Barbas, C.F., III, Burton, D.R., Scott, J.K. & Silverman, G.J. Eds. Cold Spring Harbor Laboratory Press: New York, 2000, pp 1-24. 3. Dawson, P. E.; Muir, T. W.; ...
Multivalent Peptide Dendrimers for Targeting Brett A. Helms, Ingrid van Baal, Peggy T. H. M. de GraafHeuvelmans, Maarten Merkx and E. W. Meijer Laboratory of Macromolecular and Organic Chemistry & Department of Biomedical Engineering, Eindhoven University of Technology, Postbus 513, 5600 MB, Eindhoven, The Netherlands
INTRODUCTION Phage display is a widely used technique in molecular biology for the identification of selective, peptide-based ligands for a desired proteinogenic substrate.1 Biological combinatorial libraries of the M13 bacteriophage are commonly used for this purpose (Figure 1). The M13 bacteriophage consists of a single-stranded circular piece of DNA (~6400 bases) encapsulated by 2700 copies of the pVIII major capsid protein. At the terminus of this assembly is presented five copies of the targeting peptide, usually 7-15 aa, fused at their C-terminus to pIII. The presented peptides can be randomized (or focused) at the DNA level, and the respective phage constituted in a library, e.g. ~109 unique members in a library consisting of phage with 7-mer peptides. When introduced to a substrate, in vivo2 or in vitro, members from this library may engage in substrate binding. Phage that bind to the substrate are then amplified in E. coli and reconstituted into libraries that are consequently enriched with phage that bind to the substrate. After additional rounds of selection and amplification, individual clones may be isolated and routine DNA sequencing can be used to identify the exact peptide sequence responsible. Interestingly, individual phage peptides often show only weak binding to their intended substrate (Kd ~ 100 – 1000 µM) while the phage themselves have Kd values in the nanomolar range. This superior affinity is due to the inherent, multivalent character of the phage and its association with substrates through multiple weak interactions. Nevertheless, most ligand-directed targeting strategies involving phage peptides rely on the conjugation of a single phage peptide to the active payload.
design concepts presented here will be placed in a larger context of how multivalent, dynamic, noncovalent interactions may be harnessed in biomaterials such as ours for more effective molecular medicines in the identification and treatment of disease. EXPERIMENTAL Molecular Design and Synthesis of Phage Mimics. Dendritic phage mimics were envisioned as shown in Figure 2. For these materials, polyamide dendritic wedges were prepared in a similar fashion to established protocols.6 Oligoethyleneglycol units were used to link N-terminal cysteine residues to the branched polymer support. In this manner, we were able to generate a series of related structures with controlled valency (n = 2 – 5), with the pentavalent construct providing unique access to a M13 phage analogue. A chemically orthogonal group at the focal point of the dendron allowed us to further manipulate the material so as to incorporate a wide variety of anchoring and imaging groups (e.g. LABEL = rhodamine, biotin, etc.). These materials were directly conjugated to C-terminal thioester phage peptides using native chemical ligation as described below.
Figure 1. Structure of the M13 bacteriophage: The major capsid protein, pVIII, encases the phage’s genetic information. At the head of the phage, fused to pVIII, are five copies of the pVI protein. The five presented peptides are fused to pIII via their C-terminus.
As a chemical tool for ligand-directed targeting in biomaterials, phage display has a long way to go before its full potential is realized. Our goal was twofold: (1) to bring a more fundamental understanding to the nature of multivalent phage peptide-protein interactions and (2) to generate smart biomaterials for molecular medicines and imaging from the multivalent display of phage peptides. To those ends, we will describe our most recent research towards synthetic phage mimics for applications in ligand-directed targeting and molecular imaging. In our design, synthetic phage mimics are readily derived from branched, multivalent platforms – primarily, dendrimers and dendrons. These materials are functionalized with targeting peptides identified from phage display using native chemical ligation.3 We have since applied this methodology further, towards the incorporation of fluorescent dyes and other labels in addition to the multivalent targeting scheme offered by the display of multiple peptides at the molecule’s periphery.4,5 The
Figure 2. Examples of cysteine terminated wedges for generating phage mimics. Phage Display. As a model system to validate our approach, we chose to investigate phage-binding to streptavidin. Phage display libraries with linear 7-mer (PhD-7) peptides fused to the N-terminus of the pIII protein of M13 phage were obtained from New England Biolabs (Ipswich, USA). Streptavidin and BSA were obtained from Sigma. AntiM13-HRP was obtained from Amersham Pharmacia Biotech (Roosendaal, The Netherlands). Panning and solid phase binding studies were performed as described elsewhere.7 After three rounds of panning, 6 clones were isolated and their DNA was sequenced using the -96 gIII primer (New England Biolabs) by BaseClear (Leiden, The Netherlands) to obtain the sequence of the peptide displayed by each phage clone (Table 1).
