here they are involved in the sustained chronic proliferation of cancer cells. The prominent role of the ErbB family in the development and 15155536 surviving of cancer cells was described in the 1980’s, when Sporn and Todaro established the theory of the “autocrine secretion”of DMXB-A site growth factors by cancer cells to maintain a high rate of proliferation. In many types of cancer the overexpression of ErbB receptors and enhanced ligand production allow an autocrine stimulation of the cell proliferation. Since then an enormous amount of literature has been devoted to the role of the receptors, but to a lesser extent to the ErbB ligands. After decades of high interest in the ErbB receptors as therapeutic targets, and low attention focused on the ligands, new insights have renewed the importance of the ErbB ligands, especially the EGFR ligands, as responsible for autocrine/paracrine loops and resistance to old and new therapeutic agents. We would like to suggest that “it is time to turn again our attention to the role played by the ligands”, not only the receptors, to design new therapeutic strategies against cancer. Human EGF is a small single chain polypeptide composed of 53 amino acids that adopts a well-defined three dimensional structure containing three disulfide bonds defining three loops. This scaffold is known as a TKnot and is found in a large number of functional unrelated proteins. EGF structure is tightly related to its function. There are three contact sites described between the ligand and the EGFR which in turn is divided by four domains : the B loop of EGF interacts with site 1 in domain I, the A loop of EGF interacts with site 2 in domain III, and the Cterminal region of EGF interacts with site 3 in domain III. This structurefunction relationship in EGF has been accurately examined in order to generate antagonistic analogues of EGF, or EGFR blockers. Some authors have developed shorter synthetic derivatives of EGF and TGF-a as antagonists of these growth factors. However, these strategies have not been effective because the EGF and TGF-a fragments were too short to form the tertiary An EGF Derivative as EGFR Blocker structure required for binding to the EGFR. In other studies the EGF derivative required very high concentrations to exhibit the desired responses, probably because the short peptide interacted only with one of the three interfaces of interaction, namely the site 1 of EGFR. Our group has previously described that the Potato Carboxypeptidase Inhibitor is an antagonist of human EGF. PCI is a 39-residue protein folded into a 27-residue globular core stabilized by three disulfide bonds that adopts the same conformation as EGF, the T-Knot scaffold. This similar structure probably accounts for the antagonistic activity. PCI competed with EGF for binding to EGFR, inhibited EGFR activation and cell proliferation and induced the down regulation of the receptor. In addition, PCI showed anti-proliferative activity in vitro in several human cancer cell lines and in vivo in nude mice implanted with a xenograft 22634634 of pancreatic cancer cell lines. Unfortunately, PCI’s affinity for EGFR is very low, and high concentrations were required to achieve the desired inhibitory activity. The structural and clinical interest of PCI opened the possibility of engineering new PCI-like EGF antagonists with improved ErbB affinity. The superimposition of the three-dimensional structures of EGF and PCI based on disulfide bridge topology, revealed that PCI la