Anal. Chem. 2003, 75, 6388-6393
Frontal Affinity Chromatography Combined On-Line with Mass Spectrometry: A Tool for the Binding Study of Different Epidermal Growth Factor Receptor Inhibitors Lili Zhu, Lirong Chen, Hongpeng Luo, and Xiaojie Xu*
College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, P. R. China
Frontal affinity chromatography (FAC) is a simple but powerful method to analyze molecular interactions between an analyte and an immobilized ligand by calculating the extent of retardation of the elution front. By combination of FAC with a PE-Mariner electrospray ionization mass spectrometry, a very efficient and straightforward procedure was developed herein for analyzing the binding properties of different inhibitors of the epidermal growth factor receptor (EGFR). In this study, a polyclonal antibody prepared with a known anti-EGFR inhibitor coupled with bovine serum albumin was adopted as the stationary phase in the FAC system. Using the antibody to mimic the receptor, other different anti-EGFR inhibitors as well as the small-molecule half-antigen itself were recognized directly from the crude extract of herb, which afforded us a novel promising approach for the efficient screening of lead compounds or drug candidates from natural resources. The epidermal growth factor receptor (EGFR) belongs to the superfamily of protein tyrosine kinases. This receptor catalyzes the transfer of γ-phosphate of ATP (or GTP) to the protein phenolic groups (on Tyr) and plays a fundamental role in the regulation of cell growth.1 However, under certain conditions, as a result of overexpression, mutation, or coexpression of the ligand and the receptor, EGFR can become hyperactivated; the result of this is uncontrolled cell proliferation.2-4 EGFR is known to be overexpressed in numerous tumors including those derived from the brain, lung, ovary, bladder, head, and neck.5 Selective inhibitors of this receptor are therefore of increasing interest as mediators of cell growth (e.g., in psoriasis6) and as potential anticancer drugs.7 * Corresponding author. Tel: +86-10-6275-7456. Fax: +86-10-6275-1708. E-mail:
[email protected]. (1) Fahad, A. A.; Jinzi, J. W.; Kit, S. L. Biopolymers 1998, 47, 197. (2) Plowman, G. D.; Ullrich, A.; Shawver, L. K. Drug News Perspect. 1994, 7, 334. (3) Salomon, D. S.; Brandt, R.; Ciadiello, F.; Normanno, N. Crit. Rev. Oncol. Haematol. 1995, 19, 183. (4) Gullick, W. J. Br. Med. Bull. 1991, 47, 87. (5) Woodburn, J. R. Pharmacol. Ther. 1999, 82, 241. (6) Levitzki, A. FASEB J. 1992, 6, 3275. (7) Dobrusin, E. M.; Fry, D. W. Annu. Rep. Med. Chem. 1992, 27, 169.
