-de Haen (Seelze, Germany), and potassium phosphate buffer salts from Baker (Deventer, The Netherlands). ELISA blocking reagent (EBB) was purchased from Roche (Mannheim, Germany). Boldine (2,9-dihydroxy-1,10-dimethoxyaporphine, BOL, (18) Sturzebecher, J.; Prasa, D.; Hauptmann, J.; Vieweg, H.; Wikstrom, P. J. Med. Chem. 1997, 40, 3091–3099. (19) Prezelj, A.; Anderluh, P. S.; Peternel, L.; Urleb, U. Curr. Pharm. Des. 2007, 13, 287–312. (20) Iyaniwura, T. T. Vet. Hum. Toxicol. 1991, 33, 468–474. (21) Liesener, A.; Perchuc, A. M.; Schöni, R.; Wilmer, M.; Karst, U. Rapid Commun. Mass Spectrom. 2005, 19, 2923–2928. (22) Jedinak, A.; Maliar, T.; Grancai, D.; Nagy, M. Phytother. Res. 2006, 20, 214–217.

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Figure 2. BCD system used for FIA analysis. Enzyme and substrate solutions were delivered by superloops and mixed with the eluent stream in reaction coils. (A) FIA-system: The eluent stream is delivered by a single HPLC pump. (B) Hyphenation of the BCD to the RP gradient separation using the developed countergradient (see RP-HPLC-Countergradient-BCD System section).

Figure 1), leupeptin (Ac-Leu-Leu-Arg-al.), and the enzymes trypsin (hog, 11.909 U/mg) and thrombin (bovine, 28 U/mg) were obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands). All chemicals were of the highest quality available. HPLC-BCD Instruments. The biochemical detection (Figure 2A) system comprised two superloops driven by two identical Gynkotek 300 (Separations, Ambacht, The Netherlands) HPLC pumps at a flow rate of 50 µL/min. The superloops (30 and 100 mL volume) were constructed in-house, similar to those commercially available from GE Healthcare. Superloop A contained the enzyme solution (trypsin or thrombin) and superloop B the AMC-labeled peptide as substrate. The superloops were kept on ice. Proper operation of the pumps at low flow rates was achieved as described by Kool et al.13 with slight modifications. Two 1 m pieces of coiled PEEK 1/32 in., 0.18 mm i.d. capillary (Bester BV, Amstelveen, The Netherlands) served as reaction coils and were kept at 40 °C in a model 7971 HPLC column oven (Jones, Hengoed, United Kingdom). The enzymatic conversion was monitored by a Jasco FP-920 HPLC fluorescence detector (Jasco, Tokyo, Japan) operating at 342/440 nm for the excitation and emission wavelengths, respectively. For flow-injection analysis (FIA), the samples (50 µL) were injected every 3-7 min by a model 830 autosampler (Separations, Ambacht, The Netherlands) into the eluent stream (100 µL/min) of a Gynkotek 480 HPLC pump (Figure 2A). For analysis in HPLC mode, the later parts were replaced by the countergradient system (CGS, Figure 2B) consisting of a LC20-AB gradient HPLC pump, a SIL 20 AC autosampler (Shimadzu, ′s-Hertogenbosch, The Netherlands) and an automatic MUST sixport valve (Spark Holland, Emmen, The Netherlands). The flow was split into two analytical Luna 3 µ C18(2) columns with 100 mm length, 2 mm i.d., with 3 µm particle size and 100 Å pore size (Phenomenex, Torrance, CA), as shown in I in Figure 2B serving as the separation and blind column, respectively). The countergradients were stored in a preparative HPLC column with a length of 300 mm and an inner diameter of 20 mm, packed with Luna 6766

