Modulating Angiogenesis by Proteomimetics of Vascular Endothelial Growth Factor

Abstract

Angiogenesis, formation of new blood vessels from the existing vascular network, is a hallmark of cancer cells that leads to tumor vascular proliferation and metastasis. This process is mediated through the binding interaction of VEGF-A with VEGF receptors. However, the balance between pro-angiogenic and anti-angiogenic effect after ligand binding yet remains elusive and is therefore challenging to manipulate. To interrogate this interaction, herein we designed a few sulfono-γ-AA peptide based helical peptidomimetics that could effectively mimic a key binding interface on VEGF (helix-α1) for VEGFR recognition. Intriguingly, although both sulfono-γ-AA peptide sequences V2 and V3 bound to VEGF receptors tightly, in vitro angiogenesis assays demonstrated that V3 potently inhibited angiogenesis, whereas V2 activated angiogenesis effectively instead. Our findings suggested that this distinct modulation of angiogenesis might be due to the result of selective binding of V2 to VEGFR-1 and V3 to VEGFR-2, respectively. These molecules thus provide us a key to switch the angiogenic signaling, a biological process that balances the effects of pro-angiogenic and anti-angiogenic factors, where imbalances lead to several diseases including cancer. In addition, both V2 and V3 exhibited remarkable stability toward proteolytic hydrolysis, suggesting that V2 and V3 are promising therapeutic agents for the intervention of disease conditions arising due to angiogenic imbalances and could also be used as novel molecular switching probes to interrogate the mechanism of VEGFR signaling. The findings also further demonstrated the potential of sulfono-γ-AA peptides to mimic the α-helical domain for protein recognition and modulation of protein–protein interactions.

Graphical Abstract

INTRODUCTION

Angiogenesis, formation of new blood vessels from the existing vascular network, is a vital process in early developmental processes and healing. Notably, this process is mostly inactive in healthy adults and is tightly regulated by a combination of pro-angiogenic and anti-angiogenic proteins. However, during tumor progression the angiogenesis signaling is activated and leads to tumor vascular proliferation and consequent tumor metastasis.^1–5 It is for this reason that the prospect of affecting this process has gained considerable interest in the fight against cancer. On the other hand, silenced angiogenesis could hamper tissue regeneration, resulting in other diseases such as stroke and cardiovascular disease. As such, the strategy that could manipulate the angiogenic response would be promising for applications in both molecular biology and therapeutics.

It is well recognized that sustained angiogenesis constitutes one of the hallmarks of cancer cells and is modulated through vascular endothelial growth factor (VEGF),^6,7 a potent proangiogenic factor. VEGF is a homodimeric glycoprotein that binds to three VEGF-specific receptor tyrosine kinases, VEGFR-1, VEGFR-2, and VEGFR-3, and is overexpressed in a number of cancer cells.^8–10 VEGF-A₁₆₅ represents the most predominant VEGF isoform in humans and is a potent activator of VEGFR-1 and VEGFR-2 expressed on vascular endothelial cells. The binding of VEGF with VEGFR-2 leads to VEGFR-2 dimerization and phosphorylation of intracellular kinases, which induce cell survival, migration, and proliferation.^11–15 VEGFR-1 has a 10-fold higher binding affinity to VEGF-A as compared with VEGFR-2; however its proangiogenic effect is low to none.^16,17 The exact role of VEGFR-1 in angiogenesis is debatable, but its higher affinity to VEGF-A and low proangiogenic effects and the existence of a soluble form of VEGFR-1 lacking a transmembrane tyrosine kinase domain suggest that it may serve as a decoy receptor and negatively control angiogenesis.^18,19 This is also supported by gene deletion studies where VEGFR-1 (-/-) mice died due to excessive endothelial cell differentiation, whereas VEGFR-2 (-/-) mice died due to lack of vascular formation.^20,21