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Table 1. Isolated Clones from the Phage Display to Streptavidin Isolated Peptide Clone Sequence 1 SLLAHPQ 2 NLLNHPQ 3 STHTSAQ 4 NLLNHPQ 5 SLIAHPQ 6 TLLAHPQ
1. 2.
3. 4. 5.
Solid Phase Synthesis of Phage Peptide C-Terminal Thioesters. C-terminal MPAL-thioester peptides were synthesized manually using standard tBoc-mediated SPPS with DMF as the solvent and HBTU as the coupling agent. Leucine was first coupled to an MBHA resin, followed by trityl-protected 3-mercaptopropionic acid. After deprotection of the trityl group using 95% TFA, 2.5% iPr3SiH, 2.5% H2O, the remaining peptide could be synthesized directly from the liberated thiol on the resin. Three glycine residues were inserted between the SLLAHPQ sequence identified from phage display and the MPAL thioester so that the overall peptide used for ligations was H2NSLLAHPQGGG-MPAL (i.e. the N-terminus was left unmodified, as would be the case with the phage). Cleavage of the peptide from the resin was achieved with HF, and the crude product was purified by RPHPLC. Preparation of Phage Peptide Multimers Using Native Chemical Ligation. Our group has previously described the synthesis of multivalent protein and peptide dendrimers using native chemical ligation.8 With slight modification of the protocol, we were also able to prepare multimers of the Streptavidin-binding H2N-SLLAHPQGGGMPAL precursor using the wedges shown in Figure 2. For these reactions, 1.10 – 1.25 equivalents of peptide per N-terminal cysteine on the dendritic wedge was used. MPAA was used as the transthioesterification catalyst and TCEP as the in situ reducing agent. Ligations were performed at 37 oC until the reaction was determined complete by LC-MS analysis. Crude ligation mixtures were purified by RP-HPLC. Yields 25-50%. SPR Experiments. All peptide constructs were evaluated for their binding to streptavidin (SA) using surface plasmon resonance using a Biacore T100 on sensor chips preimmobilized with SA. The SA binding sites in the reference channel were blocked using a 10 mM solution of biotin in HBS-N at a pH = 7.4. Kd values for the HPQ multimers were determined using steady state binding measurements over a concentration range of 1.0 - 1000 µM. Equilibrium responses were plotted as a function of concentration and fit to a single-site binding model to determine the Kd.
6. 7. 8.
9.
REFERENCES Smith, G. P.; Scott, J. K. Methods Enzymol. 1993, 217, 228–257. Pasqualini, R.; Arap, W.; Rajotte, D.; Ruoslahti, E. In vivo selection of phage-display libraries. In Phage Display: A Laboratory Manual Barbas, C.F., III, Burton, D.R., Scott, J.K. & Silverman, G.J. Eds. Cold Spring Harbor Laboratory Press: New York, 2000, pp 1-24. Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science. 1994, 266, 766-779. Dirksen, A.; Langereis, S.; de Waal, B. F. M.; van Genderen, M. H. P.; Hackeng, T. M. H.; Meijer, E. W. Chem. Commun. 2005, 2811. Dirksen, A.; Meijer, E. W.; Adriaens, W.; Hackeng, T. M. Chem. Commun. 2006, 1667. Newkome, G. R.; Kotta, K. K.; Moorefield, C. N. J. Org. Chem. 2005, 70, 4893 -4896. Katz, B. A. Biochemistry 1995, 34, 15421–15429 I. van Baal, H. Malda, S. A. Synowsky, J. L. J. van Dongen, T.M. Hackeng, M. Merkx, E. W. Meijer, Angew. Chem. Int. Ed. 2005, 44, 5052-5057. Krishnamurthy, V. M.; Semetey, V.; Bracher, P. J.; Shen, N.; Whitesides, G. M. J. Am. Chem. Soc. 2007, 129, 1312-1320.
RESULTS AND DISCUSSION Results of the phage display to streptavidin revealed a consensus in the binding sequence from isolated clones. The last three residues in particular form a conserved triad, i.e. HPQ, that has been observed previously in the literature.7 The monovalent peptide gave a Kd of 140 µM, in accordance with other HPQ peptides in the literature. Multimers of the HPQ peptide were successfully prepared from dendrons bearing N-terminal cysteines and H2N-SLLAHPQGGG-MPAL thioesters using native chemical ligation. The binding of these bioconjugates to SA was investigated using surface plasmon resonance. The specific details will be described elsewhere. In general, we observed affinity enhancement with increased valency, over several orders of magnitude, in good agreement with statistical models for interactions of this type.9 As a proof of principle, the HPQ-streptavidin system has provided us tantalizing results as to how multiple, weak interactions between phage peptides and their protein targets may be harnessed for the development of new biomaterials such as ours for more effective molecular medicines in the identification and treatment of disease. We further believe that the display of phage peptides by dendritic polymers can play a leading role in that development, and continuing efforts towards that goal are underway in our laboratory.
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