Ricerca | Progetti

IDENTIFICATION AND VALIDATION OF SMALL MOLECULES INHIBITING THE UROKINASE RECEPTOR

Many leukemia and cancer patients still have poor prognosis, despite prominent achievements in anticancer therapy. Mechanisms of drug resistance limit therapeutic efficacy, especially in heavily pretreated patients. Thus, identifying additional drug targets may lead to the development of novel therapeutic combinations that will help overcome resistance and disease progression. This project aims to identify, as new therapeutic agents, small molecules inhibiting uPAR activities in cancer and leukemia cells. The study is organized in four tasks (T) and activities (A). T1. Identification by SBVS of soluble organic small molecules targeting uPAR binding to VN and uPA and uPAR interactions with f-MLF-Rs and integrins. A1. Soluble organic small molecules that might inhibit uPAR interactions at the cell surface were selected trough a SBVS approach in a previous collaboration with Prof. Antonio Lavecchia, Department of Pharmacy, University Federico II, Naples, Italy. SBVS identified: - 41 lead compounds predicted to inhibit uPAR binding to VN; - 48 lead compounds predicted to inhibit uPAR binding to uPA; - 31 lead compounds targeting uPAR interaction with alpha5beta1 integrin; - 36 lead compounds targeting uPAR interaction with alphavbeta3 integrin; - 31 lead compounds predicted to inhibit uPAR interaction with f-MLP-Rs. T2. Biological selection of active hits from the lists of compounds identified in T1, in vitro evaluation of their functional effects in cancer and leukemia cell lines and in primary cell cultures. A2.1 The first biological screening of compounds predicted to inhibit uPAR activities will be performed on uPAR-negative HEK-293 epithelial cells transfected uPAR (uPAR-293 cells), using the parental cell line transfected with the empty vector (V-293 cells) as a control. Compounds able to inhibit uPAR-293 in vitro cell adhesion to VN, without exerting any effect on V-293 cells were already identified by our group. Other compounds from T1 will be tested fortheir ability to inhibit 125I radiolabeled uPA binding to uPAR-293 cells; for their ability to block uPAR interaction with integrins by in vitro cell adhesion, migration and proliferation assays; for their ability to block uPAR interaction with f-MLP-Rs by in vitro cell migration assays toward uPA and fMLF. A2.2 All compounds active on uPAR-293 cells and inactive on V-293 cells will be then tested on panels of human cancer and leukemia cell lines for their ability to inhibit cell adhesion, migration and proliferation. A2.3 Finally, active compounds will be tested on primary cell cultures derived from tumor and leukemia types more susceptible to the action of selected inhibitors. T3. Determination of IC50 and KI of the selected compounds. A3 IC50 and Ki of selected compounds will be calculated by Biacore analysis on purified receptor and by non linear regression analysis from concentration response curves in biological assays specific for each compound. Compounds active in the low micromolar range will be selected. T4. Study of the ability of compounds selected in T3 to inhibit tumor and leukemia growth and dissemination in mouse models. A4.1 To determine the pharmacokinetic parameters of selected compounds from T3, a metabolic/pharmacokinetic study in rat by high-performance liquid chromatography (HPLC) with a Nanodrop Thermo spectrophotometer will be conducted. The pharmacokinetic parameters (AUC, t1/2, tmax, cmax) will be calculated. A4.2 Cancer and leukemia cell lines more susceptible to the action of selected inhibitors, as emerged from T3, will be xenografted in Balb/c athymic nude mice and established tumors and leukemias will be treated with uPAR inhibitors. Tumor and leukemia growth and diffusion will be evaluated in trated and untreated mice. Thus, the major output of the project will be the identification of orally-available, minimally toxic small molecules to be administered in an adjuvant setting to prevent recurrences in high risk patients.