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C18(2) material of 10 µm particle size and 100 Å pore size (Phenomenex). HPLC separations were carried out with a total flow rate of 300 µL/min with 5/95 MeOH/water (v/v) as solvent A and 95/5 MeOH/water (v/v) as solvent B. Both mobile phases contained 0.1% formic acid. The following gradient was used: 0-20 min isocratic 0% B, 20-80 min linear to 100% B, 80-109 min isocratic 100% B, 109-111 min linear to 0% B, after which the six-port valve was switched (Figure 2B). For analysis, the pure inhibitors or their mixtures were dissolved in 20/80 MeOH/water (v/v). Aliquots of 20 µL were injected after 15 min of pregradient equilibration time. Subsequent to the CGS, the flows from the analytical column and the CGS were first combined in a mixing-T-piece (II in Figure 2B). Then, the flow was split 1:3 at 10 bar pressure by a mixingT-piece connected to two PEEK capillaries (64 µm i.d., Vici, Schenkon, Switzerland) (III in Figure 2B). The smaller fraction was directed to the BCD system and the larger to a MicroUVIS 20 UV detector (Carlo Erba Instruments, Milano, Italy), operating at 220 nm, or an electrospray ionization time-of-flight mass spectrometric (ESI-TOF-MS) detector (Figure 2B). For performance optimization of the CGS, three Kratos UVdetectors (Separations, Ambacht, The Netherlands) operating at 254 nm were integrated in the CGS (Figure 3A). Herewith, the organic content of the analytical gradient (detector 1), the countergradient (detector 2), and the analytical gradient recombined with the countergradient (detector 3) could be measured in parallel. To allow the UV detectors to monitor the organic content, 0.25% acetone was added to solvent B (95/5% MeOH/ water) as a tracer molecule. Detector response was calibrated by using varying concentrations of solvent B (0-100%). Hence, the solvent composition could be directly measured from its UV absorbance. In this setup, detector 1 and 2 monitor analytical and countergradient alignment, while detector 3 monitored the organic content of the analytical gradient mixed with the countergradient. Continuous Flow Biochemical Detection System. The BCD was optimized to yield a high signal-to-noise ratio and low peak

Figure 3. Countergradient system: (A) Setup for investigation of the performance of the system. Three UV-detectors operating at 254 nm measure the concentration of solvent B in the eluate of the separation column (detector I), of the preparative column (detector II), and the composition of the complete (mixed) eluate (detector III). (B) Gradient profile of the countergradient system. The six-port-valve is switched at the start (at 0 min) and the arrow indicates the time when a sample is injected in HPLC-BCD experiments.

broadening. If not stated otherwise, the following solutions were used under optimized conditions: Enzyme and substrate in the superloops were prepared in 50 mM potassium phosphate buffer pH 7.4 to mimic physiological conditions. Additionally, both solutions contained 2 g/L PEG and 2 g/L EBB as blocking reagents to prevent peak broadening and peak tailing due to wall adherence effects. The substrate concentration in superloop B was 40 µM. The enzyme concentration was optimized to yield a 70-90% conversion rate in the steady state realized by a concentration of approximately 10 U/mL trypsin and 1 U/mL thrombin, respectively, in superloop A. For FIA experiments (Figure 2A), 50/50 MeOH/water (v/v) was used as the eluent stream representing the same solvent composition as in the HPLC mode with the countergradient system (CGS). Different concentration ranges of the investigated inhibitors were prepared by serial dilution in 50/50 MeOH/water (v/v) and analyzed in triplicate. LC-MS/MS Measurements. The mass spectrometer used was a Q-ToF2, (Micromass, Wythenshawe, Manchester, U.K.) equipped with a Micromass Z-spray electrospray (ESI) source. Masslynx software was used for both control of the system and data acquisition and analysis. The apparatus was operated in positive ion mode at a 22.7 kHz frequency with a spectrum integration time of 0.5 s in “full scan” MS in the range of m/z ) 70-700. The ESI conditions were desolvation temperature 250 °C, source temperature 100 °C, and capillary voltage 2.5 kV. The cone voltage was 28 V. Nitrogen 5.0 was used at flow rates of 50 L/h for the cone-gas and at 300 L/h for desolvation. Fluorescence Microplate Reader Experiments. Microplate reader assays were used for optimization of the reaction parameters (e.g., determination of KM and Vmax values, tolerance of the enzymes toward organic modifiers) and validation of the determined IC50 values of the BCD systems. The fluorescence of AMC was measured at excitation and emission wavelengths of 355 and 460 nm, respectively, on a Victor2 1420 multilabel counter (Wallac, Turku, Finland). Alternatively, a FLUOstar plate reader (BMG LabTechnologies, Offenburg, Germany), operating at 320 and 440 nm was used. The formation of the fluorescent AMC was measured dependent on reaction time. The velocity of the conversion was determined by linear fitting of the initial increase in fluorescence within the first 10 min of the reaction. All reactions were carried out in 50 mM potassium phosphate buffer (pH 7.4)