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Recently, we prepared a molecularly imprinted polymer (MIP) with a known anti-EGFR inhibitor, quercetin, as the template, and applied the MIP in a solid-phase extraction procedure, and another inhibitor was trapped directly from the crude extract of Caragana jubata, a Chinese traditional Tibetan medicine, which was identified to be (E)-piceatannol.8 In this study, using a polyclonal antibody (PcAb) raised against piceatannol that was linked with bovine serum albumin (BSA) to be covalently bound to the chromatographic packing for mimicking the receptor, different anti-EGFR inhibitors including the half-antigen itself were recognized from the extract of this herb. Herein, frontal affinity chromatography (FAC) connected with mass spectrometry (MS) was adopted as the analytical method due to its inherent merits. A common definition of affinity chromatography is that of a liquid chromatographic technique that uses a “biologically related” agent as a stationary phase for the purification or analysis of sample components.9-13 In this chromatographic mode, a solid affinity matrix is prepared by immobilizing a ligand known to interact specifically with the solute of interest. The molecules used as biospecific or pseudospecific ligands include proteins, DNA, amino acids, and antibodies.14-16 Most affinity separations are performed in a bind-and-elute mode; however, sometimes the separation or analysis in affinity chromatography is performed in frontal mode (i.e., frontal affinity chromatography) in which the key performance criteria are breakthrough curve sharpness and residence time at the adsorption stage. FAC is a very powerful analytical tool to elucidate quantitatively the ligand-binding property of proteins or antibodies. When a large amount of an analyte solution is applied continuously to a column packed with affinity adsorbent that immobilizes a counterligand, the extent of the retardation of the elution front of the analyte reflects the strength of the interaction.17,18 (8) Lili, Z.; Xiaojie, X. J. Chromatogr., A 2003, 991, 151. (9) Walters, R. R. Anal. Chem. 1985, 57, 1099A. (10) Hage, D. S. In Handbook of HPLC; Katz, E., Eksteen, R., Miller, N., Eds.; Marcel Dekker: New York, 1998; Chapter 13. (11) Hage, D. S. Clin. Chem. 1999, 45, 593. (12) Kochan, J. E.; Wu, Y.; Etzel, M. R. Ind. Eng. Chem. Res. 1996, 35, 1150. (13) Arnold, F. H.; Blanch, H. W.; Wilky, C. R. Chem. Eng. J. 1985, 30, B9. (14) Vijayalakshmi, M. A. Trends Biotechnol. 1989, 7, 71. (15) Wilchek, M.; Miron, T. React. Funct. Polym. 1999, 41, 263. (16) Przybycien, T. M. Curr. Opin. Biotechnol. 1998, 9, 164. (17) Arata, Y.; Hirabayashi, J.; Kasai, K. J. Chromatogr., A 2001, 905, 337. (18) Kasai, K.; Oda, Y.; Nishikata, M.; Ishii, S. J. Chromatogr. 1986, 376, 33. 10.1021/ac0341867 CCC: $25.00
© 2003 American Chemical Society Published on Web 10/28/2003
Figure 2. Diagram of the frontal affinity chromatography-mass spectrometry system. The analyte solution in syringe A and the solvent methanol in syringe B are mixed in the tee valve and then detected by ESI-TOF MS.
Figure 1. Chemical structures of the compounds studied in this work.
In the current work, a very simple and sensitive procedure was applied to evaluate the differences in the “retardation” of several small molecular EGFR inhibitors existing in C. jubata in frontal affinity chromatography by using a polyclonal antibody as the affinity adsorbent. In this method, the solution of the crude extract of C. jubata, is applied by means of a sample loop to a small column packed with the antibody that was linked to the carrier, Sepharose CL-4B. Elution of the sample is then monitored continuously by MS. The combination of FAC and MS affords a straightforward and convenient approach for the identification and mensuration of the analytes. From the exact molecular weight of an analyte provided by electrospray ionization (ESI)-MS, its corresponding selected-ion frontal chromatographic trace could be obtained by using the internal software of the mass spectrometry, and thus, the extent of retardation of elution front of the analyte could be calculated from the area under the elution curve. The relative affinity of the PcAb for these anti-EGFR inhibitors could be easily compared in terms of the extent of retardation. As a consequence of these reinforcements, analysis of the binding properties of the analytes in the crude extract of herb is made extremely efficient. EXPERIMENTAL SECTION Materials. (E)-Piceatannol (3,5,3′,4′-tetrahydroxystilbene) (1) and five other compounds studied in this work were isolated from C. jubata and evaluated for their anti-EGFR inhibitory activities by our group. The structures of these analytes are shown in Figure 1. Mixtures of polyclonal antibodies against piceatannol were produced by immunization of rabbits with piceatannol conjugated to BSA. Two white male New Zealand rabbits were kindly provided by Life Science Center, Peking University. BSA, ovalbumin, and Sepharose CL-4B were bought from Sigma Bioscience. The watersoluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) was purchased from Sigma-Aldrich. Freund’s complete adjuvant and incomplete adjuvant were provided by Gibco. EGF, ATP, horseradish peroxidase (HRP), and the substrate peptide used in the enzyme-linked immunosorbent assay (ELISA) for the activities of the anti-EGFR inhibitors were also delivered by Sigma Bioscience. The solvents methanol, dimethyl sulfoxide (DMSO), epichlorohyarin, and 1,4-dioxane were HPLC grade. The sodium salts (including ClCH3COONa, NaCl, NaOH, Na2CO3, and NaHCO3 used in the preparation of the affinity columns) and acetic ammonium (NH4Ac) were analytical grade reagents. All buffers used were prepared by dissolving analytical grade purity salts in
Figure 3. Procedure for piceatannol to be linked with BSA. (*Anyone of the four hydroxyl groups could be modified to be the carboxyl derivative and this structural formula is just one of the possible results.) Reagents: (1) NaOH, DMSO, ClCH2COONa (2 h); HCl (acidification). (2) EDCI, 2 h; BSA-NH2 (in 0.1 M NaHCO3, pH 7.0).
