Discovery of New Small Molecules Targeting the Vitronectin-Binding Site of the Urokinase Receptor That Block Cancer Cell Invasion

Abstract

Besides focusing urokinase (uPA) proteolytic activity on the cell membrane, the uPA receptor (uPAR) is able to bind vitronectin, via a direct binding site. Furthermore, uPAR interacts with other cell surface receptors, such as integrins, receptor tyrosine kinases, and chemotaxis receptors, triggering cell-signaling pathways that promote tumor progression. The ability of uPAR to coordinate binding and degradation of extracellular matrix (ECM) and cell signaling makes it an attractive therapeutic target in cancer. We used structure-based virtual screening (SB-VS) to search for small molecules targeting the uPAR-binding site for vitronectin. Forty-one compounds were identified and 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. Compounds 6 and 37 selectively inhibited uPAR-293 cell adhesion to vitronectin and the resulting changes in cell morphology and signal transduction, without exerting any effect on V-293 cells. Compounds 6 and 37 inhibited uPAR-293 cell binding to vitronectin with IC 50 values of 3.6 and 1.2 mmol/L, respectively. Compounds 6 and 37 targeted S88 and R91, key residues for uPAR binding to vitronectin but also for uPAR interaction with the fMLF family of chemotaxis receptors (fMLF-Rs). As a consequence, compounds 6 and 37 impaired uPAR-293 cell migration toward fetal calf serum (FCS), uPA, and fMLF, likely by inhibiting the interaction between uPAR and FPR1, the high affinity fMLF-R. Both compounds blocked in vitro ECM invasion of several cancer cell types, thus representing new promising leads for pharmaceuticals in cancer.

Stabilizing a Flexible Interdomain Hinge Region Harboring the SMB Binding Site Drives uPAR into Its Closed Conformation

“…In some cases, circulating uPAR DI shed by proteolysis is an even more powerful prognostic biomarker than intact uPAR [47,48]. Inline with these properties, uPAR is considered a relevant translational target for intervention therapy and huge efforts have accordingly been allocated, in recent years, to development and preclinical testing of various uPAR targeting strategies including uPA-activated prodrugs [49], small-molecule inhibitors [12,31,50], targeted radiotherapy [51], and mAbs [52,53].…”

“…Importantly, this flexibility has to be taken into account if molecular modeling is used for drug discovery and refinement of small-molecule antagonists targeting these two known uPAR·ligand interfaces [12,23,30,31], as well as when docking simulations are used to build model structures of hitherto unsolved uPAR·ligand complexes [32].…”

Urokinase receptor promotes ovarian cancer cell dissemination through its 84-95 sequence

“…A possibility is to interfere with uPAR/FPRs interaction. Recently, Rea and co-workers described small molecules that impaired cell migration in virtue of their ability to target the Ser 88 and Arg 91 residues of uPAR and, consequently, uPAR/FPR interaction [48]. In this context, RERF peptide which, able to interfere with the invasive phenotype of ovarian cancer cells by inhibiting uPAR 84-95 -triggered, FPR-mediated signals, could be considered a valid prototype for the development of new anti-neoplastic therapies designed to simultaneously counteract growth and abdominal dissemination of ovarian cancer cells.…”

Therapeutics targeting the fibrinolytic system

“…Chiron Corporation screened a phage-display library to identify uPAR antagonists 105 , but no further studies were reported on the antitumor effects of these peptides. Small molecules that interrupted the interaction between uPAR and vitronectin inhibited the invasion of cancer cells in vitro 106 . The Meroueh group synthesized a series of pyrrolidinone and piperidinone derivatives that bind to the uPA-binding pocket of uPAR 107 .…”