Studio della funzione del recettore dell'urochinasi nell'adesione, migrazione e proliferazione cellulare: identificazione di un nuovo inibitore.

La degradazione della matrice extracellulare (ECM) gioca un ruolo importante in vari processi sia fisiologici, quali fibrinolisi, angiogenesi, sviluppo embrionale, che patologici, quali infiammazione e progressione tumorale. Il sistema di attivazione del plasminogeno, che porta alla formazione di plasmina, potente serin-proteasi, è coinvolto in tutti questi eventi. La molecola chiave di questo sistema, il recettore per l’urochinasi (uPAR), possiede una vasta gamma di funzioni ed è coinvolto in numerosi eventi che includono migrazione cellulare e invasione tissutale. Nelle patologie infiammatorie l’uPAR svolge un ruolo determinante nel regolare la chemotassi delle cellule del sistema immunitario nel sito di malattia attraverso la sua interazione con i recettori per l’fMLF (FPR). Abbiamo quindi ricercato piccole molecole solubili in grado di inibire l’interazione uPAR/FPR da usare come potenziali farmaci. Attualmente, le piccole molecole solubili costituiscono una delle opzioni terapeutiche più attraenti, sia per la loro bio-disponibilità per via orale sia per i bassi costi di produzione. I potenziali inibitori sono stati selezionati mediante l’utilizzo di un metodo computazionale, il “Virtual Screening”. Esso consiste in una rapida selezione di ampie librerie virtuali di composti organici solubili allo scopo di identificare le molecole che possano legarsi più favorevolmente ad un dato bersaglio molecolare. La nostra ricerca di inibitori si è focalizzata sul dominio funzionale dell’uPAR importante per le interazioni con gli FPR, ovvero la sequenza SRSRY (a.acidi 88-92), e sul dominio di legame alla vitronectina (VN). Infatti, questi due domini condividono due residui aminoacidici (R91, Y92). Inoltre, l’adesione cellulare mediata dall’interazione uPAR-VN facilita anch’essa il richiamo di cellule dell’immunità innata nei siti dell’infiammazione. Le molecole che hanno soddisfatto le interazioni chiave (legami H, interazioni ioniche e idrofobiche) sono state selezionate come possibili ligandi e i composti con lo “score” più elevato sono stati successivamente caratterizzati in vitro, attraverso saggi biologici, per i loro effetti su adesione, migrazione e proliferazione cellulare. Sono stati quindi identificati due inibitori, specifici per l’uPAR, in grado di ridurre drasticamente la chemotassi indotta sia da agonisti dell’uPAR, sia da ligandi specifici per gli FPR. Le molecole inibitorie sono state testate prima in cellule epiteliali HEK-293 transfettate con uPAR e, successivamente, in cellule della linea leucocitaria KG1 e cellule primarie da sangue periferico. Poiché l’uPAR è fortemente espresso in diverse patologie neoplastiche e infiammatorie, ci aspettiamo che una nuova terapia basata sull’utilizzo di tali piccole molecole possa essere specifica e poco tossica

uPAR REGULATES PERICELLULAR PROTEOLYSIS THROUGH A MECHANISM INVOLVING INTEGRINS AND fMLF-RECEPTORS

The expression of the urokinase-type plasminogen activator (uPA) and its receptor (uPAR) can be regulated by several hormones, cytokines, and tumour promoters. uPAR is a glycosyl-phosphatidyl inositol (GPI)-linked cell-surface protein; however, it is capable to transduce signals inside the cell by interacting with other cell-surface proteins, such as integrins and G-protein coupled (GPC) receptors. We previously reported that uPAR cell-surface expression can be positively regulated by its ligand, uPA, independently of its proteolytic activity. We now demonstrate that uPAR overexpression induces or increases uPA secretion both in uPAR-negative and in uPAR-expressing cells. Accordingly, uPAR depletion impairs uPA expression in cells which constitutively express both uPA and its receptor. uPAR exerts its regulatory effect through the activation of the ERK mitogen-activated protein kinases (MAPKs), whereas the p-38 MAPK is not involved. Overexpression of truncated forms of uPAR, lacking the N-terminal domain (DI) and not able to interact with membrane co-receptors, failed to increase uPA expression. Inhibition of uPAR-integrin interaction by the specific P-25 peptide, as well as Gi-protein inhibition by cholera pertussin toxin or depletion of the GPC receptors for fMLF (fMLF-Rs) also, impaired uPAR capability to regulate uPA expression. These findings demonstrate that uPAR, whose expression is regulated by uPA, can, in turn, regulate uPA expression through a mechanism involving its functional interaction with integrins and fMLF-Rs

Identification of chemically diverse Cdc25 phosphatase inhibitors by receptor-based virtual screening

The cell division cycle 25 (Cdc25) family of proteins are highly conserved dual specificity phosphatases that regulate cyclin-dependent kinases, the main gatekeepers of the eukaryotic cell division cycle. The three isoforms of Cdc25, including Cdc25A, Cdc25B and Cdc25C, appear to act on different cyclin-dependent kinase/cyclin complexes at different stages of the cell cycle. Overexpression of Cdc25A and/or Cdc25B, but not Cdc25C, has been detected in numerous cancers and is often correlated with a poor clinical prognosis; Thus, the inhibition of these phosphatases may represent a promising therapeutic approach in oncology [1-2]. So, a computer-aided drug design protocol involving virtual screening was performed on Cdc25B crystal structure (Figure 1) [3] in order to identify novel classes of inhibitors. In vitro experiments carried out on a selected list of 30 molecules led to the discovery of 4 compounds able to inhibit Cdc25A and B activity at low micromolar concentrations and to the significant inhibition of the MCF-7 breast cancer cell proliferation. All selected compounds also affected MCF-7 cell cycle progression. Furthermore, kinetics studies were realised on the phosphatase activity catalysed by Cdc25B in the presence of the above-mentioned compounds, in order to establish type and power of inhibition

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

Besides focusing urokinase (uPA) proteolytic activity on the cell membrane, the urokinase receptor (uPAR) is 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 chemotaxis receptors, triggering cell-signalling pathways that promote tumor progression.(1) The ability of uPAR to coordinate binding and degradation of extracellular matrix and cell signalling makes it an attractive therapeutic target in cancer. We used structure-based virtual screening (SB-VS) (2) to search for small molecules targeting the uPAR binding site for VN. 41 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 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 with IC50 values of 3.6 and 1.2 μM, 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 interaction between uPAR and FPR1, the high affinity fMLF-R. Both compounds blocked in vitro extracellular-matrix invasion of several cancer cell types and could represent new promising leads for pharmaceuticals in cancer.(3