double-distilled water and additionally purified by filtration through a 0.45-µm microfilter (Millipore). Frontal Affinity Chromatography-Mass Spectrometry. The frontal affinity chromatographic experiments were done at room temperature at a flow rate of 10 µL‚min-1 in the running buffer. The device was designed as shown in Figure 2. The antibodies were immobilized to Sepharose CL-4B and then packed wetly in the affinity columns. Both (A) and (B) are 2.5-mL syringes: (A) contains the solution (in 2 mM NH4Ac) of the sample while (B) contains methanol, and these two solutions are mixed in the tee valve and then enter the detector of the ESI-time-of-flight (TOF) MS. A Mariner ESI-TOF mass spectrometer (PE Biosystems) was used to analyze the sample. For ESI, the mass spectrometer was operated in the negative mode using the following conditions: spray tip potential, 5000 V; nozzle potential, 100 V; detector voltage, 1950 V; nozzle temperature, 140 °C; Quad temperature, 140 °C. All the other parameters were set as default values. Preparation for the Antigen. As well as we know, a small molecule by itself (which is often called a half-antigen) does not possess immunity until it is linked with some protein as a carrier (then it possesses immunity as an antigen). In this work, the compound piceatannol was thus modified and then linked to BSA before immunization. The procedure for the modification and conjugation to BSA is shown in Figure 3. There are four hydroxyl groups in the compound piceatannol, and any one of them could be reacted with ClCH2COONa (it could be assured that just one of four hydroxyl groups was altered by controlling the quantity of the reactants). Whichever of the four hydroxyl groups was modified to the carboxyl derivative would be coequal to the preparation of the final polyclonal antibodies since these four hydroxyl groups contribute to the bioactivity of this compound comparably. A total of 24.4 mg (0.1 mmol) of piceatannol (A) was dissolved in 1 mL of DMSO, and then 40.0 mg of NaOH was added to the solution. After stirring the mixture Analytical Chemistry, Vol. 75, No. 23, December 1, 2003
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for 10 min (the color of the solution changed from colorless to yellow and then brown), it was reacted with 9.3 mg (0.08 mmol) of ClCH2COONa. The product B (13.3 mg) of reaction 1 with a yield of 55% was purified by HPLC. About 3.0 mg (10 µmol) of B was dissolved in 0.5 mL of DMSO and then 2.8 mg (15 µmol) of EDCI was added to the solution as an activation reagent. Finally it was linked with 22.0 mg (0.33 µmol) of BSA that was dissolved in 2 mL of 0.1 M NaHCO3 buffer (pH 7.0). There was a total of ∼20.0 mg of the output of the antigen (C) obtained and the ratio between B and BSA was 14:1, which meant that every BSA molecule was coupled with 14 molecules of the carboxylic derivative of piceatannol (there was a total of 59 NH2 groups in a BSA molecule that could be reacted with the COOH group). The yield of reaction 2 was ∼85%. Immunity of the Rabbits. Two white male New Zealand rabbits were immunized every 10 days for 4 times. For each rabbit, 1 mg of the antigen (C) was emulsified with Freund’s complete adjuvant and used for the first immunization shot, while 0.5 mg of C was used for each of the last three applications with the emulsification in Freund’s incomplete adjuvant. After the fourth shot, both of the rabbits were had been phlebotomized from ears. ELISA test showed the potency of the serum was 80 000. Blood was collected from the artery in the rabbit’s neck one week after the last immunization. There were in total ∼50 mL of serums collected from two rabbits. Purification of the Antibodies. Using saturated ammonium sulfate solution to purify the antibody first. 