at 37 °C with a final enzyme concentration of 0.25 U/mL and were measured at least in duplicate. For determination of the IC50 values of the inhibitors Bol, SC, SP, NAPAP, and TRY, the same solutions as for the BCD system were used: Substrate solution (25 µL, 20 µM) was added to 50 µL of the eluent 50/50 MeOH/water (v/v). The reactions were started by addition of diluted (0.25 U/mL) enzyme solution. RESULTS AND DISCUSSION Development of the Countergradient System. For a constant baseline and sensitivity of BCD, the concentration of organic modifiers in the eluate has to be constant in order to maintain the enzyme activity during the complete chromatographic run. Therefore, a countergradient system has been developed to guarantee the constant solvent composition in the BCD. The new CGS is based on the retention of whole gradients in a preparative HPLC column (300 mm × 20 mm). For an optimum performance of the CGS, the flow split to the separation column and the preparative column is crucial. Therefore, the flow of a gradient HPLC system was split to two identical analytical columns (I in Figure 2B), resulting in identical backpressures and hence the same split ratios under changing solvent compositions during gradient LC runs. This resulted in a split ratio of exactly 1:1 during the runs. Only one-half of the split flow was used for separation, passing the autosampler prior to the separation column (I in Figure 2B). The other half of the flow was first directed through the analytical blind column and an automated six-port valve then led the flow to the preparative column. As the preparative column has a large void volume of 45 mL, the complete split gradient is retained inside this column. At the beginning of the second gradient run, the six-port valve is switched. This causes the preparative column to be backflushed (Figure 2B). Consequently, the retained gradient of the first run is flushed out in a reversed way, while the second gradient is retained in the other half of the preparative column simultaneously. The backflushed “retained” gradient is now mixed with the analytical gradient after the separation column. When the third gradient starts, the valve is switched again. Now, the second gradient is reversely flushed out, while the third gradient is stored on the other side of the preparative column at the same time. A constant supply of reversed gradients is obtained by looping this cycle. Hereby, a Analytical Chemistry, Vol. 80, No. 17, September 1, 2008

