Enhancing Peptide Ligand Binding to Vascular Endothelial Growth Factor by Covalent Bond Formation

Abstract

Formation of a stable covalent bond between a synthetic probe molecule and a specific site on a target protein has many potential applications in biomedical science. For example, the properties of probes used as receptor-imaging ligands may be improved by increasing their residence time on the targeted receptor. Among the more interesting cases are peptide ligands, the strongest of which typically bind to receptors with micromolar dissociation constants, and which may depend on processes other than simple binding to provide images. The side chains of cysteine, histidine, or lysine are attractive for chemical attachment to improve binding to a receptor protein, and a system based on acryloyl probes attaching to engineered cysteine provides excellent positron emission tomographic images in animal models (Wei et al. (2008) J. Nucl. Med. 49, 1828-1835). In nature, lysine is a more common but less reactive residue than cysteine, making it an interesting challenge to modify. To seek practically useful cross-linking yields with naturally occurring lysine side chains, we have explored not only acryloyl but also other reactive linkers with different chemical properties. We employed a peptide-VEGF model system to discover that a 19mer peptide ligand, which carried a lysine-tagged dinitrofluorobenzene group, became attached stably and with good yield to a unique lysine residue on human vascular endothelial growth factor (VEGF), even in the presence of 70% fetal bovine serum. The same peptide carrying acryloyl and related Michael acceptors gave low yields of attachment to VEGF, as did the chloroacetyl peptide.

Development of a Radiolabeled Irreversible Peptide Ligand for PET Imaging of Vascular Endothelial Growth Factor

“…As expected, 64 Cu-L19K-FDNB also covalently bound to mouse VEGF because of the conserved epitope recognized by the peptide in both homologs (Supplemental Fig. 10) (16,17,25).…”

“…This strategy requires the noncovalent binding equilibrium between peptide and VEGF to bring the peptide's cross-linker into close proximity to VEGF Lys-48 to facilitate a nucleophilic aromatic substitution reaction, while remaining unreactive to other nucleophilic amino acid side chain groups (Fig. 6) (17).…”

“…Cell culture reagents were purchased from Life Technologies. 64 CuCl 2 was produced in house according to previous methods (21 The purification and monitoring of reaction completion followed previous methods (17). L19K-DNP was synthesized by conjugating L19K to 1-fluoro-2,4-dinitrobenzene (FDNP) as described above.…”

“…Enhancing peptide ligand binding to its target by converting this reversible interaction with VEGF to a covalent and irreversible reaction will eliminate the peptide's dissociation from VEGF and may potentially improve target uptake. Hence, v107 was redesigned by substituting its leucine-19 residue to lysine, resulting in L19K, and was conjugated to a small library of amine-reactive cross-linkers for site-specific covalent attachment to VEGF residue lysine-48 (17). The natural affinity of the peptide for VEGF brings the peptide's cross-linker into close proximity to VEGF lysine-48 and facilitates a covalent bond formation.…”

“…The natural affinity of the peptide for VEGF brings the peptide's cross-linker into close proximity to VEGF lysine-48 and facilitates a covalent bond formation. The lead peptide, L19K-(5-fluoro-2,4-dinitrobenzene) (L19K-FDNB), was most reactive with VEGF, compared with other L19K conjugates in vitro (17). For the present studies, L19K-FDNB was modified to incorporate 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) at the peptide's N terminus (NO2A).…”