5 mL of serum was diluted with 10 mL of 0.9% NaCl in a 50-mL tube, and then 10 mL of saturated ammonium sulfate solution was added very slowly as the tube was shaken in an ice-water bath. After deposition for 30 min, the tube was centrifuged for 20 min (5000 rpm, 4 °C), and then 15 mL of 40% saturated ammonium sulfate solution was added to the tube to wash the sediment. After the mixture settled for 30 min, the tube was then centrifuged for 20 min (5000 rpm, 4 °C). The upper solution was again removed. The deposition was dissolved in 4 mL of 0.9% NaCl, and then the solution was dialyzed in 1000 mL of water at 4 °C for 16 h, with the water exchanged after 8 h. The antibodies were purified further through an affinity column of DEAE (diethylaminoethyl cellulose, DE52, bought from Whatman). The concentration of protein in the purified antibody solution was 1.8 mg/mL. ELISA test showed the potency of the purified antibody solution was 16 000. Immobilization of the Antibodies. A total of 2.4 g of wet Sepharose CL-4B beads was washed by 100 mL of 0.5 M NaCl and then 100 mL of water, and mixed with 4 mL of 2 M NaOH, 1 mL of epichlorohyarin, and 8 mL of 56% 1,4-dioxane. The mixture was kept in a water bath at 37 °C with shaking for 2 h. After activation, the Sepharose CL-4B beads were washed in a filter with 150 mL of water and 60 mL of 0.2 M sodium carbonate solution (pH 9.5) and then filtered. The filter cake was placed in a flask containing 7 mL of antibodies, which had been dialyzed in 50 mL of 0.2 M sodium carbonate buffer (Na2CO3-NaHCO3, pH 9.5) at 4 °C for 4 h before use and 2 mL of 0.2 M sodium carbonate buffer (pH 9.5), and then the flask was stirred in a water bath at 37 °C for 24 h. The Sepharose beads were washed in a filter with 80 mL of PBS (0.1 M pH 7.4 NaH2PO4-Na2HPO4 buffer + 0.5 M NaCl), 150 mL water, and 40 mL of 2 mM NH4Ac (pH 6.7). The product was kept in 2 mM NH4Ac (pH 6.7) at 4 °C. The 6390
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concentration of protein in the mixture after reaction was 4.8 mg/ mL, and the total volume was 3.2 mL, so 1 g of wet Sepharose CL-4B beads had been linked with 6.4 mg of the antibodies. Using a self-pack device (PerSeptive Biosystems) to pack the affinity columns (i.d. 2.1 × 30 mm) with the polyclonal antibodies linked covalently to Sepharose CL-4B beads. There were in total ∼100 mL of wet Sepharose beads that had been linked with PcAb filled in a column. The PcAb columns were equilibrated with 2 mM NH4Ac solution (pH 6.7) and kept in 4 °C. Preparation of the Extract Sample. Air-dried roots of C. jubata were extracted by macerating with 85% ethanol at room temperature for 2 weeks. The solvent was evaporated in a vacuum and then was partitioned between H2O, petroleum ether, CHCl3, EtOAc, and 1-butanol, successively. Bioassay-guided results (ELISA) suggested that the CHCl3 and EtOAc extracts were efficient in inhibiting the tyrosine kinase activity of EGFR. Therefore, in the following study, the mixture of these two extracts of C. jubata was investigated. The extract sample (5 mg) was evaporated to dryness in a vacuum centrifuge (RE 52A, Shanghai, China.) and then redissolved in 1 mL of methanol to a concentration of 5 mg‚mL-1; thereafter, it was diluted to 0.1 mg‚mL-1 with 2 mM NH4Ac (pH6.7) before loading on the PcAb column. Affinity Test