Enhancing Peptide Ligand Binding to Vascular Endothelial Growth Factor by Covalent Bond Formation (original) (raw)
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The angiogenic properties of VEGF are mediated through the binding of VEGF to its receptor VEGFR2. The VEGF/VEGFR interface is constituted by a discontinuous binding region distributed on both VEGF monomers. We attempted to reproduce this discontinuous binding site by covalently linking into a single molecular entity two VEGF segments involved in receptor recognition. We designed and synthesized by chemical ligation a set of peptides differing in length and flexibility of the molecular linker joining the two VEGF segments. The biological activity of the peptides was characterized in vitro and in vivo showing a VEGF-like activity. The most biologically active mini-VEGF was further analyzed by NMR to determine the atomic details of its interaction with the receptor. All biological processes are finely regulated by a network of interactions between proteins whose characterization at molecular level can promote the design of novel protein binder drugs with therapeutic and diagnostic applications. Peptides and peptidomimetics have been widely explored as protein-protein interaction modulators as they are advantageous with respect to protein-based (including antibodies) molecules and small organic compounds in spite of pharmacokinetic limitations 1. In the last years, several examples of protein-binder peptides have been reported, achieved using either structure-based design, combinatorial or computational approaches. In particular, considering that molecular recognition between proteins is often mediated by surface exposed loop or secondary structure motifs such as β-hairpins and α-helices, many efforts have been devoted to the development of peptides reproducing these protein interface elements 1,2. Less explored and more challenging is the design of protein interface mimetic peptides reproducing multiple and discontinuous binding sites. A discontinuous binding site is constituted by peptide segments which are close in the protein three-dimensional structure but distant in the primary sequence. Examples of discontinuous protein binding site/epitopes mimicry using peptides have been reported. They have been assembled by fusing into a single molecular entity two or more linear amino acid segments using molecular scaffolds or linkers 3–13 or selected by phage display from libraries of mimotopes 14. We explored the design of peptides mimicking two interacting regions of the Vascular Endothelial Growth Factor (VEGF) for its receptor. VEGF is the main regulator of angiogenesis, a fundamental process for healing, reproduction and embryonic development 15. The design of novel and effective angiogenic modulators is eliciting a considerable interest for therapeutic 16,17 and diagnostic applications 18. VEGF is a homodimeric glycoprotein, covalently bound by two disulfide bonds, characterized by a cystine knot motif 19. It binds to two receptors on the surface of endothe-lial cells (ECs) (VEGFR1 and VEGFR2). The binding of VEGF to its receptors induces receptor dimerization and phosphorylation of the intracellular kinase domain which activate the intracellular pathways ending in EC proliferation and migration 20. The analysis of the x-ray crystal structure of the complex between VEGF and the
Amino Acids, 2018
Epidermal growth factor receptor (EGFR) is overexpressed in a number of cancers and is the molecular target for several anti-cancer therapeutics. Radionuclide molecular imaging of EGFR expression should enable personalization of anti-cancer treatment. Affibody molecule is a promising type of high-affinity imaging probes based on a non-immunoglobulin scaffold. A series of derivatives of the anti-EGFR affibody molecule ZEGFR:2377, having peptide-based cysteine-containing chelators for conjugation of 99m Tc, was designed and evaluated. It was found that glutamate-containing chelators Gly-Gly-Glu-Cys (GGEC), Gly-Glu-Glu-Cys (GEEC) and Glu-Glu-Glu-Cys (EEEC) provide the best labeling stability. The glutamate containing conjugates bound to EGFR-expressing cells specifically and with high affinity. Specific targeting of EGFR-expressing xenografts in mice was demonstrated. The number of glutamate residues in the chelator had strong influence on biodistribution of radiolabeled affibody molecules. Increase of glutamate content was associated with lower uptake in normal tissues. The 99m Tc-labeled variant containing the EEEC chelator provided the highest tumor-to-organ ratios. In conclusion, optimizing the composition of peptide-based chelators enhances contrast of imaging of EGFR-expression using affibody molecules. Keywords Affibody molecules • EGFR • 99m Tc • Peptide-based chelators • Glutamate Abbreviations EGFR Epidermal growth factor receptor HER2 Human epidermal growth factor receptor type 2 HER3 Human epidermal growth factor receptor type 3 IGF-1R Insulin-like growth factor 1 receptor CAIX Carbonic anhydrase IX SPECT Single photon emission computed tomography PET Positron emission tomography ITLC Instant thin-layer chromatography SDS PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis Handling Editor: J. Pietzsch.
