Article pubs.acs.org/bc
Whole-Virus Screening to Develop Synbodies for the Influenza Virus Nidhi Gupta,‡ John Lainson, Valeriy Domenyuk,† Zhan-Gong Zhao, Stephen Albert Johnston, and Chris W. Diehnelt* The Biodesign Institute Center for Innovations in Medicine, Arizona State University, Tempe, Arizona 85287, United States S Supporting Information *
ABSTRACT: There is an ongoing need for affinity agents for emerging viruses and new strains of current human viruses. We therefore developed a robust and modular system for engineering high-affinity synbody ligands for the influenza A/Puerto Rico/8/1934 H1N1 virus as a model system. Wholevirus screening against a peptide microarray was used to identify binding peptides. Candidate peptides were linked to bis-maleimide peptide scaffolds to produce a library of candidate influenza-binding synbodies. From this library, a candidate synbody, ASU1060, was selected and affinity-improved via positional substitution using D-amino acids to produce a new synbody, ASU1061, that bound H1N1 in an ELISA assay with a KD of 90% purity) from Sigma Custom Peptide and used for subsequent synbody preparation. Synbody Conjugation. In the synthesis of a synbody, the peptide and the scaffold are separately dissolved in 30% acetonitrile in water. First, 1 equivalent of the scaffold is mixed with 2 equivalents of the peptide. The pH of the reaction mixture is then adjusted to 6.5−7.0 with the addition of dilute triethylamine (TEA) (10% in acetonitrile). The reaction is allowed to proceed at room temperature without shaking and monitored by HPLC and MALDI−mass spectrometry. The yield of the reaction is determined by integration of the peaks assigned to the desired product. SPR Screening of Synbodies. Determination of the binding of synbodies to immobilized H1N1 was performed on a Biacore 4000. The series S sensor chip CM5, amine coupling reagents, and HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20) were obtained from GE Healthcare. An amine immobilization protocol was performed at 25 °C using 10 mM NaHCO3 at pH 5.0 as the immobilization buffer. All five spots on one flow cell of the CM-5 chip were activated by a 10 min injection of a freshly prepared 1:1 solution of 400 mM EDC−100 mM NHS in water. Spots 1, 2, 4, and 5 were treated with a solution of UV-inactivated influenza A/PR/8/1934 (50 μg/mL) in 10 mM NaHCO3 (pH 5.0) for 8, 14, 16, and 10 min at a flow rate of 10 μL/min. Any residual active sites were then quenched by a 5 min pulse of ethanolamine (1 M, pH 8.5). Synbody samples at 100 μM were injected at a flow rate of 30 μL/min over the flow cell surface. Buffer injections identical to the analyte were included throughout the analysis for the purpose of double referencing. The surfaces were regenerated with one 30 s injection of pH 3.0 glycine. The binding level for each injection was determined using Biacore 4000 Evaluation Software. ELISA Binding Assay for Synbodies. Nunc MaxiSorp flat bottom 96 well ELISA plates were coated with 100 μL of 1 μg per well of UV-inactivated influenza A PR/8/1934 H1N1 in ELISA-coating buffer and kept overnight at 4 °C. Plates were washed with 1× phosphate-buffered saline with Tween-20 (PBST) followed by blocking with 200 μL of 6% BSA (ELISA grade fraction V) in 1× PBST for 2 h at room temperature. Plates were washed twice with 1× PBST, and biotin-labeled synbodies were added in ELISA dilution buffer (1% BSA + 1× PBS + 0.05% v/v Tween-20) and incubated for 1 h at room temperature. After washing, 100 μL of 1:100 000 streptavidin− HRP was added and incubated for 1 h at room temperature. Plates were washed, and 100 μL of TMB was added. Plates were incubated in the dark for 8 min at room temperature. The reaction was quenched by addition of 100 μL of 0.5 M HCl and read immediately at 450 nm using micro plate reader. ELISA dilution buffer was run as a control on influenza-coated wells. Anti-influenza NA (BEI Resources, catalog no. NR-4540) antibody (5 nM) and unrelated synbody was used as the positive and negative controls, respectively. SPR of Synbodies. To identify the target protein on influenza that the synbody binds, recombinant influenza A PR/ 8/34 hemagglutinin (HA) (Sino Biological Inc., catalog no. 11684-V08H), HA from A/New Caledonia/20/99 (Abcam, catalog no. ab69741) and nucleoprotein (NP) from A/PR/8/ 34 (Imgenex, San Diego, CA) were purchased and screened against the captured synbody using a Biacore 4000 SPR system.
