title: 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.
authors.affiliation: Department of Chemistry, §Genome Center Proteomics Core Facility, and ‡Campus Mass Spectrometry Facilities, University of California , One Shields Avenue, Davis, California 95616, United States.
authors.forename: Bernadette V
authors.initials: BV
authors.lastname: Marquez
authors.forename: Heather E
authors.initials: HE
authors.lastname: Beck
authors.forename: Tolulope A
authors.initials: TA
authors.lastname: Aweda
authors.forename: Brett
authors.initials: B
authors.lastname: Phinney
authors.forename: Cynthia
authors.initials: C
authors.lastname: Holsclaw
authors.forename: William
authors.initials: W
authors.lastname: Jewell
authors.forename: Diana
authors.initials: D
authors.lastname: Tran
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Day
authors.forename: Malalage N
authors.initials: MN
authors.lastname: Peiris
authors.forename: Charles
authors.initials: C
authors.lastname: Nwosu
authors.forename: Carlito
authors.initials: C
authors.lastname: Lebrilla
authors.forename: Claude F
authors.initials: CF
authors.lastname: Meares
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019921:Biotinylation; D002417:Cattle; D005464:Fluorobenzenes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D015335:Molecular Probes / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D042461:Vascular Endothelial Growth Factor A / Q000737:chemistry / Q000378:metabolism*
chemical_list: D005464:Fluorobenzenes; D008024:Ligands; D015335:Molecular Probes; D010455:Peptides; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D008239:Lysine