High-Affinity VEGF Antagonists by Oligomerization of a Minimal Sequence VEGF-Binding Domain
Bioconjugate Chemistry, 2012
Vascular endothelial growth factor (VEGF) neutralizing antagonists including antibodies or receptor extracellular domain Fc fusions have been applied clinically to control angiogenesis in cancer, wet age-related macular degeneration, and edema. We report here the generation of high-affinity VEGF-binding domains by chemical linkage of the second domain of the VEGF receptor Flt-1 (D2) in several configurations. Recombinant D2 was expressed with a 13 a.a. C-terminal tag, including a C-terminal cysteine to enable its dimerization by disulfide bond formation or by attachment to divalent PEGs and oligomerization by coupling to multivalent PEGs. Disulfide-linked dimers produced by Cu 2+ oxidation of the free-thiol form of the protein demonstrated picomolar affinity for VEGF in solution, comparable to that of a D2-Fc fusion (sFLT01) and ∼50-fold higher than monomeric D2, suggesting the 26 a.a. tag length between the two D2 domains permits simultaneous interaction of both faces of the VEGF homodimer. Extending the separation between the D2 domains by short PEG spacers from 0.35 kD to 5 kD produced a modest ∼2-fold increase in affinity over the disulfide, thus defining the optimal distance between the two D2 domains for maximum affinity. By surface plasmon resonance (SPR), a larger (∼5-fold) increase in affinity was observed by conjugation of the D2 monomer to the termini of 4-arm PEG, and yielding a product with a larger hydrodynamic radius than sFLT01. The higher affinity displayed by these D2 PEG tetramers than either D2 dimer or sFLT01 was largely a consequence of a slower rate of dissociation, suggesting the simultaneous binding by these tetramers to neighboring surface-bound VEGF. Finally, disulfidelinked D2 dimers showed a greater resistance to autocatalytic fragmentation than sFLT01 under elevated temperature stress, indicating such minimum-sequence constructs may be better suited for sustained-release formulations. Therefore, these constructs represent novel Fc-independent VEGF antagonists with ultrahigh affinity, high stability, and a range of hydrodynamic radii for application to multiple therapeutic targets.
Targeting VEGF receptors with non-neutralizing cyclopeptides for imaging applications
Amino acids, 2018
Pharmacological strategies aimed at preventing cancer growth are in most cases paralleled by diagnostic investigations for monitoring and prognosticating therapeutic efficacy. A relevant approach in cancer is the suppression of pathological angiogenesis, which is principally driven by vascular endothelial growth factor (VEGF) or closely related factors and by activation of specific receptors, prevailingly VEGFR1 and VEGFR2, set on the surface of endothelial cells. Monitoring the presence of these receptors in vivo is henceforth a way to predict therapy outcome. We have designed small peptides able to bind and possibly antagonize VEGF ligands by targeting VEGF receptors. Peptide systems have been designed to be small, cyclic and to host triplets of residues known to be essential for VEGF receptors recognition and we named them 'mini-factors'. They have been structurally characterized by CD, NMR and molecular dynamics (MD) simulations. Mini-factors do bind with different speci...
Biochemical and Biophysical Research Communications, 2012
Vascular Endothelial Growth Factor mimetic peptides have interesting applications in therapeutic angiogenesis. Recently, we described the proangiogenic properties of a 15 mer peptide designed on the N-terminal helix 17-25 of VEGF. The peptide was stabilized introducing well known peptide chemical tools among which N-and C-terminal capping sequence. Here, we show that the C-terminal sequence does not affect the structural and biological properties of the full-length peptide. In fact, a C-terminal truncated analog peptide resulted in a well folded and stable helix retaining the ability to bind to VEGF receptors. This study will allow to develop smaller peptidomimetic analogs able to modulate the VEGFdependent angiogenesis.
