Bioconjugate Chem. 2007, 18, 1981–1986
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Screening of Paclitaxel-Binding Molecules from a Library of Random Peptides Displayed on T7 Phage Particles Using Paclitaxel-Photoimmobilized Resin Sota Aoki,† Kengo Morohashi,‡,| Takashi Sunoki,‡,§ Kouji Kuramochi,† Susumu Kobayashi,‡,§ and Fumio Sugawara*,†,‡ Department of Applied Biological Science, Genome and Drug Research Center, and Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan. Received July 29, 2007; Revised Manuscript Received September 19, 2007
Paclitaxel (Taxol), an effective anticancer agent, is known to bind to tubulin and induce tubulin polymerization. Several other binding proteins of paclitaxel, such as Bcl-2, heat shock proteins, and NSC-1, have also been reported. Here, we describe a T7 phage-based display to screen for paclitaxel-binding molecules from a random peptide library using paclitaxel-photoimmobilized TentaGel resin. Specific phage particles that bind the paclitaxelimmobilized resin were obtained. Among them, two phage clones included the same consensus amino acid sequence (KACGRTRVTS). Analysis of the protein database using BLAST revealed that a portion of this sequence is conserved in the zinc finger domain of human NFX1. Binding affinity of paclitaxel against the partial recombinant protein of NFX1 (424aa–876aa) was confirmed by pull-down assays and surface plasmon resonance analyses.
INTRODUCTION Paclitaxel is a highly effective antineoplastic agent used in the treatment of a wide variety of tumors (Figure 1) (1). There are numerous reports in the literature describing the candidate binding proteins of paclitaxel. Paclitaxel promotes tubulin polymerization by binding and stabilizing microtubules (2–7). Heat shock proteins 70 and 90 were identified to be paclitaxel-binding proteins from a macrophage cell lysate using biotinylated paclitaxel (8). An antiapoptotic protein, Bcl-2, and a neuronal calcium sensor protein, NSC-1, were also recognized as paclitaxel-binding proteins by phage display technology (9–11). Bcl-2 was identified from a library of random phage-displayed peptides by an analysis of affinity-selected peptides (9, 10). NCS-1 was also found to be a paclitaxel-binding protein using a similar selection procedure from a human brain cDNA phage library (11) . Phage display is one of the best methods for the identification of drug-binding proteins (12). Indeed, this method has been successfully applied to the determination of drug-binding proteins for FK506 (13), doxorubicin (14), HBC (15) and paclitaxel (9–11). Moreover, phage display technology can be used to efficiently identify the specific drug-binding site within the identified protein (9, 10). The combination of screening a library of phage-displayed peptides and the analysis of affinityselected peptides is anticipated to become a powerful tool for identifying drug-binding sites (16–21). Screening phage display libraries generally entails immobilizing the drug onto a solid surface (22). Immobilization of the drug onto an affinity matrix is usually achieved by the formation of a biotinylated derivative, which can then be applied to a * Corresponding author. Phone: +81-4-7124-1501(ext. 3400). Fax: +81-4-7123-9757., E-mail:
[email protected]. † Department of Applied Biological Science. ‡ Genome and Drug Research Center. § Faculty of Pharmaceutical Sciences. | Present address: Department of Plant Cell and Molecular Biology, The Ohio State University, 106 Carmack Road, Columbus, OH 43210.
streptavidin-coated matrix. Conventional immobilization requires the presence of desirable functional groups within the drug molecule as well as a multistep process to prepare the biotinylated derivatives. However, photoimmobilization makes it possible to covalently immobilize drugs onto the solid surface without the need for derivatization. Kanoh and ourselves have reported the affinity purification of proteins using affinity matrices, in which small molecules were photoimmobilized by photoreaction (23–25). Because the photoreaction proceeds in a functional group-independent manner, the molecules are immobilized onto the solid surface in a nonoriented fashion. Thus, photoimmobilization can be a useful tool for the comprehensive analysis of drug-binding proteins. In this study, paclitaxel-binding molecules were identified from a library of random peptides displayed on T7 phage particles using paclitaxel-photoimmobilized TentaGel.
