P11 (HSDVHK) is a novel peptide ligand containing a PDZ-binding motif (Ser-Asp-Val) with high affinity to integrin αvβ3. It was identified from a hexapeptide library (PS-SPCL) using a protein microarray chip-based screening system. The integrin αvβ3 has been used as a target for gene therapy of tumors, recently.
By using a comparative pharmaco-proteomic analysis of cellular signaling proteins in P11-treated HUVECs, the inhibitory mechanism of P11 (HSDVHK) on tumor-induced angiogenesis was investigated. P11 was rapidly internalized by HUVECs via an integrin αvβ3-mediated event. In the P11 uptake process, caveolin and clathrin appeared to be involved. The suppression of bFGF-induced HUVEC proliferation was resulted by cell-penetrating P11 in a dose-dependent manner. The cell-permeable P11 inhibited phosphorylation of extracellular-signal regulated kinase (ERK1/2) and mitogen-activated protein kinase (MEK) in bFGF-stimulated HUVECs. Proteomic analysis via antibody microarray suggested that the upregulation of p53 in P11-treated HUVECs resulted in induction of apoptosis via activation of caspases-3, -8 and -9. Several lines of experimental evidence strongly indicated that the molecular mechanism of P11 inhibits bFGF-induced HUVEC proliferation via MEK/ERK inhibition as well as p53-mediated apoptosis is related with activation of caspases. Besides, additional analyses were supported to suggest the proteomic result of P11-induced upregulation of p53. However, further work is required to elucidate how the p53 expression was increased by treatment of P11 in bFGF-stimulated HUVECs.
The evaluated free energies of the binding for each hexapeptide with αvβ3 were closely related with the experimental protein chip data. The results showed that P11 was most effective in suppressing the binding of vitronectin to integrin αvβ3.
Because tumor growth requires angiogenesis while P11 inhibits angiogenesis both in vitro and in vivo, the efficacy of P11 as an inhibitor of angiogenesis-dependent tumor growth was evaluated in a mouse tumor model. It was noteworthy that P11 significantly prevented the B16F10 melanoma-induced neovascularization. These data strongly indicate that P11 should be considered as a new angiogenesis inhibitor and novel target for correcting abnormal vasculature.
Pharmacokinetics and metabolism
Chip-based binding kinectics were estimated to confirm the binding affinity of P11. Fluorescence dye labeled GRGDSP or P11 was incubated with an integrin αvβ3 microarray in a dose-dependent manner. The P11 showed a higher signal increase along with the reaction time compared to the RGD peptide in this assay. By using a conventional linear regression method, the competitive binding kinetics were also analyzed. The association rate constants (ka) of the P11 and GRGDSP were calculated to be 4.93 ± 70.99 and 2.787 ± 0.48 nM/min, respectively. According to these data, the binding mode of P11 to the RGD-binding site of αvβ3 is more kinetically stable than that of GRGDSP peptide.
1. Lee, Y., Kang, D. K., Chang, S. I., Han, M. H., & Kang, I. C. (2004). High-throughput screening of novel peptide inhibitors of an integrin receptor from the hexapeptide library by using a protein microarray chip. Journal of biomolecular screening, 9(8), 687-694.
2. Bang, J. Y., Kim, E. Y., Kang, D. K., Chang, S. I., Han, M. H., Baek, K. H., & Kang, I. C. (2011). Pharmaco-proteomic analysis of a novel cell-permeable peptide inhibitor of tumor-induced angiogenesis. Molecular & Cellular Proteomics, mcp-M110.
3. Choi, Y., Kim, E., Lee, Y., Han, M. H., & Kang, I. C. (2010). Site‐specific inhibition of integrin αvβ3‐vitronectin association by a ser‐asp‐val sequence through an Arg‐Gly‐Asp‐binding site of the integrin. Proteomics, 10(1), 72-80.