Chemistry (Weinheim an der Bergstrasse, Germany), 2018
QK peptide is a VEGF-mimetic molecule with significant proangiogenic activity. Particularly, QK is able to bind and activate VEGF receptors stimulating a functional response in endothelial cells. In order to characterize the peptide bioactivity and its molecular recognition properties, we report a detailed picture of the interaction between peptide QK and VEGF receptors. Combining NMR studies in solution on the purified receptor and in presence of intact endothelial cells we obtained a molecular description of the binding interaction between the peptide QK and VEGFR2 in the cellular context. These results reveal useful insights on the peptide biological mechanism opening the way to further optimization of this class of VEGF mimicking peptides.
2005
Modulating angiogenesis is an attractive goal because many pathological conditions depend on the growth of new vessels. Angiogenesis is mainly regulated by the VEGF, a mitogen specific for endothelial cells. In the last years, many efforts have been pursued to modulate the angiogenic response targeting VEGF and its receptors. Based on the x-ray structure of VEGF bound to the receptor, we designed a peptide, QK, reproducing a region of the VEGF binding interface: the helix region 17-25. NMR conformation analysis of QK revealed that it adopts a helical conformation in water, whereas the peptide corresponding to the ␣-helix region of VEGF, VEGF15, is unstructured. Biological assays in vitro and on bovine aorta endothelial cells suggested that QK binds to the VEGF receptors and competes with VEGF. VEGF15 did not bind to the receptors indicating that the helical structure is necessary for the biological activity. Furthermore, QK induced endothelial cells proliferation, activated cell signaling dependent on VEGF, and increased the VEGF biological response. QK promoted capillary formation and organization in an in vitro assay on matrigel. These results suggested that the helix region 17-25 of VEGF is involved in VEGF receptor activation. The peptide designed to resemble this region shares numerous biological properties of VEGF, thus suggesting that this region is of potential interest for biomedical applications, and molecules mimicking it could be attractive for therapeutic and diagnostic applications.
Structural studies of the binding of an antagonistic cyclic peptide to the VEGFR1 domain 2
European Journal of Medicinal Chemistry, 2019
Physiological and pathological angiogenesis is mainly regulated by the binding of the vascular endothelial growth factor (VEGF) to its receptors (VEGFRs). Antagonists of VEGFR are very attractive for the treatment of diseases related to excessive angiogenesis. Our previously designed C-terminal alkylated cyclic peptides [YKDEGLEE]-NHR (R = alkyl, arylalkyl) disrupt the interaction between VEGF and VEGFRs in biological assays. In this paper, we described the structural studies of the binding of one of these cyclic peptides named Peptide 3 to the VEGFR1 domain 2 (VEGFR1-D2). The molecular docking and NMR mapping identified the binding site on VEGFR1-D2. The anti-angiogenic effect of our peptide was evaluated by an experiment of VEGF-induced tube formation in two cell lines, retinal cell type RF6/A and vascular endothelial cell type HUVEC. Some new peptides were also synthesized and compared by an ELISA-based assay, in order to verify their ability to disrupt the formation of the complex VEGF-A/VEGFR1. In conclusion, the structural studies of Peptide 3 with VEGFR1-D2 will help the design of more efficient VEGFR antagonist. Moreover, Peptide 3, with improved receptor binding affinity, could be more suitable for VEGFR targeting bioimaging studies once labelled.
A distinct strategy to generate high-affinity peptide binders to receptor tyrosine kinases
We describe a novel and general way of generating high affinity peptide (HAP) binders to receptor tyrosine kinases (RTKs), using a multi-step process comprising phagedisplay selection, identification of peptide pairs suitable for hetero-dimerization (non-competitive and synergistic) and chemical synthesis of heterodimers. Using this strategy, we generated HAPs with K D s below 1 nM for VEGF receptor-2 (VEGFR-2) and c-Met. VEGFR-2 HAPs bound significantly better (6-to 500-fold) than either of the individual peptides that were used for heterodimer synthesis. Most significantly, HAPs were much better (150-to 800-fold) competitors than monomers of the natural ligand (VEGF) in various competitive binding and functional assays. In addition, we also found the binding of HAPs to be less sensitive to serum than their component peptides. We believe that this method may be applied to any protein for generating high affinity peptide (HAP) binders.