EXPERIMENTAL PROCEDURES Materials. Paclitaxel was purchased from Hoechst Marion Roussel (now known as Aventis Pharmaceuticals Inc., Paris, France). Photoaffinity beads were prepared as described previously, and paclitaxel was immobilized on the beads according to the literature (24). A 10.2 mg quantity of paclitaxel was used for immobilization onto 30 mg of photoaffinity beads. Photoaffinity beads, which were treated after UV irradiation in the absence of paclitaxel, were used for the control resin. The T7 phage library displaying a random 12 amino acid peptide was prepared as described in our previous paper (21). A sensor chip of CM5 and an Amine Coupling Kit were obtained from Biacore AB (now known as GE Healthcare, Amersham, UK). A mouse IgG-κ monoclonal antibody for anti-6His was purchased from Sigma-Aldrich (St Louis, MO). An alkaline phosphatase conjugated with a goat IgG antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Panning the Phage Library. A modified version of the biopanning technique given in the manufacturer’s instructions (Novagen, Madison, WI) was used in this study (21). One milliliter of a random 12 amino acid T7 display library (approximately 9 × 1010 pfu) was added to 100 µg of pretreated
10.1021/bc700287v CCC: $37.00 2007 American Chemical Society Published on Web 11/03/2007
1982 Bioconjugate Chem., Vol. 18, No. 6, 2007
Aoki et al. Table 1. Results of Biopanning with Paclitaxel Immobilized on Photoaffinity Beads round (th)
inputa ( × 1010)
outputa ( × 105)
recoveryb ( × 10-6)
enrichment (per round)
enrichment (total)
1 2 3
5.00 3.20 1.70
1.00 0.68 1.20
2.00 2.13 7.06
1.07 3.31
1.00 1.07 3.53
a
Figure 1. (A) Structure of paclitaxel. (B) Structure of photoaffinity beads (TentaGel).
resin in TBS (50 mM Tris-HCl at pH 8.0 and 150 mM NaCl) for 12 h at room temperature. After incubation at 4 °C for 12 h, the resin was precipitated and the phage solution removed. The resin was then washed 25 times with 1 mL of TBST buffer (50 mM Tris-HCl at pH 8.0, 150 mM NaCl, and 0.1% Tween 20). Adsorbed phage particles were subsequently removed from the resin by 15 min of incubation in 1% SDS solution. The eluted phage particles were amplified after infection into Escherichia coli strain BLT5615 as host cells. The amplified phage was applied to the resin, and subsequent rounds of panning were carried out. The titer was monitored at each panning step. The eluted phage solution at each step was used for the immediate infection of 3 mL of host cells. Incubation with shaking at 37 °C was continued until lysis was observed. Amplified phage titers of each round were estimated to be 1–6 × 1010 pfu. After each round, the eluted phage titers were compared to those of the previous round. Validation of Interaction between Monoclonal T7 Phage and Ligand. The T7 phage mixture from the final round of panning was incubated at low density on an LB plate containing 50 µg/mL of carbenicillin. Individual plaques were randomly picked and diluted into 50 µL of phage extraction buffer (20 mM Tris-HCl at pH 8.0, 100 mM NaCl, and 6 mM MgSO4). To validate the affinity between monoclonal phage and paclitaxel-immobilized resin, the isolated phage particles were amplified with E. coli BLT5615. Binding, washing, and elution procedures were carried out as described in the screening procedure. Blank resin, without immobilized paclitaxel, was prepared, and the eluted phage titers were compared to those of the binding phage. PCR Amplification and Sequencing. For PCR amplification of DNA fragments and sequencing, 5 µL of T7 phage solution was used as the template. T7 UP new (5′-TGCTAACTTCCAAGCGGACC-3′) and T7 DOWN (5′-AACCCCTCAAGACCCGTTA-3′) primers, corresponding to the sequences in the phage vector that flanks the insert, were used to amplify inserted DNA sequences. PCR was performed in a 20 µL of reaction mixture with gene Taq polymerase (Nippon gene, Tokyo, Japan). Amplified fragments were analyzed by agarose gel (1.5%) electrophoresis. After purification of the amplified fragments by ethanol-precipitation, and DNA fragments were sequenced using PRISM BigDye terminator ready reaction kit (version 3.1) (PE-ABI, Foster City, CA) and applied to an ABI3100 sequencer (Applied Biosystems, Foster City, CA). The BLAST program was used for comparing the obtained sequences with sequences in the protein database (27). Protein Expression and Purification. DNA encoding a fragment of the NFX1-type zinc finger domain of NFX1 (424aa–876aa) was amplified from a human leukocyte cDNA library using the forward primer (5′-GGGAATTCTGTGGTGAGGTTTGTAGAAAG-3′) and the reverse primer (5′CCCTCGAGACATTCACACTGTAGCTCTACC-3′). The amplified DNA was then cloned into a derivative of pET22b, constructed by insertion of the E. coli DsbC chaperone/disulfide
Plaque forming unit. b Output/input.