of the PcAb Column to Piceatannol. Compound 1 (the half-antigen piceatannol) was dissolved in DMSO at a concentration of 10 mg‚mL-1. The 2 mM NH4Ac (pH 6.7) was used to dilute the solution to concentrations of 2.5, 5, 10, and 20 µg‚mL-1. The solution of the analyte 1 was filled in syringe A and the solvent methanol was in syringe B. These two syringes were impelled synchronously by the pump of the mass spectrometer at a flow rate of 10 µL‚min-1. After each run, the column was regenerated by washing with 0.5 mL of 2 mM (pH 6.7) NH4Ac, 1 mL of the mixture 0.1 M CH3COOH + 0.5 M NaCl, and 2.5 mL of 2 mM (pH 6.7) NH4Ac until the analytes adsorbed on the column were washed out completely. Screening the Extraction of C. jubata. A 2.5-mL aliquot of the extract sample solution of C. jubata in 2 mM NH4Ac with the concentration of 100 µg‚mL-1 was filled in syringe A, while 2.5 mL of the solvent methanol was contained in syringe B, and these two liquids were pushed at a flow rate of 10 µL‚min-1 and then mixed in the tee valve (Figure 2). The mixture was detected continuously by ESI-TOF MS. Evaluation of the Potency of the PcAb with ELISA Test. Polystyrene wells (Nunc) were coated with test antigens (piceatannol linked to ovalbumin) in 100 µL of 0.01 M phosphate buffer (pH 8.0) and incubated overnight at 4 °C. The wells were washed 3 times with a washing solution (PBS, pH 7.4 + 0.05% Tween-20) and then masked with 0.07% glutin (Bio-Rad EIA purity reagent glutin) dissolved in water. A total of 100 µL of the polyclonal immune blood serum (dilution ranged between 1:100 and 1:800 000 in 0.01 M PBS) was added, and the wells were incubated at 37 °C for 2 h and washed 3 times with washing buffer. A total of 100 µL of 1:800 dilution of goat anti-rabbit/HRP conjugate (Sigma Bioscience) was added, and the wells were incubated for 2 h at 37 °C. Subsequently, the wells were washed 3 times, followed by addition of 50 µL of orthophenylenediamine‚2HCl (Sigma Bioscience). When visible results became evident (∼10 min), the reactions were quenched by the addition of 50 µL of 1.0 N sulfuric acid. Reactions were scored using a Bio-Rad plate reader with a
490-nm absorbance filter. For each ELISA test, three independent dose-response curves were done and the affinity value (potency) of the antibodies was computed. The reported value is average, and the variation was generally (10%. Evaluation for the Activities of the Anti-EGFR Inhibitors. In this study, to investigate the feasibility of using the PcAb as the receptor alternative, the binding properties of the compounds on the PcAb column were contrasted to their bioactivities against EGFR. All the six analytes were therefore evaluated for their antiEGFR tyrosine kinase inhibitory activities by ELISA. The procedure of enzyme reactions was carried out as described in the literature.19 Assays were conducted in a 96-well microtiter plate, and a Bio-Rad plate reader was used here to measure the absorbance at 490 nm. For each compound, three independent dose-response curves were done and the IC50 value was computed. The reported value is average, and variation was generally (10%. RESULTS AND DISCUSSIONS Determination of Ligand Content, Lt, and Dissociation Constant, Kd. Frontal affinity chromatographic analysis of the binding interaction between piceatannol and the PcAb revealed the specific affinity of the adsorbent (in this case, the PcAb) to the analyte. Equation 1 is the basic equation of frontal affinity chromatography:
V - V0 ) Lt/(C + Kd)
(1)
It can also be shown in another form:
