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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.


The α-helical peptide 3, its derivative 4, and the novel small-molecule dual-action ligand 5.
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fig02: The α-helical peptide 3, its derivative 4, and the novel small-molecule dual-action ligand 5.

Mentions: The reported colocalization of integrin αVβ3 and VEGFR on the surface of HUVEC upon VEGF stimulation demonstrates that a “crosstalk” between these receptors occurs.10f Following these findings, in a pioneering study, Cochran and co-workers reported that a dual specific fusion protein was able to bind simultaneously integrin αVβ3 and VEGFR-2, and to inhibit VEGF-mediated capillary tube formation in HUVEC and murine blood vessel formation within implanted Matrigel plugs.17a Inspired by this contribution, we speculated that a dual-action integrin–VEGFR small molecule ligand could also represent a novel antiangiogenic and antitumor strategy offering more effective multiple targeting to tumor cells and tumor vasculature.17b We thus designed a new dual-action ligand (compound 5 in Figure 2) targeting integrin αVβ3 and VEGFR. As integrin binding moiety, we exploited cyclo[DKP-RGD]-CH2NH2 (2) (Figure 1), a functionalized analog of ligand 1 that we had previously used for conjugation to paclitaxel18 and SMAC mimetic molecules.19 As VEGFR ligand moiety we selected the α-helical decapentapeptide 3 (Figure 2), recently reported by D′Andrea and co-workers,14 because of its simple preparation and efficacy in inhibiting angiogenesis in vivo. However, for conjugation to compound 2 we employed compound 4 (Figure 2), a derivative of 3 acetylated at the Lys13 side chain,20 in order to avoid any possible interference of the Lys13 free amino group with the “electrostatic clamp” used by the cyclo[DKP-RGD] moiety for binding to its αVβ3 integrin target. Previous studies clearly demonstrated that neither the N-terminal Lys1 side chain nor the Lys13 side chain are involved in receptor recognition.14b Accordingly, while both positions could in principle be exploited for conjugation, in this paper we used the N-terminal Lys1 side chain.


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)

The α-helical peptide 3, its derivative 4, and the novel small-molecule dual-action ligand 5.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig02: The α-helical peptide 3, its derivative 4, and the novel small-molecule dual-action ligand 5.
Mentions: The reported colocalization of integrin αVβ3 and VEGFR on the surface of HUVEC upon VEGF stimulation demonstrates that a “crosstalk” between these receptors occurs.10f Following these findings, in a pioneering study, Cochran and co-workers reported that a dual specific fusion protein was able to bind simultaneously integrin αVβ3 and VEGFR-2, and to inhibit VEGF-mediated capillary tube formation in HUVEC and murine blood vessel formation within implanted Matrigel plugs.17a Inspired by this contribution, we speculated that a dual-action integrin–VEGFR small molecule ligand could also represent a novel antiangiogenic and antitumor strategy offering more effective multiple targeting to tumor cells and tumor vasculature.17b We thus designed a new dual-action ligand (compound 5 in Figure 2) targeting integrin αVβ3 and VEGFR. As integrin binding moiety, we exploited cyclo[DKP-RGD]-CH2NH2 (2) (Figure 1), a functionalized analog of ligand 1 that we had previously used for conjugation to paclitaxel18 and SMAC mimetic molecules.19 As VEGFR ligand moiety we selected the α-helical decapentapeptide 3 (Figure 2), recently reported by D′Andrea and co-workers,14 because of its simple preparation and efficacy in inhibiting angiogenesis in vivo. However, for conjugation to compound 2 we employed compound 4 (Figure 2), a derivative of 3 acetylated at the Lys13 side chain,20 in order to avoid any possible interference of the Lys13 free amino group with the “electrostatic clamp” used by the cyclo[DKP-RGD] moiety for binding to its αVβ3 integrin target. Previous studies clearly demonstrated that neither the N-terminal Lys1 side chain nor the Lys13 side chain are involved in receptor recognition.14b Accordingly, while both positions could in principle be exploited for conjugation, in this paper we used the N-terminal Lys1 side chain.

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.