High-Affinity Peptide-Based Collagen Targeting Using Synthetic Phage Mimics: From Phage Display to Dendrimer Display Brett A. Helms, Sanne W. A. Reulen, Sebastiaan Nijhuis, Peggy T. H. M. de Graaf-Heuvelmans, Maarten Merkx, and E. W. Meijer* Laboratory of Chemical Biology and Institute for Complex Molecular Systems, EindhoVen UniVersity of Technology, P.O. Box 513, 5600 MB EindhoVen, The Netherlands Received March 23, 2009; E-mail: E.W.Meijer@tue.nl Collagens are extracellular matrix (ECM) proteins that provide mechanical strength to tissues and are important in cell attachment, differentiation, and migration. 1,2 Visualization of collagen networks in tissues is of great interest, particularly in light of the many human ailments that are characterized by ECM degradation or turnover such as angiogenesis, myocardial infarction, and atherosclerosis. 3,4 Naturally occurring collagen binding proteins such as the von Willebrand Factor (vWF) or CNA35 5-7 labeled with fluorescent dyes or collagen-specific antibodies 8 are frequently employed for collagen imaging. Protein-based targeting strategies have a few key limitations, however: tissue penetration depth decreases with increasing size, and naturally occurring collagen binding proteins are often promiscuous in binding to collagen’s many subtypes, effectively reducing the structural information gleaned with these probes. 5,6 The use of short peptides as targeting ligands presents an attractive alternative: their activity often does not require folding, and their chemical synthesis is inexpensive in comparison to, e.g., antibodies. Phage display has proven to be a particularly efficient method to rapidly screen large peptide libraries for targeting ligands to a broad variety of substrates. 9 Peptides derived from phage display, however, suffer from a serious drawback in that they typically show significantly weaker binding than their respective high affinity phage. This applies in particular to peptides targeting protein surfaces and results from the intrinsic flexible nature and limited binding interface of peptides. 10 For example, the WREPSFCALS peptide derived from vWF has been reported to bind bovine collagen type I with an apparent K d of only 100 μM. 11 In related work, Caravan et al. reported the development of a disulfide bond constrained collagen binding peptide, but its low μM affinity was obtained only after extensive affinity maturation via the incorpora- tion of non-natural amino acids. 12,13 As collagens and other ECM proteins have repetitive primary sequences and elaborate structural hierarchies permitting multivalent interactions with cells, a targeting strategy based on multivalent peptide binding is highly attractive. In fact, each phage in the widely used M13 libraries presents five peptide ligands fused to the N-terminus of the phage pIII coat protein at one end of the phage particle. Affinity selection of peptide ligands using these libraries might thus rely on not only the specific amino acid sequence displayed at the phage head but also their presentation in multiple copies capable of interacting with collagens in a multivalent fashion. While a large number of multivalent approaches have been disclosed in recent years, most of them are designed for structurally well- characterized targets such as bacterial toxins and lectins. 14,15 Here we report a multivalent approach to enhance the affinity of phage- display derived peptides through reconstruction of the phage’s multivalent architecture using carefully designed dendritic wedges as synthetic multivalent scaffolds (Figure 1). The approach is demonstrated using a collagen-specific 7-mer peptide sequence against rat tail collagen type I but is likely to be broadly applicable to other multivalent peptide or protein architectures. Collagen binding phages were identified from commercially available M13 phage libraries containing linear 7-mer, linear 12- mer, or cyclic 7-mer peptides. Initially, strong background binding was observed between the phage and rat tail collagen type I coated on 96-well plates, probably due to Coulombic interactions between the polyanionic phage and the positively charged collagen. Screen- ing of several buffer conditions revealed that this background binding was efficiently suppressed at 0.5 M NaCl. ELISA experi- ments showed clear enrichment with collagen binding phage for the linear 7-mer library after two rounds of panning, whereas little enrichment was observed for either of the other two libraries (Figure S1). Subsequent panning experiments were done with the linear 7-mer library using either low pH elution or competitive elution with the collagen binding protein CNA35. Highly homologous peptide sequences were obtained for all panning experiments with nearly all clones containing the consensus sequence H-V-F/W-Q/ M-Q-P/A-P/K (Figure S1). This newly discovered consensus sequence is markedly different from any of the previously reported collagen binding peptides. 11,13,16 To mimic the multivalent peptide presentation on the M13 phage head, a pentavalent dendritic platform was constructed, which was designed to incorporate various imaging modalities at the focal point in addition to five copies of the collagen binding phage peptide (Scheme 1). The branched core of the pentameric structure was built up via a double Michael addition of the bivalent acrylamide 10 to the linear nitroalkane 6 (Scheme 1 and Supporting Information). This AB 5 synthon is versatile, incorporating oligoethyleneglycol- based flexible linkers to peptide ligands at the periphery and nitro, biotin, or fluorescein moieties at the focal point of the wedge. Final construction of the collagen binding phage mimics was accomplished by native chemical ligation of H 2 N-H(Dnp)- Figure 1. From phage display to dendrimer display: phage display to collagen reveals a consensus binding sequence that is translated into a high affinity, versatile synthetic collagen-specific probe by mimicking the original pentavalent phage architecture on a dendritic wedge. Published on Web 07/31/2009 10.1021/ja902285m CCC: $40.75  2009 American Chemical Society J. AM. CHEM. SOC. 2009, 131, 11683–11685 9 11683 Downloaded by TECH UNIV EINDHOVEN on October 9, 2009 | http://pubs.acs.org Publication Date (Web): July 31, 2009 | doi: 10.1021/ja902285m