1 1 Kd 1 ) + C(V - V0) Lt Lt C
(2)
where V is the elution volume of the analyte, C is the concentration of the analyte, V0 is the elution volume of a controlled substance having no affinity (in the current work, BSA linked to Sepharose CL-4B was used), Lt is the total amount of immobilized ligand, and Kd is the dissociation constant. The smaller C is, the larger V is. If C , Kd, i.e., as C is negligibly small compared with Kd, V approaches its maximum value, Vm, which is independent of C and the following equation is obtained:
Vm - V0 ) Lt/Kd
(3)
We applied the present FAC-MS to test the binding specificity of the PcAb to the original half-antigen, piceatannol. A 2.5-mL solution of different concentrations of piceatannol in 2 mM NH4Ac ranging from 20, 10, 5, to 2.5 µg‚mL-1 was filled in syringe A (as shown in Figure 1) and impelled through the PcAb column, while 2.5 mL of methanol was filled in syringe B and pushed forward synchronously with (A) at the rate of 10 µL‚min-1. A totalion chromatograph (TIC) was obtained for a 200-min running time for the mixed solution through the tee valve. The selected-ion chromatography of piceatannol derived from the TIC revealed the frontal time as well as the frontal volume, which was shown in (19) David, W. F.; Alan, J. K.; Amy, M.; Linda, A. A.; James, M. N.; Wilbur, R. L.; Richard, W. C.; Alexander, J. B. Science 1994, 265, 1093.
Figure 4. Frontal affinity chromatograms of solutions of the analyte (piceatannol) at different concentrations: (a) 20, (b) 10, (c) 5, and (d) 2.5 µg‚mL-1.
Figure 5. Determination of the ligand content and dissociation constant of the PcAb. Table 1. Results of Frontal Affinity Chromatography of Piceatannol C (µg‚mL-1)
C (µmol‚l-1)
1/C (µmol/L)-1
V - V0* (µL)
1/[C(V - V0)] (µmol)-1
20.0 10.0 5.0 2.5
82.0 41.0 20.5 10.2
0.0122 0.0244 0.0488 0.0976
188.0 314.0 486.0 676.0
64.9 77.7 100.4 144.9
Figure 4. From the four traces corresponding to different concentrations of the analyte, the ligand content, Lt, and dissociation constant, Kd, could be calculated. And the results are shown in Table 1. Using the data in Table 1, the values for Lt and Kd were calculated from eq 2, which were 44.90 µg‚mL-1 and 17.14 µM, respectively. The correlative linearity was shown in Figure 5, from which it could be deduced that the antibodies did possess specific affinity toward the analyte piceatannol. Binding Study of the PcAb for Several Anti-EGFR Inhibitors in the Crude Extract Sample. Since the main object of the current work was to study the feasibility of using PcAb to mimic the receptor in recognizing the anti-EGFR inhibitors directly from the crude extract of C. jubata, the FAC-MS procedure was performed on a 2.5-mL solution of the extract sample at a concentration of 100 µg‚mL-1 in 2 mM NH4Ac. The frontal affinity chromatographic trace derived from the mass spectrum (Figure 6) was shown in Figure 7. Using the six purified components as the standards, the approximate concentrations of these analytes in the crude extract of C. jubata were detected separately. According to eq 2, the dissociation constant (Kd) for each compound could therefore be calculated since the frontal volume of each analyte had been measured (displayed in Figure 7) and Analytical Chemistry, Vol. 75, No. 23, December 1, 2003
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Table 3. Recognition Effect of the PcAb Expressed in the Contrast
Figure 6. Mass spectrum of the extract sample of C. jubata. (The six compounds displayed in Figure 1 corresponded to the six main peaks in this spectrum.)