The therapeutic relevance of inhibiting angiogenesis in cancer has been established through the extensive works done by Folkman and colleagues in the 1970s,^22–26 and concerted efforts in the following decades have produced clinically viable angiogenesis inhibitors targeting VEGF-A and VEGFRs, with Bevacizumab being the first angiogenesis inhibitor approved by the FDA in 2004, which was followed by other monoclonal antibodies and small-molecule tyrosine kinase inhibitors.^27–29 However, the success of antiangiogenic agents has been mixed across different types of cancers and has not met the high expectation of universal anticancer efficacy, underlining the need for a better understanding and more effective approach to modulate angiogenesis.³⁰

The binding interface of VEGF-A, residues (8–109), consists of two monomers linked with two disulfide bridges and two binding interfaces located opposite each other on each monomer. The crystal structure of VEGF-A bound to VEGFR-2 (3V2A) and alanine mutagenesis studies reveal that the VEGF N-terminal helix-α1 (residues 16–25) is a major binding interface for VEGF–VEGFR protein–protein interaction (PPI), in which residues Phe17, Met18, Tyr21, and Tyr25 make up the focal points of this PPI (Figure 1).³¹ For instance, alanine mutagenesis of Phe17 results in a 90-fold decrease in binding, delineating Phe17 as one of the most important residues on helix-α1 for the binding interaction.^31,32 A peptide QK, designed based on helix-α1, has been shown to have good binding affinity to VEGFR and activated angiogenesis.³³ Another peptide, MA, directly reproducing VEGF α1 helix 17–25, also demonstrated binding to VEGF receptors.³⁴ Interestingly, with differences of only two amino acid residues in the whole peptide sequence compared with QK, MA was capable of inhibiting angiogenesis.³⁴ The authors speculated that the contradictory effect of these two peptides on angiogenesis was due to the different conformations adopted by VEGFR upon binding of these peptides. Although the molecular mechanism remains elusive, their effectiveness in regulating angiogenesis renders them potential candidates for future exploration. However, it is known that peptides of natural amino acids fall short in stability toward proteolytic enzymes, hence generally have low therapeutic potentials.³⁵

Figure 1.

Structure of VEGF-A/VEGFR-2 complex, Protein Data Bank (PDB): 3V2A. Homodimeric VEGF-A (green) binding to, and dimerization of, VEGFR-2 (yellow). Key binding residues on VEGF-A helix-α1 (Phe17, Met18, Tyr21, and Tyr25) are highlighted in red.

As protein-protein interactions mediate virtually all biological processes and represent unparalleled potential targets for novel therapeutics, tremendous efforts have been undertaken in recent years toward effective PPI modulations.^36–38 Peptides and peptidomimetics offer exceptional prospects of mimicking protein domains essential for PPIs, with the latter offering unique advantages with respect to stability and bioavailability.^39,40 Some notable peptidomimetics developed so far include peptoids,^41,42 β-peptides,^43,44 α/β-peptides,^45–48 azapeptides,^49,50 oligoureas,^51,52 and others.^53–55 α/β-Peptides derived from Z domain peptides were particularly shown to bind to VEGFR and to have more resistance to proteases than α-peptides.⁴⁶

γ-AA peptides are a unique class of peptidomimetics with exceptional stability, diversity, and protein domain mimicking capability.^56–58 We have previously reported crystal structures of sulfono-γ-AA peptides adopting a left-handed helix capable of multiface recognition and binding interactions toward target proteins (Figure 2) due to their similarity to an α-helix.^59–64 Their helical propensity is even superior to the α-helix, possibly due to the synergy of both intramolecular hydrogen bonding and the curved nature of sulfonamido moieties forcing the formation of helical turns. Together with other remarkable features including chemodiversity and resistance to enzymatic degradation, we believe helical sulfono-γ-AA peptides could enable us to target a wide array of therapeutically relevant PPIs.

Figure 2.