technologies, Inc.) in ELISA coating buffer (0.1 M sodium carbonate−bicarbonate pH 9.4) and kept overnight at 4 °C. Plates were washed with 1× PBST followed by blocking with 100 μL of 3% BSA in 1× PBST for 2 h at 37 °C. After the plates were washed, biotin-labeled peptides were added in a concentration range from 0.39 to 100 μM in dilution buffer [0.1% BSA + 1× PBST + 0.05% v/v Tween20] and incubated for 1 h at 37 °C. Plates were washed, and 100 μL of 1:2000 streptavidin−HRP (Thermo Scientific, catalog no. N100) was added and incubated for 1 h at 37 °C. Plates were washed, and 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) (Thermo Scientific, catalog no. N301) was added and incubated in the dark for 15 min at room temperature. The reaction was quenched by the addition of 100 μL of 0.5 M HCl and read immediately at 450 nm using a microplate reader (Spectra MAX 190, Molecular Devices, Inc.). ELISA dilution buffer was run as a control on influenza-coated wells. Also, two peptide concentrations (0.39 μM and 100 μM) were run on noninfluenza-coated wells. Synthesis of Scaffolds Sc1 and Sc-2. All of the Fmoc− amino acids and coupling reagents were from AAPPTEC (Louisville, KY). Fmoc−Pal−PEG resin (0.2 mmol/gram) was purchased from Life Technologies (Grand Stateley, NY). Maleimidopropionic acid was from Bachem (Torrance, CA). Trifluoroacetic acid (TFA), piperidine, N,N-diisopropylethylamine (DIPEA) were purchased from Spectrum (Los Angeles, CA). The remaining reagents and solvents were obtained from Fisher Scientific. Both scaffolds (Sc1 and Sc2) were synthesized via Fmocbased solid-phase peptide synthesis. The synthesis was carried out at 0.5 mmol scale on Fmoc−Pal−PEG resin Rink amide resin (0.2 mmol/g). The sequences are shown in Figure 3. Following removal of the Fmoc-protecting group by 20% piperidine in DMF for 5 + 15 min, the stepwise assembly of the peptide sequence was performed via addition of the appropriate Fmoc-protected amino acids. The N-terminus was substituted with maleimide group manually by treating with a 5-fold excess of maleimidopropionic acid in the presence of a 5-fold excess of HOBt and diisopropylcarbodiimide in DMF. The resin was agitated at room temperature for 1 h followed by standard washings with DMF (three times, 1 min per wash), MeOH (two times, 1 min per wash), DCM (two times, 1 min per wash), DMF (three times, 1 min per wash). An aliquot of resin was taken after MeOH washing for a qualitative Kaiser Test. The Kaiser reagents were (1) ninhydrine solution, 6% in ethanol; (2) potassium cyanide in pyridine; and (3) phenol in 80% ethanol. The final protected scaffolds were treated with cleavage cocktail (TFA−H2O−TIPS, 90:5:5) for 2 h at room temperature and precipitated in cold diethyl ether. The precipitated construct was cooled for 15 min in −80 °C to ensure complete precipitation. The solid was separated from the diethyl ether by centrifugation, and the top phase was decanted off and the pellet resuspended with another addition of dry diethyl ether. The cooling and centrifugation process was done in triplicate. Upon completion, the construct was dried and dissolved in water for HPLC purification. The construct was purified on reverse-phase HPLC (Agilent 1200 Series HPLC) using a Phenomenex Luna 5u semipreparative (10 mm × 250 mm) C-18 column using a system of solvent A (0.1% TFA in H2O) and solvent B (90% CH3CN in 0.1% TFA with a linear gradient method) and 0 min, 10% B; 2 min, 10% B; 20 min, 45% B; 25 min, 95% B; 27 min, 95% B; 30 min, 100% B; 33 min, 10% B with flow rate of 4 mL/min at a wavelength of F
DOI: 10.1021/acs.bioconjchem.6b00447 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Bioconjugate Chemistry
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A CM5 sensor chip was coated with Neutravidin (Thermo Fisher Scientific) using a standard amine immobilization protocol. All five spots on one flow cell of the CM-5 dextran chip were activated by a 10 min injection of a freshly prepared 1:1 solution of 400 mM EDC−100 mM NHS in water. The activated spots were treated with a 0.1 mg/mL solution of Neutravidin prepared in 10 mM sodium acetate (pH = 5.5) for 8, 12, 16, 12, and 8 min at a flow rate of 10 μL/min. Any residual active sites were then quenched by a 5 min pulse of ethanolamine (1 M, pH 8.5). The synbody was captured by injecting a 500 nM solution of synbody followed by injection of a biotin solution to quench any remaining biotin-binding sites on the surface. Solutions of HA and NP were prepared in SPR running buffer [1× HBS-EP (GE Healthcare) + 1 mg/ML