Figure 7. Selected-ion FAC trace for six analytes existed in the extract sample of C. jubata. Traces 1-6 corresponded to the six main components displayed in Figure 6. Table 2. Results of the FAC and the Inhibitory Activities of Six Analytes in the Extract Sample compda
approx concn (%)
T (min)
V (µL)
V - V0 (µL)
Kd (M) (estimated)
1 2 3 4 5 6
7.6 5.4 2.5 2.7 2 3
45.3 21.9 19.6 8.3 7.2 5.0
453 219 196 83 72 50
409 175 152 39 28 6
1.7 × 10-5 8.1 × 10-5 9.7 × 10-5 4.2 × 10-4 5.7 × 10-4 3.6 × 10-3
a The six compounds were corresponding one-to-one to those shown in Figure 1.
the ligand content (Lt) was known as described above. The results were listed in Table 2. From the data shown in Table 2, the efficiency of the PcAb in recognizing different anti-EGFR inhibitors according to their different Kd values could be observed. To make clear the correspondence between the bioactivities and the relative binding affinities of these inhibitors, an auxiliary coefficient factor, relative binding strength (Sr), was evaluated for each compound using the half-antigen (piceatannol) as the standard, which means the Sr value for piceatannol was set to be 1.00, and those for other analytes were calculated according to the ratio between the Kd value for piceatannol and that for the analyte. In a frontal affinity chromatography system applied to study the binding property of a Caenorhabditis elegans galectin,17 Yoichiro et al. also used the relative binding strength to compare the different binding affinities of recombinant C elegans galectins for ligands. The results are listed in Table 3. The correspondence between the relative binding strength and the bioactivity (expressed in IC50 value) for each analyte indicated clearly the effect of the PcAb for recognizing different anti-EGFR inhibitors directly from the crude extract of C. jubata: the smaller was the IC50 value of the inhibitor, the longer was the frontal time for the compound to be eluted from the PcAb column, which 6392 Analytical Chemistry, Vol. 75, No. 23, December 1, 2003
compd
Sr (rel binding strength)
IC50 (against EGFR)
1 2 3 4 5 6
1.00 0.21 0.18 0.04 0.03 0.005
4.9 µM 10 µM 15 µM 1.1 mM 1.2 mM 59.0 mM
meant stronger specific affinity between the analyte and the PcAb. Especially for the three compounds (1-3) that possess relatively high inhibitory activities against EGFR, the recognition effect of the affinity adsorbent was more satisfactory. The values of relative binding strength for piceatannol, butein, and quercetin were 1.00, 0.21, and 0.18, respectively, which were consistent with the order of their bioactivities: the higher the bioactivity of the inhibitor, the larger the relative affinity strength of PcAb for the analyte. While for 6, which displayed rather low anti-EGFR inhibitory activity, the frontal volume was so small that it could be deemed that little binding affinity existed between this analyte and the sorbent (the relative affinity strength was 0.005). Therefore, it could be concluded that as the PcAb with specific binding affinity was used to mimic the receptor, not only could the inhibitors displaying relative high bioactivities be separated from those bearing low activities but also these analytes could be screened out according to their different biocharacteristics (in this case IC50, indicating activity was used) directly from the crude extract of herb. The development of what is essentially an “antiidiotype” antibody studied herein allowed it to mimic the binding site of EGFR and thus to recognize different EGFR inhibitors directly from the crude extract of a Chinese traditional herb efficiently, which afforded us a promising technique for an initial screen of inhibitory activities from natural resources. The current application of PcAb has rarely been reported. In the present system, as lower concentrations (10 and 2.5 µg‚mL-1) of the extract solution were tested for recognition ability of the PcAb, better results were obtained but the running time for analysis was prolonged (about 4-6 h) accordingly. It was not surprising considering that the frontal time got longer at lower concentrations of the analyte, and then the differences of retardation between them were more obvious as well as the recognition ability getting better. Just as described above, as the concentration of the analyte (C) is negligibly small compared with