(A) Structure of sulfono-γ-AA peptide building block. “a” and “b” denote chiral side chain and sulfono side chain, respectively. (B) Schematic representation of side chain distribution in a left-handed sulfono-γ-AA peptide helix scaffold. (C and D) Crystal structure of sulfono-γ-AA peptide side view and top view, respectively.

Herein we report rationally designed sulfono-γ-AA peptide based peptidomimetics of VEGF-A. We designed a few unnatural sulfono-γ-AA peptide foldamers to mimic the helical binding domain of VEGF-A: helix-α1 (16–25). These helical mimics were designed based on the X-ray crystal structure of VEGFR-2/VEGF-A (3V2A) and the alanine mutagenesis data to mimic the three-dimensional spatial orientation of the key binding residues: Phe17, Met18, Tyr21, and Tyr25.⁶⁵ These helical mimics were shown to bind VEGFR1 and VEGFR2 with high affinity and specificity. Cell-based studies demonstrated that these sulfono-γ-AA peptide mimics of VEGF-A could effectively downregulate and, with a subtle change of a side chain, upregulate angiogenesis. On the basis of our results, we reasoned the inverse angiogenesis signaling was due to the selective binding of helical mimetics toward either VEGFR1 or VEGFR2, respectively. Moreover, these helical mimetic sequences demonstrate exceptional proteolytic stability, highlighting their potential as molecular probes to unravel mechanisms of angiogenesis and therapeutic agents targeting angiogenesis.

RESULTS AND DISCUSSION

Design of Sulfono-γ-AA Peptide Mimics of VEGF.

The design of VEGF-mimicking sulfono-γ-AA peptides was based on the positioning of the key binding residues (Phe17, Met18, Tyr21, and Tyr25) on the helical binding domain, helix-α1 of VEGF-A, featured on the crystal structure of the VEGFR-2/ VEGF-A complex (Figure 3A, PDB: 3V2A). Phe17, Tyr21, and Tyr25 are positioned on the same face of the helical binding domain (Figure 3B). As shown in Figure 2B, these functional groups could be reproduced by the side chains at 3a, 5a, and 7a positions in sulfono-γ-AA peptides, respectively. The additional residue Met18 could be mimicked with a hydrophobic and relatively sized sulfono isobutyl group at position 4b. An overlay of a sulfono-γ-AA peptide V3 (Table 1) with the helical domain of VEGFR2 shows that the designed side chains on the positions overlap with Phe17, Met18, Tyr21, and Tyr25 very well (Figure 3B). To this end, a series of sulfono-γ-AA peptides (V1-V4) were designed and synthesized along with a negative control sulfono-γ-AA peptide bearing alanine-like side chains (V5)(Table 1).

Figure 3.

(A) Binding interaction of key residues on helix-α1 of VEGF-A (green) with VEGFR-2 (yellow), PDB code: 3V2A. (B) Structures of helix-α1 of VEGF-A (green), sulfono-γ-AA peptide mimic V3 (magenta), and overlay of key binding residues.

Table 1.

Structures of Selected Sulfono-γ-AA Peptide Helical Mimics (V1–V5)^a

	Structure	Kd (μM)
		VEGFR-1	VEGFR-2
V1		12.9	5.1
V2		0.46	2.3
V3		7.7	0.63
V4		>100	>100
V5		>100	>100
QK	Ac-KLTWQELYQLKYKGI
MA	Ac-KLTWMELY QLAYKGI

^aCritical residues for binding are shown in red and pink. Binding affinity (K_d) of sequences to VEGFR-1 and VEGFR-2 as determined by SPR The chemical structures of two reported peptides QK and MA are also included.

Binding Assays.

