Discovery of new small molecules targeting the vitronectin binding site of the urokinase receptor that block normal and leukemic cell migration

Besides focusing urokinase (uPA) proteolytic activity on the cell membrane, the urokinase receptor (uPAR) is an adhesion receptor able to bind vitronectin (VN), via a direct binding site. Furthermore, uPAR interacts with other cell surface receptors, such as integrins, receptor tyrosin kinases and G-protein coupled chemotaxis receptors, triggering cell-signalling pathways that promote cell proliferation and migration. The ability of uPAR to coordinate binding and degradation of extracellular matrix and cell signalling makes it an attractive therapeutic target. We used a structure-based virtual screening (SB-VS) approach to search for small molecules that might target the uPAR binding site for VN. 41 compounds were identified and further tested on uPAR-negative HEK-293 epithelial cells transfected with uPAR (uPAR-293 cells), using the parental cell line transfected with the empty vector (V-293 cells), as a control. Two compounds, 6 and 37, selectively inhibited uPAR-293 cell adhesion to VN and the resulting changes in cell morphology and signal transduction, without exerting any effect on V-293 cells. 6 and 37 inhibited uPAR- 293 cell binding to VN at micromolar concentrations, showing IC50 values of 3.6 and 1.2 mM, respectively. Compounds 6 and 37 targeted S88 and R91, key residues for uPAR binding to VN but also for uPAR interaction with the f-MLF family of chemotaxis receptors (fMLF-Rs). As a consequence, 6 and 37 impaired uPAR-293 cell migration toward FCS, uPA and f-MLF, likely by inhibiting the structural and functional interaction between uPAR and FPR1, the high affinity fMLF receptor. Both compounds blocked in vitro cell migration of normal blood cells as well as of several acute myeloid leukemia cell lines; therefore they represent new promising leads for pharmaceuticals in inflammatory diseases and leukemia.