Discovery of New Inhibitors of Cdc25B Dual Specificity Phosphatases by Structure-Based Virtual Screening

Abstract

Cell division cycle 25 (Cdc25) proteins are highly conserved dual specificity phosphatases that regulate cyclin-dependent kinases and represent attractive drug targets for anticancer therapies. To discover more potent and diverse inhibitors of Cdc25 biological activity, virtual screening was performed by docking 2.1 million compounds into the Cdc25B active site. An initial subset of top-ranked compounds was selected and assayed, and 15 were found to have enzyme inhibition activity at micromolar concentration. Among these, four structurally diverse inhibitors with a different inhibition profile were found to inhibit human MCF-7, PC-3, and K562 cancer cell proliferation and significantly affect the cell cycle progression. A subsequent hierarchical similarity search with the most active reversible Cdc25B inhibitor found led to the identification of an additional set of 19 ligands, three of which were confirmed as Cdc25B inhibitors with IC(50) values of 7.9, 4.2, and 9.9 μM, respectively.

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

“…Indeed, uPAR-293 cell adhesion to increasing concentration of vitronectin, in the absence 50 , resulted in a significant loss of uPAR-293 cell binding to vitronectin (Fig. 5B).…”

“…Therefore, we investigated, by pull-down assays, whether compounds 6 and 37 were able to block Rac-1 activation in response to vitronectin. Serum-starved uPAR-293 cells were plated on vitronectin-coated wells for 20 minutes in the presence of both ligands, at 2Â IC 50 , or DMSO, as a negative control. Active Rac-1 (Rac-1/GTP) was precipitated from cell lysates using the p21-binding domain (PBD) of its target, PAK1, bound to agarose beads; Rac-1/GTP was eluted from beads and estimated by Western blot analysis with a monoclonal antiRac-1 antibody.…”

“…uPAR-coimmunoprecipitates with FPR1; treatment with compounds 6 or 37 significantly reduced uPAR association to FPR1. C, highly invasive HT1080 sarcoma cells were treated with DMSO (À) or with compounds 6 or 37 (INH), at their 2Â IC 50 , and allowed to invade Matrigel, using as chemoattractants cell culture medium without serum (ÀFCS), to evaluate spontaneous invasion, or with 10% serum (þFBS). Invading cells on the lower side of the membrane were fixed, stained, and photographed; a representative image is shown.…”

“…Invading cells on the lower side of the membrane were fixed, stained, and photographed; a representative image is shown. D, HT1080 sarcoma cells ( ), HCT human colon cancer cells ( ), PC3 human prostate cancer cells ( ), and MDAMB231 human breast cancer cells ( ) were treated with DMSO (À) or with compounds 6 or 37, at their 2Â IC 50 , and allowed to invade Matrigel, using as chemoattractants cell culture medium without serum (ÀFCS) or with 10% serum (þFBS). Invading cells on the lower side of the membrane were fixed, stained, counted (magnification, Â200), and the results were expressed as a percentage of the invasion in the absence of chemoattractant (ÀFBS).…”

Antileukaemic effect of PI3K‐mTOR inhibitors in acute myeloid leukaemia‐gene expression profiles reveal CDC25B expression as determinate of pharmacological effect

“…We therefore investigated the CDC25 inhibitor NSC 95397 (Larsson et al, 2010;Lavecchia et al, 2012), which caused a dose-dependent inhibition of cytokine-dependent AML cell proliferation (data not shown). This drug had an additive antiproliferative effect to GDC-0941 or rapamycin only for two (K562, HEL) out of the five leukaemic cell lines examined (not for HL60, KG1a and EOL-1; data not shown).…”