isomerase gene. A truncated form of NFX1 (424–876) containing an N-terminal His-tag was coexpressed with DsbC after induction with 0.4 mM IPTG at 16 °C. The harvested cells were lysed by sonication, and cell debris was removed by centrifugation at 12000g for 10 min. The crude extract was then loaded onto a Ni-NTA column, and the bound proteins were eluted with a solution of 200 mM imidazole (pH 7.4). The eluted proteins were then dialyzed against PBST buffer. Pull down Assay and Western Blotting. Small molecule pull-down assays were carried out as described previously (24). NFX1 adsorbed on the resin was detected using the Bradford assay and Western blot analysis. Western blotting was performed with a mouse IgG monoclonal antibody for anti-6His (used at 1:1000 dilution). A goat antimouse IgG conjugated with alkaline phosphatase was used as the secondary antibody at a dilution of 1:1000. Immunoreacted signals were detected with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. Surface Plasmon Resonance (SPR) Analysis. SPR analysis was performed on a BIAcore X (Biacore AB, Sweden, now known as GE Healthcare). The truncated form of NFX1 (424aa–876aa) with an N-terminal His-tag was covalently immobilized to a hydrophilic carboxymethylated dextran matrix on a CM5 sensor chip using a standard amine coupling reaction in 10 mM CH3CO2Na (pH 4.0) at a level of approximately 7500 resonance units (RU). Binding analysis was carried out in PBST buffer (20 mM, 150 mM NaCl, 10 µM ZnCl2, and 0.001% Tween 40) containing 8% DMSO at a flow late 20 µL/min. Appropriate concentrations of paclitaxel were injected over the flow cell. The bulk effects of DMSO were subtracted by using reference surfaces. To derive binding constants, data was analyzed by means of global fitting by using the BIA evaluation software (version 3.1).
RESULTS Selection of T7 Phage Associated with Paclitaxel. A library of random peptides displayed on phage particles was screened for the ability to specifically bind paclitaxel-immobilized resin. Phage particles having affinity for paclitaxel-immobilized resin were efficiently enriched (Table 1). The ratio of eluted phage titer increased in each successive round of screening. We anticipated that the eluted phage particles from the third round would display high affinity for paclitaxel. Thus, 32 phage clones were randomly selected from plates and then sequenced. To estimate the binding specificities of the peptides displayed on phage particles, each single phage clone was amplified, and the resultant phage clones were used for a binding assay (Table 2). The affinity titers of eight selected phage clones (numbers 13, 15, 18, 20, 21, 26, 27, and 29) from paclitaxel-immobilized resins were more than 6.7 times higher than those of the background phage from the control resin. Phage clones 18, 20, and 21 displayed a series of peptides of more than 12 amino acids and had specific affinity for paclitaxel immobilized on the resin. The sequence of clones 20 and 21 were identical for the C-terminal 10 amino acid residues (KACGRTRVTS). Recombinant Protein Interacts with Paclitaxel. A homology search using the BLAST program demonstrated that the amino acid sequence of the consensus peptide (KACGRTRVTS)
Screening of Paclitaxel-Binding Molecules
Bioconjugate Chem., Vol. 18, No. 6, 2007 1983
Table 2. Affinity Titers and Selected Amino Acid Sequences of Peptide Displayed on the Phage after the Third Round of Biopanninga clone
sequence
relative enrichment ratiob
#13 #15 #18 #20 #21 #26 #27 #29
VYCVL* FLFGLFFFTCRS* FRIVTCATCVILKAFMGWHYHISRSRS* CDSFARSCVRGVGIMKACGRTRVTS* LFTDMALSGKVLVKACGRTRVTS* LFLCCMGFHGPL* LLLSHVSLVCRL* CFLLSFVVLGYD*
12.5 9.2 11.3 49.0 6.7 27.3 34.4 7.3
a Estimation of binding specificities for selected phage clones by affinity titers. The titers of the input phages were adjusted to 1.0 × 10 10 cfu/mL. Binding assays were performed on paclitaxel-immobilized resins (paclitaxel +) or control resins (paclitaxel –). b The relative enrichment ratio is determined as the output phages from paclitaxel-beads divided by the output phages from control beads.
