Miniaturizing VEGF: Peptides mimicking the discontinuous VEGF receptor-binding site modulate the angiogenic response

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.

Peptide cleavage from resin and deprotection were carried out using a mixture of trifluoroacetic acid (TFA) /triisopropylsilane (TIS) /H 2 O /ethanedithiol (EDT) (Sigma-Aldrich, Milan, Italy) in the ratio 94/1/2.5/2.5 (v/v/v/v) for 3.5 h under stirring at room temperature. The resin was finally filtered and the crude peptide was precipitated in ice-cold diethyl ether (Sigma-Aldrich, Milan, Italy). Each peptide was purified by RP-HPLC performed on a AXIA RP-MAX Synergi column (4 µ, 80 Å, 50 x 21.2 mm; Phenomenex, Torrance, US) applying a linear gradient of CH 3 CN (0.1% TFA) in H 2 O (0.1% TFA) from 5% to 50% in 20 min using a flow rate of 20 mL/min. Pure peptides were finally lyophilized. Peptides purity and identity were verified by LC-MS on an Agilent 1200 Infinity Series (Agilent Technologies, Santa Clara, CA, US) chromatographic system equipped with a diode array combined with an electrospray ion source and a time-of-flight mass analyzer using a C18 Jupiter column 150 x 2 mm, 300 Å, 3 μm (Phenomenex, Torrance, US) and applying a gradient of CH 3 CN (0.1% TFA) in H 2 O (0.1% TFA) from 5 to 70% in 20 min at a flow rate of 0.2 ml/min (Table S1).
Coupling reactions were performed with 5 equiv of Fmoc-amino acid, 5 equiv of HATU and 10 equiv of DIPEA for 40 min at room temperature; capping steps (1x5 min) were performed with a Thioesterification reaction. Carboxylic group on the side chain of glutamic acid, exposed by allyl deprotection, was then thioesterified. 10 equiv of benzyl mercaptane (BzSH, Sigma-Aldrich, Milan, Italy) and then 10 equiv of N',N'-diisopropylcarbodiimide (DIC, Sigma-Aldrich, Milan, Italy) were added to the resin suspended in DCM. Reaction was left overnight, at room temperature and it was repeated once again. Thioester peptide was cleaved from resin and all the protecting groups removed by incubation in a solution of TFA/BzSH/H 2 O/TIS (94/2.5/2.5/1 v/v/v/v) for 3 h. AcHx(thioester) peptide was precipitated in ice-cold diethyl ether and was finally purified by RP-HPLC on RP-MAX Synergi column (4 µ, 80 Å, 50 x 21.2 mm) using a linear gradient of CH 3 CN (0.1% TFA) in H 2 O (0.1% TFA) from 10% to 60% in 20 min performed at 20 ml/min. Peptide purity and identity were verified by analytical RP-HPLC on an Agilent 1200 Infinity Series (Agilent Technologies, Santa Clara, CA, US) chromatographic system equipped with a diode combined with an electrospray ion source and a time-of-flight mass analyzer using a C18 Jupiter column 150 x 2 mm, 300 Å, 3 μm (Phenomenex, Torrance, US) and applying a gradient of CH 3 CN (0.1% TFA) in H 2 O (0.1% TFA) from 5 to 70% in 20 min performed at 0.2 ml/min (Tab. S1). Jupiter C4 column 5 µ, 300 Å, 250 x 10 mm (Phenomenex, Torrance, US) using a linear gradient of CH 3 CN (0.1% TFA) in H 2 O (0.1% TFA) from 5% to 70% in 40 min performed at 5 mL/min. Peptide purity and identity was verified by analytical RP-HPLC on an Agilent 1200 Infinity Series (Agilent Technologies, Santa Clara, CA, US) chromatographic system equipped with a diode combined with an electrospray ion source and a time-of-flight mass analyzer using a C18 Jupiter column 150 x 2 mm, 300 Å, 3 μm (Phenomenex, Torrance, US) and applying a gradient of CH 3 CN (0.1% TFA) in H 2 O (0.1% TFA) from 5 to 70% in 20 min performed at 0.2 ml/min (Tab. S1 and Fig. S1). EPs peptides purity was > 95% based on the analytical HPLC area revealed at 210 nm.

VEGFR1D2 expression and purification
Reagents used for preparation of buffers and growth media of Escherichia coli and the reagents for polyacrylamide gels electrophoresis (Acrylamide, APS, TEMED, SDS, Tris, Glycine) were

Expression, refolding and purification of human VEGFR1D2
VEGFR1D2 gene (DNA fragment encoding amino acids 129-229 of the human VEGFR1) was cloned as reported elsewhere 2 . For bacterial expression, the plasmid construct was transformed into E. coli BL21 Codon Plus (DE3) RIL cell strain (Agilent Technologies) carrying an inducible T7 RNA polymerase gene. Bacterial culture of 1L of prewarmed LB medium containing 50 μg mL -1 kanamycin and 33 μg mL -1 chloramphenicol was grown in shaking flasks at 37 °C until reaching 0.7/0.8 OD 600 . Then, they were induced with 0.7 mM IPTG. After 4-5 h, the cells were harvested by centrifugation; the pellet was dissolved in 50 mM Tris-HCl, pH=8 containing protease inhibitors cocktail (Roche), to avoid proteins degradation, and the suspension was sonicated for 6 min, by using a Misonix Sonicator 3000 apparatus (Misomix, Farmingdale, US) with a micro tip probe and an impulse output of 1.5/2 (=9/12 Watt). Bacterial lysate was then centrifuged (17000 rpm, 30 min, 4°C) and the supernatant (soluble fraction) and the pellet samples were analyzed by SDS-PAGE.
For purification of VEGFR1D2 , solubilized protein was applied on Ni 2+ -NTA resin (45 min, 20°C) in the presence of 300 mM NaCl. The His-tagged protein was refolded by equilibrating the resin in 50 mM Tris-HCl, 10 mM imidazole, 300 mM NaCl, pH 8 with decreasing concentrations of urea and then it was eluted with increasing imidazole concentration from 100 to 300 mM. The 15 Nlabeled recombinant VEGFR1D2 2 was prepared according to Sambrook et al. 3 Figure S4. In vitro angiogenic properties of EP6 and AcHx peptides. HUVEC were plated onto a layer of basement membrane matrix (Matrigel) and incubated at 37 °C for 18 h in the presence of EP6 (50 ng/ml) and AcHx (50 or 26 ng/ml). After treatment, tubular structures and photomicrographs were quantified as angiogenic index, calculated as the number of complete circles counted/field by microscope image analysis (Bottom). *p<0.05 vs control.