Synthesis, Characterization, and Biological Evaluation of a Dual-Action Ligand Targeting αvβ3 Integrin and VEGF Receptors

Abstract

A dual-action ligand targeting both integrin αVβ3 and vascular endothelial growth factor receptors (VEGFRs), was synthesized via conjugation of a cyclic peptidomimetic αVβ3 Arg-Gly-Asp (RGD) ligand with a decapentapeptide. The latter was obtained from a known VEGFR antagonist by acetylation at the Lys13 side chain. Functionalization of the precursor ligands was carried out in solution and in the solid phase, affording two fragments: an alkyne VEGFR ligand and the azide integrin αVβ3 ligand, which were conjugated by click chemistry. Circular dichroism studies confirmed that both the RGD and VEGFR ligand portions of the dual-action compound substantially adopt the biologically active conformation. In vitro binding assays on isolated integrin αVβ3 and VEGFR-1 showed that the dual-action conjugate retains a good level of affinity for both its target receptors, although with one order of magnitude (10/20 times) decrease in potency. The dual-action ligand strongly inhibited the VEGF-induced morphogenesis in Human Umbilical Vein Endothelial Cells (HUVECs). Remarkably, its efficiency in preventing the formation of new blood vessels was similar to that of the original individual ligands, despite the worse affinity towards integrin αVβ3 and VEGFR-1.

Keywords: Angiogenesis; VEGFR; dual-action ligands; integrins; ligand conjugation.

Figure 1

The potent and selective integrin α_Vβ₃ ligand cyclo[DKP-RGD] (1), and its derivative cyclo[DKP-RGD]-CH₂NH₂ (2).

Figure 2

The α-helical peptide 3, its derivative 4, and the novel small-molecule dual-action ligand 5.

Scheme 1

Reagents and conditions: a) SPPS: 1) 25 % piperidine in DMF, 2) Fmoc-AA-OH (4 eq), DIC, HOAt, DIPEA, DMF, 3) 25 % Ac₂O in DMF; b) CH₂Cl₂/TIS/TFA 94:5:1 v/v/v, r.t., 12×2 min; c) 4-pentynoic acid, HATU, HOAt, DIPEA, DMF, r.t., o/n; d) succinic anhydride, DMAP, DIPEA, CH₂Cl₂, r.t., 18 h, 96 %; e) N-hydroxysuccinimide, DIC, DMF, r.t., 2 h, then 2, CH₃CN, phosphate buffer, pH 7.3–7.6, 0 °C, 18 h, 65 % over 2 steps; f) 6+7, CuI, sodium ascorbate, DIPEA, DMF, 72 h, r.t.; g) TFA/EDT/H₂O/TIS 94:2.5:2.5:1 v/v/v/v, 3 h, r.t., 5 % (4, over 16 steps) and 6 % (5, over 19 steps). Mtt=4-methyltrityl.

Figure 3

Bifunctional PEG₈ amino azide 10 (A), and the distance between the two ligand moieties of conjugate 5 (B).

Figure 4

CD spectra of peptide 4 in water, 2,2,2-trifluoroethanol (TFE) and methanol (MeOH) (0.1 mm).

Figure 5

CD spectrum of 2 (left) and 7 (right) in H₂O (0.1 mm).

Figure 6

The preferred intramolecular hydrogen-bonded pattern proposed for compound 1 on the basis of NMR spectroscopic data. The arrow indicates a significant nuclear Overhauser effect (NOE) contact. Computational studies assessed that more than 90 % of the conformations sampled during restrained mixed-mode Metropolis Monte Carlo/Stochastic Dynamics simulations adopted an extended arrangement of the RGD sequence characterized by a pseudo-β-turn type II at DKP−Arg and the formation of the corresponding hydrogen bond between the NH−Gly and C(5)=O.

Figure 7

CD spectrum of 5 (solid line) in H₂O (0.1 mm) superimposed to the sum CD spectrum of 4+7 (dashed line).

Figure 8

CD spectra of 5 in TFE, MeOH and H₂O (0.1 mm).

Figure 9

The potent α_Vβ₃ integrin ligand c[RGDfV] (13) (see Ref. 23).

Figure 10

Representative phase contrast photomicrographs of HUVEC plated on Matrigel in the presence of: A) VEGF₁₆₅ (10 ng mL⁻¹); B) VEGF₁₆₅ (10 ng mL⁻¹)+5 (1 μm); C) VEGF₁₆₅ (10 ng mL⁻¹)+14 (1 μm). Images were elaborated by phase-contrast microscopy using a fluorescence microscope. Frames are approximately 10 μm wide × 10 μm tall.

Figure 11

Effect of incubation of HUVEC for 5 h with the ligands 1 (A), 3 (B), 4 (C), 5 (D), and 1+4 (E) on VEGF-induced morphogenesis. Tube formation was evaluated as length of branches. Data are presented as mean±S.D. of 4–10 separate experiments (*=p<0.05 and **=p<0.01 vs. VEGF alone).

Figure 12

Inhibitory effects exerted by incubation with the different ligands on VEGF-induced morphogenesis. Results are presented as % of the effect normalized to VEGF alone and data are expressed as mean±S.D. for 4–10 separate experiments.