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total carrier flow of constant organic composition is produced, while having gradient LC separations. To align the countergradient with the analytical gradient, the solvent composition of the eluates of the separation column, the preparative column, and the mixed flow was monitored by UVdetection (Figure 3A). As starting conditions, a gradient program was selected, which consisted of 20 min isocratic 100% solvent A followed by a 60 min gradient to 100% solvent B, after which a “postgradient” equilibration time of 20 min isocratic 100% solvent B was applied. After this equilibration time, the CGS was backflushed. As the countergradient is produced by eluting a retained portion of this analytical gradient in reversed order, the start of the reversed gradient is determined by the “postgradient” time span of the analytical gradient (Figure 3B). In order to synchronize the countergradient elution with the start of the analytical gradient, various incremental postgradient equilibration time spans were evaluated, while the “pregradient” equilibration time of 100% solvent A and the gradient itself remained constant. A flow rate of 300 µL/min was applied (Figure 7A,B). An optimum alignment of the countergradient was observed visually at a postgradient equilibration time of 31 min (arrow in Figure 7B). Thus, the best time program consists of 20 min isocratic 100% solvent A, then a 60 min linear gradient to 100% solvent B, followed by 31 min of 100% isocratic solvent B. When mixed with the analytical gradient, a constant organic content of 50% (±7%) was obtained (Figure 7C) This mixed carrier flow remained virtually constant until 50 min runtime. Thereafter, the organic content slightly drops during the last 10 min of the gradient. This was probably due to diffusion of organic modifier inside the preparative column. As the organic modifier concentration of the analytical gradient exceeds 80% in this time frame, the instability was acceptable; as most analytes already have passed the BCD before the organic content reaches this concentration. This optimized time program was then applied to other gradient programs of 40 and 50 min, respectively, in order to evaluate the characteristics of the system. To maintain optimum alignment, both the pre- and postgradient equilibration time of the 40 and 50 min gradient programs were shortened by 33% and 16%, respectively (Figure 7D). The organic content monitored by detector 3 showed an identical profile when compared to the 60 min gradient (Figure 7E). To examine if the system is applicable to different flow rates, 100, 200, 300, and 400 µL/min were evaluated. During these runs, gradient alignment and organic content of the mixed carrier flow remained stable. The long-term stability of the CGS was determined by looping the countergradient retention and elution cycle (Figure 7F). No change in the solvent composition was observed for at least 15 cycles. Development of the BCD System for Serine Protease Inhibitors. Strategy. A basic property of the BCD system is the continuous mixing of the enzyme, nonfluorescent substrate (AMClabeled peptide), and eluent from a carrier flow in the reaction coils (Figure 2). In the reaction coils, a continuous formation of free AMC takes place, which is measured by fluorescence detection at the end of the second reaction coil. The fluorescence of the substrate itself, where AMC is bound covalently to arginine, is negligible. When inhibitors elute from the carrier solution (in FIA mode) or from the HPLC column (in HPLC mode), they bind 6768

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to the active site of the protease in the first reaction coil. Hence, they temporarily decrease the formation of free fluorescent AMC, observable as a negative peak in the fluorescence detection (for example Figure 6A). In the following section, optimization of the system comprising the composition of the eluate as well as the substrate and the enzyme solution is described. Thereafter, a small library of inhibitors is analyzed with the BCD system in FIA mode, and the results are compared to plate reader assays. Substrate Concentration KM-Values. To optimize the bioassay conditions, the KM values for the conversion of the substrate by the two enzymes under the selected buffer conditions were determined in plate reader assays. The two tested enzymes showed Michaelis-Menten kinetics at ascending concentrations up to 250 µM of the substrate (data not shown). KM values were calculated by plotting the determined velocities in a Hanes plot. Values of 13.8 ± 1.7 µM for trypsin and 5.0 ± 0.5 µM thrombin were determined (n ) 4) as KM in 50 mM potassium phosphate buffer (pH 7.4). As a compromise, a final substrate concentration of 10 µM for the BCD (40 µM in superloop B) was selected. This concentration is close to the determined KM value for the two tested enzymes and therefore allows a sensitive detection of all inhibitors in the same assay setup. Eluent Modifiers. The proteases and the lipophilic compounds may in principle adhere to the reaction coils and therefore lead to peak broadening. In order to reduce the adsorption, blocking reagents and organic modifiers have been successfully applied in other BCD methods.12 Prior to use of these substances, their effect on the enzyme activity has to be evaluated. Therefore, plate reader assays with different concentrations of blocking reagents were carried out. Buffer (50 µL) containing the modifier was mixed with 25 µL of substrate solution (final concentration, 10 µM), and the reaction was started by addition of 25 µL enzyme solution. No influence on the enzyme activity of trypsin and thrombin was found for PEG 6000 at concentrations of 0.01, 0.05, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 4, and 5 g/L and for hydrolyzed milk protein of the EBB at 0.01, 0.04, 0.1, 0.2, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, and 2 g/L. Therefore, both reagents were used at a concentration of 2 g/L for the assay to prevent any kind of unspecific adsorption effects on the surface of the capillary. In other BCD methods,12 detergents like Tween 20 have been applied to reduce peak broadening due to wall coating effects as well. Plate reader experiments with Tween 20 showed a significant decrease of the reaction velocity of the two enzymes. Although the reaction velocity in the presence of 4 g/L remained higher than 80% of the control experiments for all enzymes, Tween 20 was excluded as an eluent modifier. Organic modifiers like MeOH and ACN are known to lower enzyme activity by denaturation. In contrast, it is known that moderate concentrations of some organic solvents, e.g., MeOH, slightly increase the enzymatic activity of trypsin.23,24 The use of either MeOH or ACN is helpful in the FIA experiments to prevent peak broadening of lipophilic compounds and is routinely applied in the case of the RP-HPLC. Therefore, the influence of MeOH and ACN on the enzymatic activity of trypsin and thrombin was investigated in plate reader experiments in a concentration ranged (23) Park, H.; Chi, Y. M. J. Microbiol. Biotechnol. 1998, 8, 656–662. (24) Compton, P. D.; Coll, R. J.; Fink, A. L. J. Biol. Chem. 1986, 261, 1248– 1252.

