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Synthetic Peptides Derived from the Angiostatin K4 Domain Inhibit Endothelial Cell Migration

Angiogenesis, the process by which new capillaries are formed from preexisting blood vessels, represents a vital function for the growth of normal tissues during embryogenesis as well as for the pathological growth of tumors. Experimental evidence has shown that both primary and metastatic tumors need to recruit angiogenic vessels for their growth, and form their own circulatory system by up-regulating angiogenic stimulators and down-regulating angiogenesis inhibitors. Blocking of positive regulators or utilization of negative regulators to suppress angiogenesis results in a delay or regression of induced tumors. In particular, negative regulators of angiogenesis, such as angiostatin, endostatin, and antagonists for integrin v 3, displayed profound antitumor activities in vivo. Angiostatin is a 38 kDa internal fragment of plasminogen, produced by proteolytic cleavage, and consists of four triplydisulfide-bridged kringle structures. In mice, angiostatin inhibits primary tumor growth as well as angiogenesis-dependent growth of metastases. In vitro, angiostatin inhibits endothelial cell migration and reduces endothelial cell growth in proliferation assays. Furthermore, it has been shown that angiostatin induces endothelial cell-specific apoptosis in vitro. Despite intense research efforts, the exact mechanisms by which angiostatin down-regulates neovascularization remain to be determined. Little is also known about the nature of receptors mediating inhibitory effects on endothelial cells, although several receptors for angiostatin have been identified. ATP synthase has been described as a high-affinity angiostatinbinding protein, and antibodies to ATP synthase have been reported to inhibit the antiproliferative activity of angiostatin on endothelial cells. Plasminogen itself has been shown to bind to other cell-surface proteins such as annexin II and alpha enolase. Recently, angiomotin has been identified in a yeast two-hybrid screen as a functional angiostatin-binding protein that mediates the inhibition of endothelial cell motility and tube formation in vitro. A fundamental step for deciphering the biology of angiostatin is to understand the role of the kringle functional domains in the structure/function of angiostatin. Each kringle is composed of approximately 80 amino acids in a double-loop conformation, held together by three disulfide bonds. Angiostatin was originally described as consisting of kringles 1 ± 4, although the COOH terminus was identified later on by Soff and co-workers, who described an angiostatin consisting of kringles 1 ± 4, plus approximately 85% of plasminogen kringle 5. Early attempts to generate recombinant angiostatin indicated the importance of the disulfide-bonded kringle in the inhibition of angiogenesis. Several other findings suggested that the kringle structure itself confers a large part, if not all, of the antiangiogenic activity. While sharing structural similarity and sequence homology, kringle domains appear to have distinct inhibitory profiles on angiogenesis-associated endothelial cell activities. Specifically, the in vitro activity of angiostatin in endothelial-cell proliferation assays resides in kringles 1 ± 3, with kringle 1 being the most potent inhibitor, while kringle 4 is relatively ineffective. In contrast, K4 is the smaller fragment with the most potent antimigratory activity. Finally, Cao and co-workers demonstrated that K5 of plasminogen presents the most potent activity against endothelial-cell proliferation. The reasons why some of the kringle domains are significantly more potent than others and the mechanisms explaining the dissociation of antiproliferative and antimigratory activity attributed to K4, compared with other kringle domains, are still unknown. However, comparisons of different angiostatin-related proteins require careful scrutiny since differences in activity between different kringle domains, or different angiostatin isoforms with varying kringle compositions, may reflect artifacts of the recombinant technology, and not necessarily an inherent difference in the kringle composition. To investigate the different contribution of K4 regions to the antimigratory activity, three linear peptides that mapped the entire K4 sequence were designed (Figure 1), synthesized, and tested for biological activity, the quantification of the biological activity being regarded as the first step in the process of identification of the minimum active sequence in the design of peptides of reduced length. All linear peptides present Nand C-terminal Cys residues protected by Acm or tButhio groups. In [19] Another receptor, Gua-Glu(OBnz)-Trp-Glu(OBnz)-NH2, which is one of the weak binding receptors according to the on-bead assay (Kass 90M ), did not give any reasonable data. The binding constant was too low to be measured accurately in free solution by UV titration, which is again in good agreement with the rather poor binding affinity of this specific receptor observed in the on-bead assay. [20] M. Conza, H. Wennmers, Chem. Commun. 2002, 866 ± 867. [21] F. Mohamadi, N. G. J. Richards, W. C. Guida, R. Liskamp, M. Lipton, C. Caufield, G. Chang, T. Hendrickson, W. C. Still, J. Comput. Chem. 1990, 11, 440 ± 467. [22] Because of the limited solubilities both of the free peptide and of the receptors at concentrations 10 mM in aqueous solvents, the NMR studies had to be performed in DMSO. Although the relative importance of H-bonds/ion-pairing versus hydrophobic interactions should be shifted towards the hydrophobic interactions in water, the observation of the H-bonded pattern required for a sheet in DMSO is also a necessary prerequisite for its occurrence in water.