Novel high-throughput screening system for identifying STAT3–SH2 antagonists

Synthesis of new dithiolethione and methanethiosulfonate systems endowed with pharmaceutical interest

“…The effects of the final compounds on STAT3 dimerization were determined by the AlphaScreen-based assay, 24 an in vitro competitive binding test used to identify compounds able to directly inhibit the binding of SH2-containing proteins to their correspondent phosphopeptides. To check the selectivity on STAT3, the new products were tested also on the highly homologous (78%) STAT1.…”

Synthesis, structure–activity relationships and stereochemical investigations of new tricyclic pyridazinone derivatives as potential STAT3 inhibitors

“…Binding of biological partners brings donor and acceptor beads into close proximity and as a result, a uorescent signal between 520 and 620 nm is produced. The alphaScreen-based assays 18 were performed in a nal reaction volume of 25 mL of the assay buffer containing 10 mM HEPES-NaOH (pH 7.4), 50 mM NaCl, 1 mM EDTA (pH 8.0), 0.1% NP-40, and 10 ng mL À1 BSA in a 96-well microtiter plate at 25 C. Phospho-Tyr (pTyr) peptide probes used in this study were 5-carboxyuorescein (FITC)-GpYLPQTV for STAT3, FITC-GpYDKPHVL for STAT1, and FITC-PSpYVNVQN for Grb2. Firstly, 75 nM of each SH2-containing protein was incubated with the test compound for 15 min.…”

Biological and computational evaluation of an oxadiazole derivative (MD77) as a new lead for direct STAT3 inhibitors

“…Binding of biological partners brings donor and acceptor beads into close proximity and as a result, a fluorescent signal between 520 and 620 nm is produced. The AlphaScreen-based assays 13 were performed in a final reaction volume of 25 mL of the assay buffer containing 10 mM HEPES-NaOH (pH 7.4), 50 mM NaCl, 1 mM EDTA (pH 8.0), 0.1% NP-40, and 10 ng mL À1 BSA in a 96-well microtiter plate at 25 C. Phospho-Tyr (pTyr) peptide probes used in this study were 5-carboxyfluorescein (FITC)-GpYLPQTV for STAT3, FITC-GpYDKPHVL for STAT1, and FITC-PSpYVNVQN for Grb2. Firstly, 75 nM of each SH2-containing protein was incubated with the test compound for 15 min.…”

“…To investigate the direct binding properties of several 3,4-disubstituted-1,2,5-oxadiazoles 12 to the SH2 domain we performed the AlphaScreen-based assay, 13 as described in the Experimental section. In particular, besides STAT3, other SH2-containing proteins, such as STAT1 and Grb2 (''Growth factor receptor-bound protein 2''), having a high degree of sequence homology to STAT3 (78% and 65%, respectively) were tested.…”

“…The interesting results led us to investigate if these compounds were able to directly interact with STAT3. Therefore, they were submitted to the AlphaScreenbased assay, 13 an in vitro competitive binding test used to identify compounds able to directly inhibit the binding of SH2-containing proteins to their correspondent phosphopeptides, the physiological ligands. Quite unexpectedly, only N-[4-(4-chlorophenyl)-1,2,5-oxadiazol-3-yl]-4-(trifluoromethyl)benzamide (MD77) proved to be able to significantly interact with the SH2 domain.…”