Figure 5. Fluorescence signal of system qualification of the BCD system. In section A, 50 µL substrate solution from superloop B and 50 µL water from superloop A are added to the eluent (100 µL/min). In section B, enzyme solution (10 U/mL trypsin) is pumped into the BCD system by superloop A (steady state). In section C, the flow is stopped (100% conversion).

Figure 4. Influence of the organic modifiers MeOH (A) and ACN (B) on enzyme activity. The relative activity in comparison to the control experiments is plotted against the concentration of organic modifier.

between 0-70%. (Figure 4). Both MeOH and ACN exhibit a strong influence on the activity of all tested enzymes. At concentrations levels of 60% MeOH and 40% ACN, none of the enzymes remained significantly active. Trypsin maintained at least 80% of the initial activity at concentrations up to 30% of MeOH and 25% ACN. Thrombin showed a similar stability toward ACN. With MeOH, it showed an increase of the enzymatic activity, up to 150%, compared with the control experiment. As no significant differences in the fluorescence activity of AMC in different concentrations of MeOH could be observed, these findings could not be explained by spectroscopic reasons. MeOH was selected as an organic modifier for both FIA and HPLC experiments because of the lower adverse effects on enzymatic activity than ACN. In order to realize a better comparison of the FIA experiments with the HPLC-BCD, a solution of 50% MeOH was selected as a solvent stream. By combination of the flows of enzyme and substrate (Figure 2A), a dilution occurs, resulting in an effective concentration of 25% MeOH in the assay zone. At 25% MeOH, trypsin and thrombin showed an activity of approximately 110-120% of the controls (Figure 4). In the case of gradient elution, the MeOH concentration in the assay zone is raised up to 50% organic modifier leading to a strong influence on the activity of the enzymes, which underlines the necessity of the countergradient in gradient RP-HPLC analysis. Setup and Stability of the BCD System. Before the BCD system was started, it was checked for proper operation by the following “system qualification” standard protocol (Figure 5): First, the superloop A was filled with water instead of enzyme solution. After addition of substrate solution by superloop B to the eluent stream either from the FIA pump or the CGS, the fluorescence signal of

the substrate alone was measured as the blank (A in Figure 5). Then, superloop A was run with enzyme solution, and the signal of the steady state of the product formation was observed (B in Figure 5). To determine the signal of 100% conversion of the substrate to product, the eluent flow was stopped by unscrewing the fittings from the reaction capillary (C in Figure 5). In this case, the enzyme converts the substrate completely within the detection cell. After subtraction of the blank signal from both other signal intensities, the conversion rate results from dividing the fluorescence intensity at steady state by the signal of 100% conversion. If the conversion rate was not in the range of 70-90%, the enzyme concentration was adjusted accordingly. The conversion rates were adjusted everyday, and enzyme solutions of about 10 U/mL in superloop A (final concentration in the assay zone, 2.5 U) were found be optimal. To determine the stability of the detector response, the intraday and interday variability of the BCD signal were measured exemplarily for trypsin as follows: Intraday variability was determined by injecting TH (300 µM) in triplicate in the morning, at noon, and in the evening under optimized conditions without changing the content of the superloops. For interday variability (expressed as relative standard deviation, RSD), the same solution of inhibitor TH (300 µM) was injected daily, in triplicate for 3 days, using fresh solutions in the superloops each day. Intraday variability as RSD was found to be 4.3%, and interday variability was 3.6%. Validation of Determined IC50 Values. The BCD system was validated in FIA mode. Therefore, different known serine protease inhibitors were analyzed, and the results were compared to those from conventional plate reader assays. Three competitive inhibitors of the benzamidine type, TH, SP, and TRY (see Figure 1), were analyzed in concentrations ranging from 100% to 0% inhibition. Each dilution was injected at least in triplicate into the BCD system running with trypsin and thrombin, respectively. A typical result, obtained from the inhibitor TH in the BCD-thrombin system is shown in Figure 6A. The peak heights of the injections were plotted against the concentration in logarithmic scale yielding Analytical Chemistry, Vol. 80, No. 17, September 1, 2008