Surface plasmon resonance (SPR) assays were carried out to determine the binding affinities of our VEGF mimic sequences toward the extracellular domains of VEGFR-1 and VEGFR-2 (Figures S3, S4). Excitingly, V1, V2, and V3 demonstrated good binding affinities toward VEGFRs. Particularly, with only one residue difference at the position 3a, V2 and V3 displayed remarkable selectivity for VEGFR1 and VEGFR2, revealing submicromolar binding affinity to VEGFR-1 and VEGFR-2, respectively. As expected, V4, which lacks a critical binding residue at position 3a to mimic F17 in VEGF-A-helix-α1 and V5, with all residues replaced with alanine, had no detectable binding. The binding studies demonstrated that sulfono-γ-AA peptides could mimic VEGF-A-helix-α1 and exhibit good binding specificity and affinity toward VEGFRs.

Circular Dichroism (CD) Measurements.

CD studies were next conducted to assess the helicity of our VEGF-mimicking sulfono-γ-AA peptides. The studies were carried out in PBS buffer and monitored between 190 and 260 nm. In line with our previous reports,⁶⁰ sulfono-γ-AA peptides V1-V5 adopted a left-handed helical conformation with a Cotton effect maximum between 200 and 215 nm. For comparison, the reference peptide QK, an agonist reported by D’Andrea et al.,³³ was also synthesized and included as a control. Indeed, QK exhibited a characteristic double minimum Cotton effect, confirming a right-handed helix conformation, consistent with previous reports (Figure 4).

Figure 4.

CD spectra of sulfono-γ- AA peptides V1-V5 and QK measured at 100 μM, room temperature in PBS buffer.

In Vitro Angiogenesis Assays.

Activation of VEGFR-2 leads to endothelial cell proliferation, migration, and vascular formation.⁶⁶ To assess the effect of our VEGF mimics on the VEGF signaling pathway, we conducted in vitro angiogenesis assays and examined the effect of these helix-mimicking sequences on cellular responses mediated through the activation of VEGFR-2. To ascertain that the observed effects were the result of our sulfono-γ-AA peptides interacting with VEGFR-2, pVEGR-2 and a key VEGF downstream signal (Akt) were monitored with Western blotting.

Cell Migration Assay.

Endothelial cell migration is an essential component of angiogenesis mediated by the VEGF pathway and can be used to assess the effect of drugs on the VEGF pathway. A Transwell migration assay was used to test the effect of our mimic sequences on the migration of human umbilical endothelial cells (HUVECs). In the presence of VEGF₁₆₅, the number of HUVECs migrating was significantly lowered by V1 and V3, suggesting both sequences inhibited VEGF-stimulated VEGFR2 signaling, possibly because these sequences mimicked VEGF helix-α1 and blocked the binding of VEGF to VEGFR2. However, intriguingly, V2 markedly increased the number of HUVECs migrating. It was noted that QK also had a strong migration-stimulating effect, in line with its previously reported pro-angiogenic effect.³³ The results suggested that V2, similar to QK, could somehow enhance VEGFR2 signaling (Figure 5). Given the findings that V2 has a much stronger binding affinity for VEGFR1 than VEGFR2 (Table 1), we speculated that this stimulatory effect may be due to its specific binding to VEGFR1, the decoy receptor. It is plausible that inhibition of VEGF-A binding to VEGFR-1 leads to an increased level of VEGF-A to bind and activate VEGFR-2, an indirect activation of the main angiogenic receptor.⁶⁷

Figure 5.

Transwell migration assay of HUVECs following treatment with VEGF mimics in the presence of VEGF₁₆₅. (A) Increased migration of HUVECs treated with V2 and QK. Marked inhibition of migration is observed in cells treated with V1 and V3. (B) Percentage of HUVECs migrating, compared to control, following treatments with VEGF mimics in the presence VEGF₁₆₅. Concentration of treatments: VEGF₁₆₅ (50 ng/ mL), all other peptidic sequences (10 μM), control (no treatment). Results presented as mean ± SD (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001.

Wound Healing Assay.