Figure 3. Binding analysis of the recombinant NFX1 (424aa–876aa) with the paclitaxel-immobilized resins (PCT-resins) and control resins (Control resins). (A) Quantitative analysis of proteins bound to 10 mg of resin using the Bradford assay. Note that 348 µg of the recombinant NFX1 (424aa–876aa) was applied to the binding assay. (B) Western blot analysis of the His-tagged zinc finger domain of NFX1 bound to the resin.
cillin showed significant interaction with immobilized NFX1 (424aa–876aa) (Figure 4B).
DISCUSSION Figure 2. Alignment between obtained amino acid sequence and human NFX1. (A) The similarity site (marked by upward arrow/underline) is contained in the NFX1-type zinc finger domain of NFX1. This region affects the DNA binding of NFX1. (B) The obtained amino acid sequence indicated similarity to C463-T469 of human NFX1. The numbers show amino acid positions in NFX1. The asterisk indicates identical amino acids.
is similar to a part of the zinc finger domain of human NFX1 (Figure 2) (20, 21, 26). This region affects the DNA binding of NFX1 (28). A truncated recombinant NFX1 (424aa–876aa) was overexpressed and purified for the binding assays (28). The recombinant NFX1 (424aa–876aa) contains an amino acid sequence similar to that of the consensus sequence selected by phage screening. The amount of truncated NFX1 (424aa–876aa) bound to the paclitaxel-immobilized resin (10 mg) or control resin (10 mg) was determined using the Bradford assay by monitoring absorbance at 595 nm (Figure 3). Approximately 30% of the applied recombinant NFX1 (424aa–876aa) was recovered from thepalitaxel-immobilizedresin.TherecoveredNFX1(424aa–876aa) from the palitaxel-immobilized resin was detected by Western blotting using an anti-6His monoclonal antibody (Figure 3). By contrast, only a small amount of NFX1 bound to the control resin. Binding analysis between paclitaxel and the truncated form of NFX1 (424aa–876aa) was performed by surface plasmon resonance. The recombinant NFX1 (424aa–876aa) was fixed on a CM5 sensor chip using an amine coupling reaction. The dissociation constant of paclitaxel with NFX1 (424aa–876aa) was determined to be 8 × 10-7 M, using BIA evaluation 3.1 software (Figure 4A). Specificity of binding was confirmed by analyzing the interaction of NFX1 (424aa–876aa) with doxorubicin and ampicillin, whose structures are different from that of paclitaxel. Unlike paclitaxel, neither doxorubicin nor ampi-
Identification of binding partners to biologically active small molecules is an important step in elucidating the corresponding mechanism of action. Phage display is a versatile method for the detection of small molecule-binding proteins. One advantage of phage display is that the technique can also be used to identify binding sites within the target protein itself. Indeed, several binding sites for bioactive compounds have been elucidated by screening with phage display. In the conventional method of phage display, small molecules are usually converted into the biotinylated derivatives and immobilized on a streptavidin-coated matrix. Preparation of biotinylated derivatives can be a laborious and time-consuming process. Furthermore, biotinylation of a small molecule might interfere with binding to the cognate binding partner(s). By contrast, immobilization of small molecules on a solid support using UV irradiation can be readily achieved without the need for derivatization. Using this procedure, the molecules will be immobilized in a nonoriented manner. Therefore, photoimmobilization can eliminate serious problems associated with biotinylation, which could potentially abolish the functional groups required for protein binding. In the present study, we isolated phage clones that show affinity for paclitaxel-photoimmobilized TentaGel resin. Two of the selected clones have the consensus amino acid sequence KACGRTRVTS. A comparison of this short peptide sequence with the protein database revealed similarity to residues 463–469 of human NFX1. NFX1 is known to be a transcriptional regulator of the human major histocompatibility complex (MHC) class II gene, although it is also expressed in MHC class IInegative cells (28). Analysis of NFX1 shows that it possesses a central DNA-binding domain that is subdivided into repeated Cys-rich motifs (437–868 amino acids of NFX1). The motif is similar to, but distinct from, LIM and RING finger domains. NFX1 binds to the conserved X-box motif of MHC class II genes via a repeated zinc finger domain and represses their expression to regulate the duration of an inflammatory response (28). The RING-type zinc finger domain also interacts with a ubiquitin-conjugating enzyme (E2) and facilitates ubiquitination (29).