CMD + 0.05% Surfactant P-20 (GE Healthcare)] and injected for 120 s association and 300 s dissociation times. The surface was regenerated with one 30 s injection of pH 3.0 glycine. Data was then analyzed using Biacore 4000 Evaluation Software. Determination of the synbody binding affinity to NP was performed on a Biacore 4000. A pair of different conditions were used to generate binding data. In the first, the synbody was captured on a Neutravidin-coated CM5 chip, prepared in the same manner as in SM8. A pair of different levels of synbody were captured to produce Rmax values of less than 100 RU. Recombinant NP (Imgenex) was prepared in SPR running buffer and injected at 0, 0.781, 1.56, 3.13, and 6.25 nM for a 120 s association time and a 300 s dissociation time. The surfaces were regenerated with one 30 s injection of pH 3.0 glycine. Data from both surfaces were then combined and globally fit to a 1:1 binding model using Biacore 4000 Evaluation Software. In the second condition, the synbody was captured on an SA1 streptavidin chip (GE Healthcare). The synbody was captured at three different levels (high, medium, and low) to produce surfaces with Rmax values of less than 100 RU. Recombinant NP (Sino Biological, catalog no. 11675-V08B) was prepared in a running buffer [20 mM Tris−HCl, 300 mM NaCl, 0.0025% P20 surfactant, pH 7.4] shown to eliminate NP binding to streptavidin.23 The 1 nM NP solution was injected for 600 s, and dissociation was monitored for 900 s. Data from each spot were fit to a 1:1 binding model using Biacore 4000 Evaluation Software. Pull-Down Assay. UV-inactivated A/PR/8/34 H1N1 (Advanced Biotechnologies, Inc.) was lysed by mixing an equal volume of virus with lysis buffer (0.1% Triton X-100, 50 mM Tris−HCl pH 7.4, 150 mM NaCl, 2 m M EDTA, 10 ug/ mL phenylmethanesulfonylfluoride (PMSF) in H2O) and vortexing for 1 min. The sample was centrifuged and stored at 4 °C until use. Synbodies were captured on 30 μL of streptavidin-coated M280 Dynabeads (Life Technologies, catalog no. 11205D) by mixing at room temperature for 2 h at 1000 rpm. Synbody-coated beads were washed three times with 1 mL of 1× PBST and incubated overnight at 4 °C with gentle shaking with 100 μL of 0.082 mg/mL of lysed virus. After binding, beads were washed five times with 1 mL of 1× PBST and heated at 90 °C in 30 μL of LDS loading buffer (Life Technologies) for 10 min. The samples were analyzed by Western Blot detection using a 1:1000 dilution of anti-NP antibody (Abcam, catalog no. ab128193), followed by detection with a 1:1000 dilution of goat anti-mouse AF647 (Invitrogen, catalog no. A21235). The WB was then imaged on a GE Healthcare Typhoon Trio.
Article
ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00447. Additional figures showing peptide and synbody binding data including ELISA, SPR, and pull-down experiments; and the characterization of ASU1060, ASU1061, ASU1062, and ASU1063. A table showing the amino acid sequences of influenza-binding peptides. (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Present Addresses ‡
Merz Aesthetics, Mesa, AZ, USA. Caris Life Sciences, Phoenix, AZ, USA.
†
Notes
The authors declare the following competing financial interest(s): The authors declare competing financial interest. N.G., V.D, Z.Z, S.A.J. and C.W.D. are inventors on a patent application for the influenza ligands described in this report.
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ACKNOWLEDGMENTS We thank Dr. J.B. Legutki, M. Blynn, L. Phillips, A. Carpenter, Dr. P. Stafford, D. Shepard, K. Kotlarczyk, and A. Kombe for their assistance with this work. We also thank Dr. P.E. Belcher for his advice on SPR characterization of NP binding. This work was funded by a grant from the Defense Advanced Research Projects Administration 7-Day Biodefense Program (grant number: W911NF-10-1-0299) and discretionary funding to S.A.J.
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ABBREVIATIONS USED CMD, carboxymethyldextran; DMF, N,N-dimethylformamide; TFA, trifluoroacetic acid; AcCN, acetonitrile; MeOH, methyl alcohol; DCM, dichloromethane; HOBt, 1-hydroxybenzotriazole; HBTU, 2-(1-H-benzotroazole-1-yl)-1,3,3-tetramethyluronium hexafluorophosphate; NMM, N-methylmorpholine; TFA, trifluoroacetic acid; DIPEA, N,N-diisopropylethylamine; TIPS, triisopropylsilane; DoDt, 3,6-dioxa-1,8-octane-dithiol; ivDde, 1(4,4-dimethyl-2,6-dioxo-cyclohexylidene)-3-methyl-butyl; Fmoc, fluorenylmethoxycarbonyl
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DOI: 10.1021/acs.bioconjchem.6b00447 Bioconjugate Chem. XXXX, XXX, XXX−XXX