Wound healing is facilitated by the VEGF pathway’s effect on cell migration and proliferation.¹⁴ The effect of our helical mimic sequences on cell migration and invasion was tested on HUVECs in the presence of VEGF₁₆₅ using a scratch-wound motility assay. Consistent with the cell migration assay, the results (Figure 6) show marked prevention of cell migration by V1 and V3 upon stimulation with VEGF, in which V3 virtually completely blocked wound motility at 10 μM, consistent with its strongest binding affinity toward VEGFR2. However, V2 and QK did not exhibit inhibition of migration in this assay.

Figure 6.

Wound healing assay. (A) Migration of HUVECs following treatments with VEGF mimics in the presence of VEGF₁₆₅ observed at 0 and 24 h. (B) Percentage of wound area remaining high (inhibition of migration) after 24 h in HUVECs treated with V1 and V3. However, V2 and QK, as compared to cells treated with VEGF₁₆₅ only, did not activate migration. Concentration of treatments: VEGF₁₆₅ (50 ng/mL), VEGF mimic sequences (10 μM), control (no treatment). Results presented as mean ± SD (n = 3), ***P < 0.001.

Capillary Tube Formation Assay.

The ultimate effect of angiogenesis is the formation of vasculature that can support healing, survival, and proliferation. Next, we used HUVECs to investigate the effect of our AA peptide sequences on the formation of capillary-like tube structures in vitro in the presence of VEGF₁₆₅. Again, similar to findings from previous assays, the results show strong stimulation of tube formation in response to V2 and QK (Figure 7A,B) in the presence of VEGF₁₆₅, with the stimulatory effects significantly higher as compared to cells treated with only VEGF₁₆₅. In contrast, substantial inhibition of capillary tube formation is observed in cells treated with V1 and V3 in the presence of VEGF₁₆₅ (Figure 7C,D).

Figure 7.

Formation of capillary tube structures of HUVECs following treatment with VEGF mimics in the presence of VEGF₁₆₅ observed after 24 h. (A) Increased capillary tube formation in cells treated with QK and V2. (B) Graphical representation of percentage count, compared to control, of capillary tube nodes of HUVECs for (A). (C) Decreased capillary tube formation in cells treated with V1 and V3. (D) Graphical representation for (C). VEGF₁₆₅ (50 ng/mL), VEGF mimic sequences (2 μM), negative control (no treatment). Results presented as mean ± SD (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001.

Western Blot Analysis.

The effect of VEGF-mimicking sequences on VEGF-A-mediated activation of Akt, a key VEGF downstream signal,⁶⁸ and VEGFR-2 was then examined by Western blot (Figure 8). V1 (Figure 8A) and V3 (Figure 8B) exhibited dose-dependent inhibition of phosphorylation of VEGFR-2 (P-VEGFR-2) and Akt (P-AKT), with V3 particularly showing potent inhibition even at concentrations as low as 100 nM. On the contrary, V2 (Figure 8C) and QK (Figure 8D) increased the levels of phosphorylation of VEGFR-2 and Akt in a dose-dependent manner. However, the activation of the VEGF pathway by V2 and QK is dependent on the presence of VEGF-A (Figure 8E). In the absence of VEGF-A stimulation, none of the sequences demonstrated activation of VEGFR signaling. The results of the Western blot analysis of VEGFR-2- and VEGF-dependent pathways were congruent with the other in vitro angiogenesis assays, suggesting activation of angiogenesis signaling by V2 is due to its specific binding to the decoy receptor VEGFR-1.

Figure 8.

Western blot data on the levels on phosphorylation of VEGFR-2 and Akt in HUVEC lysates following treatment with (A) V1 (0.1, 0.5, 1, 10 μM), (B) V3 (0.1, 0.5, 1, 10 μM), (C) V2 (0.1, 0.5, 1, 10 μM), and (D) QK (0.1, 0.5, 1, 10 μM) and (E) QK, V1, V2, and V3 (20 μM) with or without VEGF₁₆₅.

Immunofluorescence Assay.