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Figure 6. Inhibition of thrombin: (A) The FIA-BCD for analysis of the inhibitor TH. The observed peak heights were plotted against the concentration yielding a typical dose-response plot shown in part B. The observed dose-response plots of FIA-BCD (C) for the three inhibitors SP, TRY, and TH were compared to the results of plate reader experiments shown in part D.

typical sigmoidal dose-response plot (Figure 6B). Comparing the dose-response plots of the three inhibitors to the results from the plate reader assay (Figure 6C,D), it can be seen that both assays give qualitatively similar results. TH is the inhibitor with the highest affinity for thrombin in both assays, whereas TRY and SP similarly showed a lower inhibitory activity. This is well in line with literature data, where TH is described as the most active thrombin inhibitor of the benzamidine type.18) The IC50 values of the dose-response curves were calculated with Origin 7.0 (OriginLab Cooperation, Northampton, MA). To compare the IC50 values of the BCD system with those measured by plate reader experiments, the dilution factor of the injected inhibitor solution in the BCD by diffusion and enzyme and substrate solution has to be taken into account. A dilution factor of 20.4 was calculated assuming ideal Gaussian peaks with an average full width at half-maximum height of 2.4 min. The calculation is described in the Supporting Information in detail. With this dilution factor, the IC50 for the three inhibitors toward the two serine protease from the BCD are similar to those from conventional plate reader assays (Table 1) and in the range of literature data, where an IC50 value of 6 nM was found for TH toward thrombin.18 Additionally, the observed differences between the two assays are within the range reported in literature for the comparison of BCD with plate reader assays.12 The method therefore allows to semiquantitatively distinguish between highly active, less, or inactive compounds. Furthermore, the observed LODs for the compounds measured are in the same range as for the plate reader assay underlining the sensitivity of the BCD. In contrast to plate reader assays, BCD systems can be hyphenated to HPLC separations and therefore are not limited to the analysis of pure substances. As the developed BCD system 6770

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Table 1. Comparison of IC50 Values and LODs Determined in Plate-Reader Format and in the Developed BCD Assaya Trypsin flow assay

plate reader

inhibitor IC50 value [µM] LOD [µM] IC50 value [µM] LOD [µM] TRY SP TH

1.5 ± 0.1 1.5 ± 0.3 0.8 ± 0.3

TRY SP TH

1.1 ± 0.1 3.2 ± 0.2 0.03 ± 0.003

1 1 1

1.4 ± 1.3 1.1 ± 0.8 1.1 ± 0.8 Thrombin

1 1 0.03

3.1 ± 1.8 5.2 ± 2.9 0.03 ± 0.015

0.3 0.3 0.3 0.3 0.1 0.001

a For BCD determination of the IC50 values, the injected concentration is divided by the calculated dilution factor, whereas for the LOD, the injected concentration is provided. IC50 values of BCD and plate reader were determined at least by triple injections at each concentration of the inhibitors and three independent experiments, respectively. LOD was the concentration with a signal of the 3-fold noise in BCD and a significant reduction in the slope of fluorescence signal in the plate reader assay, respectively.

