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uPAR promotes cell associated proteolysis
uPAR promotes cell-associated proteolysis by binding to uPA which locally converts plasminogen into active plasmin [10]. Ligand-engaged uPAR also acts as a potent regulator of cell migration and matrix attachment. Being a membrane receptor, uPAR needs to interact with extracellular matrix (ECM) proteins such as vitronectin, and transmembrane receptors, including G protein-coupled formyl peptide receptors (FPRs), integrins and receptor tyrosine kinases, regardless its catalytic activity [11], [12], [13], [14], [15], [16], [17]. FPRs are a family of 7 transmembrane domains, G-protein coupled receptors that exert multiple functions in many pathophysiological processes because of their capacity to interact with a variety of structurally diverse ligands [17]. Human FPR1, originally identified in neutrophils, monocytes and macrophages, elicits many responses upon ligation to formyl-peptide ligands derived from bacteria and/or mitochondria of eukaryotic cells, including morphological polarization, locomotion, production of reactive-oxygen species and release of cytokines and proteolytic Adriamycin HCl synthesis [17]. Therefore, the inhibition of the crosstalk between uPAR and FPR1 represents an attractive target to inhibit the metastatic processes sustained by an altered cell migration. uPAR is a glycosylphosphatidylinositol (GPI)-anchored receptor arranged in three domains (DI, DII, and DIII), from the N-terminal, linked by short sequence regions. The three domains pack together into a concave structure that binds uPA [18], [19], [20]. Plasmin generated by uPA or uPA itself can cleave intact uPAR, releasing DI. The remaining GPI-anchored DIIDIII can be left on cell surface or be secreted in the extracellular milieu following cleavage of the anchor [21]. The domain boundary between DIDII is more flexible than the DIIDIII domain boundary and includes the protease-sensitive signalling region uPAR(84–95) [22], [23]. Both uPAR(84–95) sequence and the synthetic shorter peptide (uPAR(88–92) Ser88-Arg-Ser-Arg-Tyr92, SRSRY) retain chemotactic activity and trigger directional cell migration and angiogenesis in vitro and in vivo[14], [15], [24]. Mechanistically, uPAR(84–95) sequence as well as SRSRY exert these activities through their interaction with the formyl peptide receptor type 1 (FPR1) which, in turn, activates the vitronectin receptor with an inside-out type of mechanism which involves protein kinase C and extracellular signal-regulated kinase phosphorylation [15], [24]. The multi-domain uPAR protein may reversibly acquire specific conformational states [22], [23]. The equilibrium of such conformations may be sensitive to mutations, and has proved to be pivotal in its function and role [20], [22], [23]. In this light, uPAR(84–95) sequence, mainly projected on the external surface of uPAR, possesses a structural flexibility in both membrane-associated and soluble forms of uPAR [22], [23]. Structure-activity relationship studies of the chemotactic peptide Ser88-Arg-Ser-Arg-Tyr92 have been reported [25], [26], [27], [28], [29]. We have previously identified the key residue Ser90 as positioned in a critical “hinge”, where it can influence the conformation of nearby residues. Indeed, replacement of Ser90 with a glutamic acid residue in the full length membrane-associated uPAR prevents the uPAR/FPR1/vitronectin receptor cross-talk, thereby blocking cell migration and invasion both in vitro and in vivo experiments [25]. Furthermore, synthetic linear penta- and tetra-peptides carrying Ser90 substituted with a glutamic acid residue or peptidomimetic analogs carrying Ser90 substituted with Cα-methyl-α-aminoisobutyric acid (Aib) revealed inhibitory activity of cell migration toward several chemoattractants, including uPAR(88–92) and the bacterial-derived N-formyl-methionyl-leucyl-phenylalanine peptide (fMLF) [26], [27], [28], [29]. Based on such information, we have recently developed the head-to-tail cyclic analog [SRSRY] (1), a new potent and stable inhibitor that prevents the in vitro and in vivo recruitment of monocytes into inflamed tissues and trans-endothelial migration of sarcoma cells [30], [31]. The [SRSRY] peptide competes with both SRSRY and fMLF for binding to FPR1, thus preventing agonist-induced FPR1 activation and internalization, leading to the inhibition of cell migration [30], [31]. Interesting activities of [SRSRY] prompted us to develop novel cyclic and linear analogs with the aim to broaden the knowledge about structure-activity relationships of peptide 1 (Scheme 1, Table 1). Herein we report their synthesis, effects on cell migration, FPR1 internalization, conformational and docking analyses.