On the basis of the assays conducted, we speculated that distinct cell signaling and angiogenic response are due to our sequences V2 and V3 binding to different VEGFR subtypes. To further characterize the binding profiles of V2 and V3 to VEGFR-1 and VEGFR-2, immunofluorescence studies were carried out using HUVECs and antibodies specific to each receptor. Cells treated with V2 and V3 (10 μM) were fixed and treated with anti-VEGFR-1 and anti-VEGFR-2 antibodies, followed by FITC-labeled secondary antibody and a DAPI counterstain. Cells treated with V2 had markedly lower fluorescence when incubated with anti-VEGFR-1 antibody (Figure 9A,C) and a negligible reduction in fluorescence when incubated with anti-VEGFR-2 antibody (Figure 9B,D). On the other hand, V3-treated cells exhibited a significant reduction of fluorescence when incubated with anti-VEGFR-2 antibody (Figure 9B,D) but showed almost no effect on intensity of fluorescence in cells incubated with anti-VEGFR-1 antibody (Figure 9A,C). The findings further demonstrated that V2 binds to VEGFR-1 more tightly than VEGFR-2, whereas V3 binds more specifically to VEGFR-2 over VEGFR-1 on cells.

Figure 9.

Characterization of binding profiles of V2 and V2 toward VEGFR-1 and VEGFR-2 with immunofluorescence. HUVECs treated with no drug (control), V2, and V3, stained with anti-VEGFR-1 (A) and anti-VEGFR-1 (B) antibody, and detected by FITC-labeled goat anti-rabbit IgG secondary antibody followed by DAPI counterstain. (C and D) Bar graph of relative fluorescence intensity of V2 and V3 compared to control in cells treated with anti-VEGFR-1 and anti-VEGFR-2 antibodies. Results presented as mean ± SD (n = 3), **P < 0.01, ***P < 0.001.

DISCUSSION

Angiogenesis has profound physiological and structural effects that are implicated in the invasive nature of cancer cells. Being a central process to the metastatic pathway and a hallmark of cancer cells, angiogenesis has garnered tremendous interest in anticancer drug development efforts.^6,69 Besides in cancer, inhibition of amplified angiogenesis also has therapeutic applications in retinopathy. On the other hand, stimulation of angiogenesis has demonstrated therapeutic benefits in ischemic heart disease, organ repair, and wound healing.^4,5 In this regard, the controlled inhibition and activation effects on angiogenic cellular responses is very exciting.

In this study, we used our sulfono-γ-AA peptides-based helical foldamers to mimic a critical binding domain on VEGF-A (helix-α1). Recently we have successfully employed sulfono-γ-AA peptides as a new helical framework to design helical domain mimetics and modulate a range of medicinally relevant PPIs, owing to their similarity to mimic α-helix, and reproduce the functionalities on multiple faces of the α-helix. The current design was based on the crystal structure of VEGF-A bound to VEGFR-2. As the most critical residues of VEGF-A, Phe17, Met18, Tyr21, and Tyr25 are involved in binding with VEGFR-2, a few sulfono-γ-AA peptides were designed to reproduce these functionalities using the side chains at 3a, 4b, 5a, and 7a, respectively. For side chains of sulfono-γ-AA peptides not directly involved in the recognition of VEGFR2, we employed a negatively charged Glu side chain and a positively charged Lys side chain at positions 4a and 6a, respectively, in the hope of establishing a salt bridge to stabilize the helical scaffold, as well as enhancing the solubility. Other positions were accommodated with a few common hydrophobic or hydrophilic side chains. The design was very effective, as V1, V2, and V3 all demonstrated good binding affinity toward VEGFR-1 and VEGFR-2. Particularly, V2 showed good selectivity for the binding of VEGFR1, whereas V3 revealed a higher selectivity for VEGFR2. V4, which lacks a critical side chain to mimic F17 and, V5, bearing alanine-like side chains (similar to alanine mutagenesis), did not exhibit any binding activity. CD studies also suggested that our mimic sequences adopt stable left-handed helices as anticipated. The peptide QK, as previously reported, was synthesized and demonstrated significant α-helical folding propensity as well. The results demonstrated that sulfono-γ-AA peptides could be successfully designed to mimic VEGF N-terminal helix-α1.