allows the sensitive detection of inhibitors for trypsin and thrombin, it can be concluded that it is a promising approach for a general HRS system for serine protease inhibitors. RP-HPLC-Countergradient-BCD System. A small library of inhibitors was screened with the BCD hyphenated to RP-HPLC applying the new countergradient. Figure 8 shows the gradient separation of a mixture of the substances SC, BOL, Leupeptin (Leu), SP, TRY, and TH (each 2 nmol) with BCD detection based on trypsin as the enzyme. Five peaks are observed with UV detection and could be assigned to SP (peak 1) eluting with the injection peak, SC (peak 2) eluting as a broad peak, BOL (peak

Figure 7. Optimization of countergradient alignment: (A) solvent composition of 60 min countergradients eluting from the preparative column after postgradient equilibration times of 20, 27, 31, 33, 35, and 40 min and (B) the corresponding mixed eluate. (C) Countergradient (I), analytical gradient (II), mixed eluate composition (III) under optimized conditions. (D) Countergradient with gradient times of 40 (gray), 50 (opaque), and 60 min (black), and (E) the corresponding mixed eluate. (F) Reproducibility of the countergradient system with a 60 min gradient. The gray line represents the analytical gradient; the black line is the countergradient. In the first run, there is no gradient retained in the filling column, thus no countergradient results.

3), TRY (peak 4), and TH (peak 5), respectively. Leupeptin (Leu) was not detectable with UV-detection in the used concentrations (up to 300 µM). The baseline of the BCD was stable over the gradient of the separation, thus proving the applicability of the newly developed countergradient system. Eluting inhibitors are detected as negative peaks, whereas the peak height indicates the inhibitory activity. By comparison of the UV signal with the BCD signal, SP (peak a), TRY (peak d), and TH (peak e) could be identified as active compounds. For the broad peak of the irreversible inhibitor SC, only a very small response in the BCD was observed. SC irreversibly inactivates the proteases by sulfonylation of the serine group at the active site of the enzymes with a bimolecular reaction kinetics.25,26 Because of the slower reaction speed, the short postcolumn online incubation in BCD did not allow sensitive detection of this irreversible inhibitor. No inhibitory activity for the natural antioxidant BOL (peak 3) could be detected, (25) Markwardt, F.; Walsmann, P.; Richter, M.; Klocking, H. P.; Drawert, J.; Landmann, H. Die Pharmazie 1971, 26, 401–404. (26) Walsmann, P.; Richter, M.; Markwardt, F. Acta Biol. Med. Ger. 1972, 28, 577–585.

as no corresponding peak was observed by BCD. This is well in line with the literature, where BOL has not been described as a protease inhibitor.27,28 Similar results were obtained by HPLCBCD analysis for these five potential inhibitors for thrombin. The BCD signal of the mixture (Figure 8) showed two additional peaks (peaks b and c) without corresponding signals in the UV trace. These peaks could both be assigned to Leu. Because of equilibrium among the three forms in solution, Leu causes multiple peaks in HPLC separations.29 In a confirmative HPLC-ESI(+)-MS analysis of the Leu solution used, the total ion current (TIC) chromatogram showed at least three peaks (Figure 9). These peaks all show the molecular ion ([M + H]+) masses of Leu (427 amu) and/or the methanol adduct of Leu (459 amu). The inhibitory activity toward trypsin measured with the HPLC-BCD (27) O’Brien, P.; Carrasco-Pozo, C.; Speisky, H. Chem. Biol. Interact. 2006, 159, 1–17. (28) Wink, M.; Latz-Bruning, B.; Schmeller, T. In Principles and Practices in Plant Ecology; CRC Press: Boca Raton, FL, 1999; pp 411-422. (29) Saino, T.; Someno, T.; Miyazaki, H.; Ishii, S. Chem. Pharm. Bull. 1982, 30, 2319–2325.

