Evaluation of peptide combinations from different protein families. Two peptides from three different protein families were combined serially in the proliferation assay, and the efficiency of the various peptide combinations was evaluated after calculating the isobolograms and C.I. for each of the combinations. (A) The isobolograms for the combination of a CXC chemokine-derived peptide and a TSP1 repeat-derived peptide. For each of the tested conditions (the individual peptides and the peptides tested in combination), proliferation dose response sigmoidal curves were estimated by fitting the data to sigmoidal Hill curves. Equally effective dose pairs in the combination experiments were then matched to the same level of inhibition obtained with a single peptide. The red highlighted areas designate peptide concentrations at which the activity is synergistic. The isobolograms for the combination of a collagen IV-derived peptide and a CXC chemokine and for a collagen IV-derived peptide and a TSP1 repeat-containing protein-derived peptide are included in Fig. S5. (B) Graphical interpretation of the most potent synergistic strategies, based on the calculations from the combinatorial experiments. The red lines indicate strong synergism and the blue lines antagonism.

Outline of the computational bioinformatic algorithm. (A) The similarities between conserved domains from previously identified antiangiogenic proteins were calculated after performing multiple sequence alignments of their amino-acid sequences. The blue color designates the amino acid conservation (the darker the color, the more conserved the amino acid). (B) In cases in which a protein contained more than one copy of a conserved domain, those that were primarily similar to the known antiangiogenic domains (pink highlight) were used. (C) Using the sequences from the known conserved domains, we determined their similarity to other such domains from the whole human proteome. (D) In addition to identifying large domains showing sequence similarity, we used the information from shorter known antiangiogenic fragments (pink highlight) and searched for sequences similar to those of the fragments. The blue highlight designates conservation as in A. These short fragments were derived from a variety of protein families, including the somatotropins, CXC chemokines, collagen IV fibrils, serpins, and TSP1 repeat-containing proteins.