Kd, the frontal volume (V) will be independent of C, and thus, the recognition ability of the PcAb will be dependent mostly on the dissociation constants of the analytes. The current efficient screening of different anti-EGFR inhibitors derived from the affinity of the PcAb is reasonable from the viewpoint of the basic intermolecular interactions (hydrophobic, electrostatic, hydrogen bonding) determining the behavior of chemical compounds in both biological and chromatographic environments. Those inhibitors against some receptor often possess several common characteristics, such as some hydrophobic groups at the special position or some hydrophilic groups (e.g., OH, NH2, COOH) at other sites. Since the specific interactions between the antigen and antibodies are similar to those
between the inhibitors and the binding pocket of the receptor, we could use the antibodies against one representative inhibitor of the receptor to mimic its binding site and thus to recognize other different inhibitors possessing similar intermolecular interactions with the receptor. The present work has validated primarily the efficiency of this novel application of polyclonal antibodies. Although there were only six analytes recognized from the extract sample of herb in this study, a more complete and successful work has been accomplished by our group in which 13 compounds with inhibitory activities against hepatitis C virus (HCV) were screened out directly from Phyllanthus urinaria L, another traditional Chinese medicine.20 CONCLUSIONS The current work afforded us a feasible approach for screening the effective anti-EGFR inhibitors using a PcAb as the sorbent material directly from the crude extract of C. jubata. FAC-MS was used here as a powerful tool for the detection and evaluation of the specific affinity of the PcAb for the analytes. Combined with our previous work where a MIP was used for the direct extraction of anti-EGFR inhibitors from natural resources,8 it can be concluded that as the complementary methods to the traditional isolation using conventional materials (e.g., silica gel, polyamide, ion-exchange types, and reversel-phase column), both of these potent techniques (MIP and PcAb) could be effectively applied to mimic the enzyme for separating and recognizing active antiEGFR inhibitors directly from natural resources and, therefore, could improve the efficiency for the discovery of novel inhibitors. (20) Hongpeng, L.; Zhensheng, D.; Zhengquan, L.; Lirong, C.; Xiaojie, X. Chem. Lett., submitted. (21) Owens, P. K.; Karlsson, L.; Lutz, E. S. M.; Andersson, L. I. Trends Anal. Chem. 1999, 18, 146.
These two materials (MIP and PcAb) both possess inherent predetermined specific binding affinity to the template (in MIP) or antigen (in PcAb), and those compounds are chemically similar to the target molecule, which enable them to simulate the receptor for directly screening the analytes of interest from natural resources. Contrasted to MIP, the application of PcAb has its own merits and demerits. More (often 1 mmol) of the template molecule was demanded in molecular imprinting, while usually only a trace amount (0.01-0.1 mmol) of half-antigen (in this case, piceatannol) was needed to produce PcAb. So the biological technology (preparing PcAb) would be more favorable as the target molecule was expensive or difficult toobtain, especially for some drug candidates or lead compounds that existed in only trace contents in natural resources, while MIP possesses better chemical and physical robustness than PcAb, and it is more inert toward acids, bases, metal ions, and organic solvents as well as elevated temperature and pressure than the latter.21 These factors provide more extensive applications for MIP. However, as the complementary methods to traditional isolation following with bioassay guidance, both of these two techniques displayed potential efficiency for high-throughput screening of analytes of interest. It was expected that the novel application of these two materials (MIP and PcAb) would be beneficial for efficient screening of lead compounds or drug candidates from both natural resources and synthetic combinatorial libraries and, thus, would help to accelerate future drug development.
Received for review February 25, 2003. Accepted April 30, 2003. AC0341867
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