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reduced (dihydrogenated) derivative of TH ([M + H]+ ion of 522 amu). Here, the benefit of HPLC-BCD compared with classical HTS carried out in plate reader format becomes obvious. In conventional HTS techniques, impurities are not detected and may lead to false positive results. In contrast, in HPLC-BCD techniques, the compound of interest can be separated from possible impurities and matrix constituents, which may interfere with inhibitory activity measurement. Furthermore, the potential inhibitory activity of impurities is directly determined. This renders the HPLCBCD not only the method of choice for the screening of complex mixtures, but it also offers new opportunities for the screening of (synthetic) substance libraries. Figure 8. HPLC-UV-BCD separation of a mixture of inhibitors (each 2 nmol) applying the developed countergradient. UV signal of the inhibitors (black line) and fluorescence signal from BCD of trypsin inhibiton (gray line). The separation of a mixture containing SP (peaks 1,a), SC (peak 2), BOL (peak 3), Leu (peaks b,c), Try (peaks 4,d), and TH (peaks 5,e) is shown. Insert: Superimposed sector of an HPLC-UV-BCD chromatogram of TH (6 nmol) and a present impurity.

Figure 9. HPLC separation of Leu (2 nmol): (A) BCD signal of the inhibition of trypsin applying the countergradient. (B) Total ion chromatogram (TIC) of HPLC-ESI(+)-MS separation of Leu under the same conditions.

correlated well to the ESI-MS chromatogram (Figure 9). Therefore, the isoform eluting at the retention time of peak 1, as well as the isoform(s) of Leu eluting in peak 2 are trypsin inhibitors. No inhibitory activity was determined for the significantly smaller ESI-MS peak 3, probably caused by its lower concentration. De Boer et al. 7 investigated the inhibitor Leu toward cathepesin in a HPLC-BCD system, too. They only described one peak for Leu in MS as well as in the BCD system. In contrast, the resolution power of our developed method allows the separation of the isomers. As the isoforms are formed in equilibrium in solution,29 no other technique apart from online coupling of BCD to a HPLC separation offers the opportunity to identify their activity. Additionally, the application of the developed HPLC-BCD lead to the detection of an inhibitory active impurity in the inhibitor TH. At high concentrations (>300 µM), different small peaks are observed additionally to the main TH peak in the UV chromatogram. The peak closely eluting prior to TH showed a corresponding peak in the BCD (insert in Figure 8). It could therefore be identified as an active compound. This peak shows ions at 524 amu in the ESI(+)-MS analysis, implying that it might be a 6772

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CONCLUSION A new countergradient system for RP-gradient separations requiring no additional HPLC pumps has been developed. It is based on the new principle of retaining gradients in a preparative column to generate a countergradient in order to obtain isocratic conditions in the eluate of gradient separations. The system was systematically evaluated and optimized. It proved to be so stable that it offers new opportunities for the coupling of gradient separations to BCD and any analytical detection technique depending on isocratic conditions (e.g., inductively coupled plasma-MS, electrochemical or refractive index detection). Furthermore, a new HPLC-based postcolumn online biochemical detection system suited for the detection of serine protease inhibitors was developed. The BCD system was successfully applied to trypsin and thrombin as model proteases in order to demonstrate its applicability to various proteases. It was validated in FIA mode, and the observed IC50 values for three inhibitors for all tested enzymes were in good agreement with conventional plate reader assays. Although the activity of the proteases is strongly influenced by the MeOH concentration, the developed countergradient system assures the applicability of the BCD for serine protease inhibitors with a straight baseline even in gradient elution from 5 to 95% MeOH. When applied to mixtures, the activity of various compounds could be determined simultaneously with the developed HPLC-BCD system. This was demonstrated by the screening of a small library of known serine protease inhibitors. ACKNOWLEDGMENT N.H.S. and F.H. contributed equally to this work. We thank Ben Bruyneel, Mark Eggink, and Niels Jonker for their help with the MS measurements and Ronald Maul, Andy Scheffer, Lineke van der Sneppen, and Nicole Amecke for helpful discussions. The “Studienstiftung des Deutschen Volkes” (Bonn, Germany) and Pentapharm GmbH (Basel, Switzerland) are gratefully acknowledged for financial support in form of a Ph.D. scholarship for Nils Helge Schebb and for providing substrates and inhibitors, respectively. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review May 21, 2008. Accepted June 30, 2008. AC801035E