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

Zanella S, Mingozzi M, Dal Corso A, Fanelli R, Arosio D, Cosentino M, Schembri L, Marino F, De Zotti M, Formaggio F, Pignataro L, Belvisi L, Piarulli U, Gennari C - ChemistryOpen (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Chimica, Università degli Studi di Milano Via C. Golgi 19, 20133, Milan, Italy.

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.

No MeSH data available.


Bifunctional PEG8 amino azide 10 (A), and the distance between the two ligand moieties of conjugate 5 (B).
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fig03: Bifunctional PEG8 amino azide 10 (A), and the distance between the two ligand moieties of conjugate 5 (B).

Mentions: The dual-action ligand 5 (Figure 2) was obtained from the two fragments 6 and 7, that were joined by copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction (Scheme 1). Alkyne 6 was obtained from the resin-supported peptide 8, which was readily synthesized by microwave-assisted solid phase peptide synthesis (SPPS). Treating the on-bead decapentapeptide resin with a 94:5:1 dichloromethane/triisopropylsilane/trifluoroacetic acid (v/v/v) cleavage mixture resulted in the selective removal of 4-methyltrityl (Mtt) protecting group from Lys1 side chain. The free amine moiety in 9 was reacted on solid phase with 4-pentinoyc acid, after activation of the carboxylic acid moiety in the presence of the condensing agents, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) and 1-hydroxy-7-azabenzotriazole (HOAt), and N,N-diisopropylethylamine (DIPEA) as base, obtaining fragment 6. Fragment 7 was prepared starting from the commercially available bifunctional PEG8 compound 10 (Figure 3 A), which was selected because of its monodisperse structure profile.21 Amino azide 10 was first elongated with succinic anhydride, affording the azido acid 11. Cyclo[DKP-RGD]-CH2NH22 (Figure 1) was coupled with the spacer 11 in acetonitrile/phosphate buffer, controlling the pH of the medium in order to have the free benzylic amine of 2 acting as nucleophile. Finally, the synthesis of the dual-action ligand 5 was achieved by coupling fragments 6 and 7 on solid phase via CuAAC reaction in the presence of copper iodide and sodium ascorbate, and then by cleaving the adduct 12 from the resin.


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

Zanella S, Mingozzi M, Dal Corso A, Fanelli R, Arosio D, Cosentino M, Schembri L, Marino F, De Zotti M, Formaggio F, Pignataro L, Belvisi L, Piarulli U, Gennari C - ChemistryOpen (2015)

Bifunctional PEG8 amino azide 10 (A), and the distance between the two ligand moieties of conjugate 5 (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608532&req=5

fig03: Bifunctional PEG8 amino azide 10 (A), and the distance between the two ligand moieties of conjugate 5 (B).
Mentions: The dual-action ligand 5 (Figure 2) was obtained from the two fragments 6 and 7, that were joined by copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction (Scheme 1). Alkyne 6 was obtained from the resin-supported peptide 8, which was readily synthesized by microwave-assisted solid phase peptide synthesis (SPPS). Treating the on-bead decapentapeptide resin with a 94:5:1 dichloromethane/triisopropylsilane/trifluoroacetic acid (v/v/v) cleavage mixture resulted in the selective removal of 4-methyltrityl (Mtt) protecting group from Lys1 side chain. The free amine moiety in 9 was reacted on solid phase with 4-pentinoyc acid, after activation of the carboxylic acid moiety in the presence of the condensing agents, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) and 1-hydroxy-7-azabenzotriazole (HOAt), and N,N-diisopropylethylamine (DIPEA) as base, obtaining fragment 6. Fragment 7 was prepared starting from the commercially available bifunctional PEG8 compound 10 (Figure 3 A), which was selected because of its monodisperse structure profile.21 Amino azide 10 was first elongated with succinic anhydride, affording the azido acid 11. Cyclo[DKP-RGD]-CH2NH22 (Figure 1) was coupled with the spacer 11 in acetonitrile/phosphate buffer, controlling the pH of the medium in order to have the free benzylic amine of 2 acting as nucleophile. Finally, the synthesis of the dual-action ligand 5 was achieved by coupling fragments 6 and 7 on solid phase via CuAAC reaction in the presence of copper iodide and sodium ascorbate, and then by cleaving the adduct 12 from the resin.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Chimica, Università degli Studi di Milano Via C. Golgi 19, 20133, Milan, Italy.

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.

No MeSH data available.