Among these sulfono-γ-AA peptide foldamers, in vitro angiogenesis assays show that V3 is the most potent inhibitor of angiogenesis; however V2 is a potent activator of angiogenesis. The complete reversal of the effects of these helical mimics to be either pro- or anti-angiogenic is very intriguing because their only difference is the phenyl side chain at the position 3a in V2 vs an indole side chain at the same position in V3. In previous reports, QK and MA peptides, which are derived from the same helix domain of VEGF-A, also demonstrated a counteracting effect in angiogenesis, with differences of only two amino acid residues.^33,34 The contradictory effect was attributed to the different receptor conformation upon binding to these two peptides.³⁴ Based on our results, including binding specificity of V2 and V3 toward VEGFR1 and VEGFR2, respectively, and cell-based studies such as cell migration, Western blotting, and immunofluorescence, we hypothesized an alternative mechanism of angiogenesis modulation. The sulfono-γ-AA peptide-based VEGF-mimicking sequences mimic only a small portion of VEGF-A (helix-α1) and cannot achieve the same overall binding interaction and receptor dimerization as VEGF-A required for receptor activation; thus it is unlikely that the proangiogenic effects observed with V2 and QK are the results of direct binding activation. Western blot assays also show that none of the sequences activate VEGFR-2 and Akt in the absence of VEGF-A (Figure 8E), indicating that the proangiogenic effects observed are likely due to VEGF-A. It is well known that VEGFR-2 is the main receptor responsible for VEGF-A-mediated angiogenic effects; however, VEGFR-1 is largely believed to be a decoy receptor for circulating VEGF-A, although it may be involved in angiogenesis under certain conditions.^18–21 Moreover, VEGF-A binds more strongly to VEGFR-1, and it was expected that developing a VEGF mimic targeting VEGFR-2 selectively would require some modifications. Based on the binding affinity studies, which reveal a higher affinity of V2 toward VEGFR-1, the “decoy” receptor, and a selective binding of V3 toward VEGFR-2, the main angiogenic receptor, we hypothesize that the pro-angiogenic effect of V2 is the result of selective/higher binding of V2 to VEGFR-1, which would increase the levels of VEGF-A available to bind and activate the main angiogenic receptor, VEGFR-2. This is indeed consistent with previous findings, in which protein ligands specific for VEGFR-1 led to activation of VEGFR-2-mediated signaling.⁶⁷

In order to gain insight into the binding selectivity of V2 and V3, we next conducted computational modeling. Crystal structures of VEGF-A bound to VEGFR-1 (PDB: 1FLT) and VEGFR-2 (PDB: 3V2A) exhibit some subtle but probably crucial differences in the binding surfaces of the receptors; notably the hydrophobic pocket interacting with VEGF Phe17 is much smaller in VEGFR-1 and would be less accommodating to larger groups (Figure 10B,C,D). The indole group replacing the phenyl group on V3 would be presumably too big for this pocket; however this pocket is much larger in VEGFR-2, and this larger indole group of V3 would be expected to produce a more favorable interaction (Figure 10A). This might explain the possible selectivity of V2 and V3 to VEGFR-1 and VEGFR-2 respectively. The modeling could also rationalize the different binding affinities of V1 and V2, which bear same critical binding groups, but V1 exhibited a much weaker binding capability toward VEGFR1. As shown in Figure 10C, sulfono-γ-AA peptides form longer helices than the helical domain of VEGF-A. When binding to VEGFR1, the N-terminus of the helix has to tilt away from the binding site slightly to avoid direct clash with the top edge of the binding site (Figure 10D), which may weaken the interaction of V1 with VEGFR1. In contrast, the positively charged side chain at position 2b of V2 could form an electrostatic interaction with the negatively charged Glu141 of VEGFR1 at the edge, which instead could enhance the binding (Figure S5). However, the binding site on VEGFR2 is not as protruding as the one on VEGFR1 (Figure 10A), and as a result, the side chains on 2b are much less relevant. Next, immunofluorescence studies further verified the selective binding activities at the cellular level. In this study V2 demonstrated a much stronger inhibition of anti-VEGFR-1 antibody than anti-VEGFR-2 antibody, whereas V3 had near complete selectivity toward inhibition of anti-VEGFR-2 antibody, without significant inhibition of anti-VEGFR-1 antibody. This investigation further supports our hypothesis that the selectivity of V2 and V3 for either VEGFR-1 or VEGFR-2 leads to pro- and antiangiogenic effects, respectively. A healthy level of angiogenesis is maintained through a set of pro-angiogenic and antiangiogenic factors, a process termed as angiogenic switch.⁷⁰ Having control of this process gives a tremendous opportunity to intervene in several disease conditions that are caused due to imbalances in angiogenesis (Figure 10E). In this regard, the pro-angiogenic and anti-angiogenic activities of V2 and V3, respectively, offer a dynamic control of the angiogenic switch.