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Figure 4. SPR analysis of the binding between paclitaxel and partial recombinant NFX1 (424aa–876aa). Analytes in PBST buffer (containing 8% DMSO) were injected over flow cells on the immobilized NFX1 (424aa–876aa). The background resulting from an injection of running buffer was subtracted from the data before plotting. Response units (RU) were generated by subtraction of the background signal generated simultaneously on the control flow cell. (A) Binding of paclitaxel on immobilized NFX (424aa–876aa) at various concentrations (1, 7.5 µM; 2, 5 µM; 3, 2.5 µM; 4, 1.25 µM). Kinetic studies were performed with BIA evaluation software. (B) Estimation of binding specificity. A 5 µM solution of paclitaxel (1), doxorubicin (2), or ampicillin (3) was loaded.
Interaction between paclitaxel and the partial recombinant NFX1 (424aa–876aa) was confirmed by pull-down assays and surface plasmon resonance analyses. Recombinant NFX1 (424aa–876aa) selectively binds the pactitaxel-photoimmobilized resin by the pull-down assay. Paclitaxel bound the immobilized recombinant NFX1 (424aa–876aa) with a dissociation constant (Kd) of 8 × 10-7 M. By contrast, doxorubicin and ampicillin
did not show significant interaction with the immobilized NFX1 (424aa–876aa). Sengupta et al. measured the binding of N-dibenzoyl-N-[3-(dimethylamino)benzoyl]-paclitaxel, a fluorescent derivative of paclitaxel, to tubulin. Data from these experiments gave an equilibrium dissociation constant of 4.9 × 10-5 M for the binding of paclitaxel to unpolymerized tubulin (30). Rodi et al. reported that paclitaxel binds to Bcl-2 with a dissociation
Screening of Paclitaxel-Binding Molecules
constant of 4 × 10-7 M (9). Although we have not examined whether paclitaxel influences the activities of NFX1 and its resultant effects, these results verify that NFX1 is one of the binding proteins of paclitaxel. Interestingly, our experiments established that paclitaxel binds to the zinc finger domain of NFX1, which requires NFX1–DNA interaction. Considering that NFX1 functions as a repressor, paclitaxel might inhibit the repressive effect of NFX1 for MHC class II gene expression, suggesting that paclitaxel activates MHC class II gene expression. Indeed, this hypothesis is consistent with a previous report, which demonstrated that paclitaxel induced MHC class II genes in dendritic cells (31). A homology search of our selected consensus peptide by BLAST gives other candidate binding proteins of paclitaxel. For example, tropoelastin has an amino acid sequence (779KACGRKR785) similar to that of the consensus sequence in the C-terminal region (32). Furthermore, placenta-specific 1-like protein precursor contains a segment of sequence (76CGIRTRVVS84) that displays similarity to the selected peptide sequence (33). Surprisingly, none of our isolated clones showed similarity to known paclitaxel-binding proteins such as tubulin, heat shock proteins, Bcl-2, or NCS-1. One possible explanation for these results is that our original phage library was not sufficiently diverse. The titer of the primary clones in the library used in this study was 4 × 106 pfu/mL (21). The use of a high diversity library would provide a greater amount of information on paclitaxel-binding peptides. Alternatively, interaction of paclitaxel with proteins such as tubulin may involve multiple binding motifs, which despite being far apart in the primary amino acid sequence are spatially close together in the three-dimensional structure. Indeed, paclitaxel binds to a pocket in the second globular domain of β-tubulin facing the microtubule. The use of a highly diverse library would also contribute to more comprehensive information on the binding peptides. In summary, we have used a T7 phage display screening method to identify paclitaxel-binding molecules from a random peptide library using paclitaxel-photoimmobilized resin. Two of the selected clones have the consensus peptide KACGRTRVTS, which displays similarity to a part of the zinc finger domain of human NFX1. Binding between paclitaxel and recombinant NFX1 was confirmed by pull-down and SPR analyses. Our results demonstrate that this methodology can be used to identify novel binding proteins of small molecules. Studies on the biological effect of paclitaxel on human NFX1 as well as further application and optimization of this methodology are currently underway.
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