Figure 5.

Identification of pan-SHIP inhibitors. (A–C) (i) Structures of 1PIE (A), 2PIQ (B) and 6PTQ (C). (ii) Fluorescence polarization assay shows inhibition of recombinant SHIP1 activity by 100 μmol/L 1PIE (A), 2PIQ (B) and 6PTQ (C). The decrease in mean polarization units without compound (–) is inhibited by adding 1PIE, 2PIQ or 6PTQ to the reaction mix (+). (iii) Comparison of efficiency of recombinant SHIP1 and SHIP2 inhibition by increasing concentrations of inhibitors, as determined by either Malachite Green assay (2PIQ, B) or fluorescence polarization assay (1PIE [A] and 6PTQ [C]). (D) Recombinant SHIP2 and SHIP1 precipitated from OPM2 cell lysates were allowed to dephosphorylate PtdIns(3,4,5)P₃ in the absence (DMSO) or presence of inhibitors. OCRL, precipitated from OPM2 cell lysates, was allowed to dephosphorylate PtdIns(4,5)P₂ in the presence of inhibitors or DMSO control. Free phosphate groups were detected with Malachite Green reaction. The percentage inhibition of phosphatase activity in two independent immunoprecipitation experiments is represented (means ± SEM).