Figure 10.

(A) Superimposition of V3 (teal) with critical residues of helix-α1 (green) on the binding surface of VEGFR-2 (yellow). (B) Binding interaction of key residues on helix-α1 of VEGF-A (green) with VEGFR-1 (bronze), PDB: 1flt. (C) Structures of helix-α1 of VEGF-A (green), sulfono-γ-AA peptide mimic V2 (magenta), and overlay of key binding residues. (D) Superimposition of V2 (magenta) with critical residues of helix-α1 (green) on the binding surface of VEGFR-1 (bronze). (E) Modulation of the angiogenic switch with sulfono-γ-AA peptide mimics of VEGF-A.

One of the major bottlenecks in the development of peptide-based molecular probes or drug candidates is their inherent susceptibility to degradation with proteolytic enzymes. Next, we assessed the stability of our lead mimics V2 and V3 and the control model peptide QK in Pronase, a broad-specificity mixture of proteases extracted from Streptomyces griseus. The sequences were incubated in Pronase for 24 h and analyzed with HPLC (Figure 11). Our sulfono-γ-AA peptide mimics (V2 and V3) were remarkably stable and did not exhibit any noticeable degradation. However, QK, bearing natural amino acid residues, was completely degraded.

Figure 11.

Stability studies of VEGF-A mimics. HPLC traces of indicated control sequences and sequences incubated in Pronase for 24 h.

CONCLUSION

We have successfully designed unprecedented unnatural helical foldameric mimetics of a critical binding domain of VEGF-A (helix-α1). Cell-based angiogenesis assays show that these mimicking sequences could be either pro- or anti-angiogenic and upregulate or downregulate angiogenesis, thus effectively modulating the angiogenic switch. We believe the distinct angiogenesis signaling is due to the specific binding of helical mimetics toward VEGFR-1 or VEGFR-2, respectively. Targeting VEGFR-1 specifically (V2) is expected to free more VEGF from VEGFR-1 binding and shift the dial for VEGFR-2 interaction, leading to amplified angiogenesis. Specific binding to VEGFR-2 (V3), on the contrary, would be capable of inhibiting VEGF-A/VEGFR-2 PPI and therefore block the angiogenesis signaling pathway. Therefore, V2 and V3 represent promising unnatural peptidomimetics for the intervention of disease conditions arising due to angiogenic imbalances and could be used as a tool for chemical biology. Moreover, the study further manifested the versatility of sulfono-γ-AA peptides to mimic protein helical domains. This is a remarkable feat for helical sulfono-γ-AA peptides considering the significance of helices in PPIs, where 62% of multiprotein complexes in the Protein Data Bank involve a helix at the interface.⁷¹