br Results br Discussion Immune mediated chronic
Discussion Immune-mediated chronic inflammatory diseases (e.g. rheumatoid arthritis, psoriasis, multiple sclerosis, and inflammatory bowel disease) are characterized by a dysregulated migration of leukocytes from blood vessels into inflamed tissues. To date, several new targets and inhibitors that mediate leukocyte motility are being subjects of study , . However, the currently available therapies are often not efficient enough or may even induce undesired effects . uPAR plays an important role in the regulation of leukocyte trafficking . In particular, we have shown that the capability of uPAR to trigger cell migration depends on its Ser88-Arg-Ser-Arg-Tyr92 chemotactic sequence even in the form of a synthetic, linear peptide (SRSRY) , , , , , , , , , . Also, we reported that the residue Ser90 is positioned in a critical “hinge”, which influences the conformation of the nearest residues. Indeed, the substitution of Ser90 in the full length, membrane-associated uPAR with a glutamic Gemcitabine residue prevents the complex uPAR/FPR1 cross-talk, thereby blocking in vitro and in vivo cell migration . Accordingly, uPAR-derived linear, tetra, and penta peptides carrying the Ser90 substituted with a glutamic acid residue elicited inhibitory effect on cell migration in virtue of their ability to prevent agonist induced FPR1 internalization , , . More recently, we found that the cyclization of the Ser88-Arg-Ser-Arg-Tyr92 chemotactic sequence of uPAR generates a peptide [SRSRY] (1) exerting an opposite effect on cell migration, as compared to its linear form . [SRSRY] behaves as new potent and stable inhibitor of cell migration that prevents the in vitro and in vivo recruitment of monocytes into inflamed tissues and trans-endothelial migration of sarcoma cells , . The mechanism by which [SRSRY] interferes with fMLF binding to FPR1 has been deeply investigated in rat basophilic leukemia RBL-2H3/ETFR cells expressing high levels of constitutively activated FPR1: [SRSRY] inhibits fMLF-induced, FPR1-mediated cell migration by blocking FPR1 internalization , . In this paper, novel analogs of both linear SRSRY and cyclic [SRSRY] (1) peptides were developed and tested for their inhibitory effect on both FPR1 internalization and cell migration (Table 1, Fig. 1, Fig. 2, Fig. 3). We found that, like peptide 1, the peptides 5, 6, 8 and 11 appeared highly effective in reducing the binding of fMLF to FPR1. Analysis of their capability to promote or inhibit fMLF-directed cell migration allowed us to investigate whether a structure/function relationship does exist. Serine residues were variously replaced by negatively charged phosphorylated serine or glutamate since it is well known that replacement of Ser90 with a glutamate switches the peptide activity from chemotactic to inhibitory of cell migration. This point was confirmed by our experiments that revealed the strong inhibitory activity of peptide 5 (Table 1) in which the Ser90 residue was replaced by Ser(P). As for most of the tested peptides, the inhibitory effect on cell migration of peptide 5 well correlated with its inhibitory effect on the FPR1 internalization. The same mutation (Ser90 to Glu) in the cyclic peptide 1 did not improve the inhibitory activity of the resulting peptide. Thus, the activity switching observed passing from SRSRY to SRSPRY is not based on indispensable interactions of the Ser(P) residue with the receptor but on conformational variation of the peptide upon phosphorylation. In fact, peptide SRSRY has a complete random coil conformation in DPC solution (Fig. 4), hence its bound conformation with the FPR1 receptor, triggering the pro-migratory effect, must be stabilized by induced fit events. In contrast, peptide 5 should have the suitable 3D structure to bind the receptor without evoking the migratory effect. Conformational analysis demonstrated that peptide 5 has a relatively stable conformation when interacting with DPC micelles. It can be described as a 310-helix structure encompassing the last four residues of the peptide (Fig. 5b). This result is in accordance with that obtained with the potent antagonists RERF and UPARANT. Both RERF and UPARANT adopt helicogenic-turned structures in solution; an α-turn (type I-αRS) is found for RERF  and an incipient 310-helix for UPARANT . Moreover, in peptide 5 structure, the side chains of Arg91 and Tyr92 are close in the space while that of Arg89 points in opposite direction and is close to Ser(P)90 with which it establishes a salt bridge in many conformers. Considering peptide 1, it cannot fold as a helix due to its head-to-tail cyclic structure (Fig. 5a) but the side chain orientation of important residues Arg89, Arg91 and Tyr92 is very similar to that observed in peptide 5 (Fig. 9a) and this result gives a structural explanation of the similar activity of the two peptides. An important consequence of the last observation is that the particular side chains orientation more than backbone secondary structure is determinant for the (antagonist) activity. As matter of fact, the reported structure of RERF and UPARANT also showed similar orientation and proximity of the corresponding side chain couples .